Control of ciliary activity in Paramecium: An analysis of chemosensory transduction in a eukaryotic unicellular organism

Progress in Neurobiology Vol. 16. pp. 1-115

0301-0082/81/0601-0000505.00,0

© Pergamon Press Lid 1981. Printed in Great Britain

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM: AN ANALYSIS OF CHEMOSENSORY T R A N S D U C T I O N IN A EUKARYOTIC UNICELLULAR ORGANISM MIKE J. DOUGHTY* and STANISLAWDRYLf *Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221, U.S.A. f Department of Cell Biology, M. Nencki Institute of Experimental Biology, 3, Pasteur Street, 02-093 Warsaw, Poland [Received 18 September 1980)

Contents 1. Use of unicellular organisms for the study of the mechanisms of sensory transduction 1.1. Molecular mechanisms of sensory transduction: A perspective 1.2. Paramecium: A free-living, motile sensory cell with an excitable membrane 2. Localization and structural components of the sensory transduction system in Paramecium 2.1. Electrical excitability in Paramecium, its existence, its relationship to cell behavior and the localization of the electrical excitability in the ciliary membrane 2.2. Structure, protein composition and activity of the ciliary membrane 2.3. Functional role of the ciliary membrane 2.4. Structure, protein composition and enzyme activity of the ciliary axoneme 3. Passive behavior, ciliary activity and electrical properties of Paramecium 3.1. Swimming characteristics of Paramecium in different chemical environments 3.2. Characteristics of ciliary motion 3.3. Membrane resting potential in different environments 3.4. Cation fluxes across the surface membrane of Paramecium 4. Active behavioral responses, ciliary activity and electrical responses following depolarizing stimuli under different physical and chemical conditions 4.1. Ciliary reversal behavior in Paramecium to inorganic cation salts 4.2. Inorganic cation-induced ciliary reversal behavior in Paramecium: A pharmacological analysis 4.3. Ciliary reversal and the Ca-regenerative response 4.4 Ca 2 +-gating: Activation, inactivation and control 4.5. Ca 2 +-modification of the ciliary mechanochemical cycle 4.6. Renormalization of ciliary activity: Evidence for a Ca 2+ pump and its control 4.7. Ciliary metachrony: Hydrodynamic vs neural control? 5. Summary and perspectives Acknowledgements References

1 1 3 7 7 11 20 26 33 33 40 44 48 55 55 63 71 75 79 88 92 93 98 98

1. Use of Unicellular Organisms for the Study of the Mechanisms of Sensory Transduction 1.1. MOLECULAR MECHANISMS OF

SENSORYTRANSDUCTION:A PERSPECTIVE

The last 25 years research in molecular and cell biology have resulted in a rapid development of our knowledge and understanding of the physiological, pharmacological and biochemical responses of a variety of chemically and electrically excitable membranes from a wide variety of tissue types and cells in higher phyla. The choice of cells or tissue types for such studies has largely been determined by both current interests and, perhaps more importantly, as a result of the availability of material that was readily amenable to electrophysiological, pharmacological and physiological analysis. Sensory membranes are distributed at key points throughout a multicellular organism and include sites of intersynaptic relay, synaptic junction relay to either skeletal or smooth muscle types, and sensory perception at either olfactory, gustatory or photoreceptive organelles. In short, all of these sites exhibit one common phenomenon--that of sensory tranduction. This process can generally be described in which an incident physiological signal is converted to another with, usually, concommitant regulation of both the J.P.~. 16,,I--A

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FIG. 1. Theoretical components of an excitable sensory system and their localization in Paramecium. Based on Grundfest (1966) and Dryl (1974).

signal frequency and amplitude emergent from the "output" side of the system. The "input" side of the system is often characterized by the presence of certain, often specific, receptor molecules that are suitably sited and arranged so as to maximize perception and processing of incoming signals whether these be ions, biogenic amines, other amino acids, odorant molecules or photons. Such receptor molecules are necessarily functionally coupled to the operation of an excitable membrane. As a result of the operation of this membrane, an "output" signal is produced which is invariably of a different nature to the stimulus signal. The produced chemical messenger then acts on either internal components of the same localized system or may act upon the input component of an adjacent sensory cell. Such receptor-effector molecular mechanisms are generally considered as being derived from early ancestral forms and preserved throughout evolution (Grundfest, 1966). For the majority of excitable membrane systems, the input component is generally stimulus (chemo-) sensitive and acts as a transducer to convert the stimulus information into an electrical response of a depolarizing or hyperpolarizing type. Changes in the resting potential of the cell membrane often appear as graded receptor potentials which can lead to generation of active responses (spike potentials) of either a graded or all-or-nothing nature. Associated with the occurrence of active potential changes is release of chemical messengers (Fig. 1). Rapid progress has been achieved in our understanding and knowledge of both the electrophysiological characteristics and pharmacological sensitivity of some of these sensory membranes in addition to knowledge of the nature of the chemical messengers associated with their operation. However, we still have only a limited understanding of the molecular mechanisms responsible for such delicately controlled information processing. Problems have been encountered in both the isolation and, more particularly, in the isolation in an active state of either the receptors or ion channelling proteins of the membranes. The major problems are of a two-fold nature. Firstly, most sensory membranes upon which molecular biochemistry studies have been carried out are located within widely differentiated tissue and it has proved very difficult from a technical point of view to isolate the membrane of interest from both other membranes and tissue components. Secondly, those tissues that have consistently been the choice material for electrophysiological and pharmacological characterization have, to date in the few cases where even reasonable purification has been achieved, yielded only small amounts of material for biochemical and molecular analysis (e.g. axonal membranes). Studies on isolated nerve endings (synaptosomes) have resulted in considerable development of our understanding of the metabolic and energy dependence of ion and neurotransmitter release and similar work has been carried out on tissue slices from a variety of sensory loci. Several uniquely specialized tissues have provided, and will continue to provide, material for analysis of sensory perception and transduction (e.g. stimulus-secretion coupling) and provide material for biochemical analysis of such phenomena at the molecular level in terms of the receptor molecules and enzymes mediating such phenomena.

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

3

The electric organ (electroplax) has proved to be one such source of material for analysis of chemical control of ion permeability (flux) activity as well as more recently providing material for molecular dissection of both receptor and channel components of an excitable membrane. Successful isolation, electrophysiological analysis, pharmacological and biochemical studies on nerve cell lines in culture should prove instrumental in elucidation of the molecular components and activity of excitable membranes. A variety of membranes have been used as source material for the purification and reconstitution of a variety of transport proteins for ions and other species. The characteristics of such isolated components in artificial (lipid) membranes have been studied in pursuit of an understanding of the molecular operation of such components in their natural membrane. Application of these techniques to, e.g., isolation and reconstitution of components from either electroplax or isolated excitable cell lines, should lead to elucidation of the character and action of components associated with the generation of active potential changes in the model sensory system detailed above and thus supplement information obtained on the receptor and secretory sides of the system determined largely on other systems. We can anticipate that, through such systematic studies of the molecular components, molecular form and biochemical activity of sensory membranes both as isolated and as reconstituted back into defined artificial systems, will lead to elucidation of the molecular mechanisms underlying the physiological response of sensory membranes from a variety of sources. In the past few years especially, attention has turned to the use of free-living unicellular eukaryotic cells for such molecular dissection studies of sensory perception and sensory transduction in addition to their already exploited use as model systems for studies on cell development, cellular biochemistry related to the natural cell cycle and a variety of other biochemical studies relating to the biochemical energetics and overall metabolism in a eukaryotic cell. Molecular dissection of sensory transduction should be facilitated by use of a free-living sensory cell, since studies on sensory recognition and perception, studies on membrane transduction at both electrophysiological and molecular biochemical levels and on the physiological response of the sensory system can be carried out on the same cell. The eukaryotic unicell Paramecium, a holotrichous ciliated protozoan, is one of such cells receiving considerable interest in recent years for both the molecular dissection of sensory transduction and for the molecular analysis of a gated calcium ion ionophore in an excitable membrane. 1.2. PARAMECIUM: A FREE-LIVING, MOTILE SENSORY CELL WITH AN EXCITABLE MEMBRANE

Since the turn of this century, various workers have studied the structure, physiology, pharmacology and sensory behavior of single-celled eukaryotic organisms in the belief that such unicellular organisms would serve as simple model systems for the study of a variety of processes related to the physiological, pharmacological and other sensory responses shown by organs in higher phylogenetic species. In such studies, often additionally prompted by a desire to show uniformity across phylogenetic classes in terms of the manner in which cell development (growth) or organelle activity was controlled, it was reasoned, in one way or another, that the sensory responses of unicells were uncomplicated by the presence or overt control of either a central nervous system or a compartmentalized endocrine system (Abderhalden and Schiffman, 1922; Ball, 1926; Bauer, 1926; Worley, 1934; Wense, 1934, 1935; Bramstedt, 1937; Seaman and Houlihan, 1951; Aaronson, 1963). An equally popular concept prevailed in the earlier part of this century, especially in that it was widely believed, at least amongst protozoologists and some neurophysiologists, that eukaryotic unicellular organisms did in fact possess an intricate nervous system (Sharpe, 1914; Yocum, 1918; Taylor, 1922; Rees, 1922; Parker, 1929). The free-living ciliated protozoan cell, Paramecium, was consistently the organism of choice in such studies and also for widespread studies on the behavior of protozoa in general, probably in view of its widespread occurrence and availability. Paramecium is

4

M . J . DOUGHTY and S. DRYL

favored now largely due to the extensive nature of such behavioral studies. In addition, the biology, physiology and genetics of this cell have been extensively studied over the last 80 years, as evidenced by the appearance of three monographs on the cell (Kalmus, 1931 ; Wichterman, 1953; Van Wagtendonk, 1974). Paramecium are free-swimming and prescribe very characteristic swimming paths through their aqueous habitat as a result of the coordinated activity of many cilia which are distributed over the entire body surface (Jennings, 1897; Alverdes, 1922; Bullington, 1925, 1930). The activity of these body (somatic) cilia alone is responsible for the locomotion of Paramecium. Non-lethal removal of the cilia (deciliation) results in immobilization of the cells (Alverdes, 1922; Kuznicki, 1963a; Kennedy and Brittingham, 1968). Upon regrowth of the cilia, the cells become motile again (Kuznicki, 1963a; Dunlap, 1977). The cilia thus enable the cell to move quickly from one environment to another. Regulation of either the frequency or direction of ciliary activity permits the cell to either avoid unfavorable conditions (extremes of pH, temperature, light; toxic chemicals or metabolic waste products or conditions of ionic strength unsuitable for normal osmoregulation of the cells' cytoplasm: see Jahn and Bovee, 1968), or to linger longer in favorable conditions (abundance of bacterial food, optimum pH, temperature and chemicals for growth and ionic conditions suitable for osmoregulation). In response to external stimuli (pH, temperature, chemicals and electrical current), the cilia can both change their frequency of beating and their relative orientation with respect to the long axis of the cell and effect marked, often rapid, alterations in the swimming behavior of the cells (Massart, 1891; Jennings, 1897, 1899a, b; Pearl, 1901 ; Dale, 1901; Bancroft, 1906a, b; Vieweger, 1912; Alverdes, 1922; Mast and Nadler, 1926; Oliphant, 1938, 1942). These behavioral responses (motor responses) have a finite duration that is dependent on both the type and strength of the applied stimulus and as such can be considered as the sensory response of the cell. An analysis of the cells' behavior in response to defined stimuli under controlled conditions allows analysis to be made of the sensory responses of the cells with respect to the sensory stimuli in their environment. Early studies indicated that the surface membrane of Paramecium was electrically responsive (Vervorn, 1889; Ludloff, 1895; Pearl, 1901; Jennings, 1905; Bancroft, 1906a, b). Later studies first showed the existence of a resting potential across the plasma membrane of the cell (Kamada, 1934) and showed that there was a close correlation between the cells' swimming velocity, ciliary activity and this resting potential (Yamaguchi, 1960a; Kinosita et al., 1964a-c, 19651. More recent studies have revealed that the surface membrane of Paramecium was in fact electrically excitable and coul~t show both graded (Naitoh and Eckert, 1968a; Naitoh et al., 19721 or all-or-nothing electrogenesis. The active electrogenesis of the surface membrane could be correlated with concommitant alterations in ciliary activity (Kinosita et al., 1964b, c, 1965; Machemer and Eckert, 1973). Paramecium (Fig. 2) can thus be viewed as a motile, free-living sensory cell capable of perception of changes in its extracellular environment and in turn showing defined sensory responses--the characteristics and duration of which are governed by the electrical activity of the surface membrane. Alterations in the orientation of Paramecium in time and space are achieved without the overt control of a central nervous system as in higher phyla and apparently without the use of a neuromotor system. Protozoan cells, and in particular Paramecium, can be conveniently grown (on defined media if required) in large quantities and at a relatively low cost compared to other sources of excitable membranes. In short, since the excitable membrane constitutes a significant portion of the cells' overall composition, Paramecium provides us with a potential, bulk and readily available source of material for electrophysiological, pharmacological, biochemical and molecular studies on an excitable membrane. If, during such an analysis, close attention is paid to the viability, sensitivity and sensary behavior of the ceils in vivo with respect to the electrophysiological, pharmacological and biochemical sensitivity in vitro of isolated fractions or components, we can perhaps be a little more certain that the isolated components of this excitable membrane either on their own or reconstituted back into an artificial membrane will show an activity that is physiologically relevant.

CONTROL t)F CILIARY ACTIVITY IN P4R4'~IE(I{ ~,l

FiG. 2. Paramecium: Interference contrast, light micrograph of a living cell of Paramecium multimicronucleatum. Cell is approx. 300/am long. Cells bathed in 9 mM CaCI> 3 mM KCI, pH 7.1, and thus show normal ciliary activity and metachrony (see Section 3.2). The cilia can be seen in profile at the edges of the cell the different positions and the grouping are the cilia at different stages of their beat cycle and the metachronal waves respectively. Photograph (provided courtesy of the author) from Machemer (1972b), and reproduced with permission of J. Mechanochem. Cell Motility, Gordon Breach Sci. Publ. Ltd.

5

CONTROLOF CILIARYACTIVITYIN PARAMECIUM

7

We shall now endeavor to present Paramecium as a sensory system, to detail what we currently know of its components and their activity and to demonstrate how analysis of the behavior, electrophysiological responses and biochemical activity of the components of this sensory system should enable us to carry out a molecular dissection of sensory transduction. We unfortunately do not know whether the molecular mechanisms underlying the operation and control of sensory transduction in this cell are the same as those in higher phyla. However, an analysis of a sensory transduction pathway at a molecular level, greatly facilitated by use of a unicell that is hopefully largely uncomplicated" by intracellular and intercellular controls of a type ubiquitously found in sensory tissues of higher phyla, should provide a sound basis for development of our understanding of the molecular mechanisms operative in any sensory transduction pathway. In addition, studies on Paramecium should further our understanding of the molecular mechanisms underlying Ca 2 +-dependent electrogenesis in excitable membranes. Studies on ciliary activity and its control in Paramecium are pertinent to our understanding of ciliary activity and its control in both tracheal cilia and also in other specialized ciliated epithelia such as found in the oviduct, brain ventricles and olfactory processes. Studies on Paramecium compliment studies on both the specialized epithelia noted above and on metazoan ciliated epithelia. In addition, it is perhaps timely to reiterate that some common morphological properties exist between olfactory cilia (Andres, 1975; Menco, 1977); a wide variety of sensory organelles (Barber, 1974); the transition zone of retinal rod cells (Rohlich, 1975, Matsusaka, 1976; Duncan, 1976) and the axoneme structure in cilia of protozoa (Ehret and McArdle, 1974: Dute and Kung, 1978) as well as the flagellar axoneme of protozoa and invertebrate and vertebrate spermatozoa (Summers, 1965).

2. Localization and Structural Components of the Sensory Transduction System in Paramecium 2.1. ELECTRICAL EXCITABILITY IN PARAMECIUM: ITS EXISTENCE, ITS RELATION TO CELL BEHAVIOR AND THE LOCALIZATION OF THE ELECTRICAL EXCITABILITY IN THE CILIARY MEMBRANE

The earliest studies reported using conventional glass microelectrode recording techniques on Paramecium showed that a potential difference of approximately 20 mV (inside negative) was maintained between the inside of the cell and the buffered, inorganic cation salt-containing solution in which the cell was bathed (Kamada, 1934). Such a result was not surprising to Kamada ('As might be expected, a measurable potential difference is found between a pair of microelectrodes, one of which is inside a Paramecium and the other in the external medium': 1934). Apart from a precedent for such a potential difference from measurements on plant cells, earlier results on Paramecium had resulted in discussion of the role of cation permeability in the control of ciliary activity. Many workers, prior to Kamada's report, had noted that Paramecium showed defined, finite duration, behavioral responses to an oriented, externally applied d.c. field (Vervorn, 1889; Ludloff, 1895; Mouton, 1889; Pearl, 1901; Greeley, 1903; Jennings, 1905: Dale, 1901; Bancroft, 1906a, b). Vervorn (1889), amongst others, noted that Paramecium swam towards the cathode when a d.c. signal was applied through the medium in which the cells were suspended. He also noted that, at low current strengths, only a small number of cells showed net cathodal migration and that the number of migrating cells increased in a manner proportional to the applied current intensity (Vevorn, 1889; Kinosita, 1939). The time course for expression of net galvanotaxis was also noted to vary with the applied current strength (Ludloff, 1895; Kamada, 1931b). In forward swimming cells, the cilia are observed to be pointing obliquely backwards from the anterior end of the cell, whereas in backward swimming cells the cilia point towards the anterior end (Vervorn, 1889; Ludloff, 1895; Jennings, 1897; Alverdes, 1922; Kamada, 1931a, b). In the galvanotaxis experiments, if the cells were initially obliquely oriented with respect to.

8

M.J. DOUGHTY and S. DRYL

the lines of the applied field, ciliary reversal was observed only on the cathodal side of the cell (the 'Ludloff Phenomena': Ludloff, 1895). The stronger the applied current, the greater the proportion of the body cilia observed in reversed beating mode (Vervorn, 1889; Ludloff, 1895; Dale, 1901; Kamada, 1931a, b). The cathodally directed, spiral, swimming paths of the cells were observed to be different from the swimming paths of cells in the absence of the applied field (forward swimming behavior) and additionally found to be influenced by the ionic strength of the medium (Pearl, 1901). Such results were in accord with those of Loeb and Budgett (1897) who noted an anodic, as opposed to cathodic, migration tendency of Paramecium in salt solutions. In addition to these reports of variation in the galvanotactic tendency of cells as a result of changes in the salt concentrations in the cells' medium, Bancroft (1906a) noted that in bathing solutions low in free-extracellular calcium ions ('devoid of Ca 2 ÷ ions') but containing low concentrations of either K ÷, Na ÷ or Ba 2÷ ions, Paramecium showed preferential migration to the anode in an applied d.c. field--i.e, in the absence of significant extracellular Ca 2 ÷ ions, other cations had somehow reversed the galvanic sensitivity of the cells (see also Mayeda, 1928; Schwab-Bonaventure, 1955). As the extracellular Ca 2 ÷ ion concentration was raised, so the occurrence of cathodal galvanotaxis increased, to become prevalent in the presence of millimolar concentrations of this ion (Bancroft, 1906a, b). Kamada (1929b) noted that cells in 100 mM KCl-containing solutions, in the presence of calcium ion concentrations above 6.25mM, showed rearward swimming behavior (the K ÷-induced ciliary reversal response:see Section 4.1). In these solutions, the distribution of cells between the cathode or anode was approximately equal. In the presence of lower concentrations of calcium ions, however, the cells swam forward on transfer to K ÷/Ca 2÷ solutions and showed anodal galvanotaxis predominantly. Similar results were obtained with K+/Sr 2÷ solutions. With Na+/Ca 2÷ solutions, anodal galvanotaxis was only observed in the presence of less than 1.5 mM CaC12. The anodal galvanotaxis, induced by monovalent cations in the presence of low concentrations of Ca 2÷ ions, is not permanent. Bancroft (1906a) notes rapid or gradual loss of anodal galvanotactic tendency in salt solutions. Kamada (1929a, b) notes gradual change from anodal to cathodal galvanotaxis in salt-treated cells and found that the rate of change appeared to be influenced both by the applied field strength and the salts present (Fig. 3). Later studies (Kamada, 1931a) showed that d.c. stimulation of Paramecium bathed in high concentrations of Ba 2 ÷ salts (25-50 mM), effected a temporary suppression of the action of the Ba 2 ÷ ions I00

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FIG. 3. Gatvanotaxis in Paramecium: Temporal changes in ciliary activity, and thus cell swimming direction, induced by external, d.c., electrical stimulation of Paramecium caudatum. Influence of applied voltage and cations. Left: 26 V; right: 6 V. Solutions contain (approx.) 0.t25 mM CaCI 2 and 100m~ NaCI----O, II or 0.02ram CaCI 2 and 83mM KCI-----@, rq. 15-17°C. The index of galvanotaxis = C/C + A where A is the number of cells with their anterior end facing the anode and C is the number of cells with the anterior end facing the cathode. Data taken from Kamada, 1929b.

CONTROL OF CILIARY ACTIVITY IN P4RAMECIUM

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0.80 mA FIG. 4. Galvanotaxis in Paramecium: Changes in ciliary orientation induced by d.c. stimulation of Paramecium caudatum. Effects of current strength. Cells in 20-50 mM Na2CO3, < 10 -4 M CaCI2 (estd.), and 25-50 mM BaCl2 21-26°C. The unstimulated cell (diagram inserted for comparison) has a ciliary orientation of a type shown by rearward swimming cells. Ciliary orientation given for the current strengths indicated. Diagrams taken from Kamada, 1931a.

on the cell (induce ciliary reversal): cilia on the anodal side of the cell could be reversibly restored to forward beating by the d.c. current with the galvantotactic orientation being proportional to the applied current strength to the extent that high current intensities reverse the forward beating cilia on the cathodal end of the cell to reversed beating (Fig. 4). Virtually all cations tested, unless severely toxic, could induce anodal gaivanotaxis in cells 'normally' cathodally galvanotactic (Bancroft, 1906a, b; Kamada, 1929b). Anions were generally found to have little effect. Notable exceptions amongst the cations tried were Ca 2+ and Sr 2+ (Bancroft, 1906a; Kamada, 1929b), but, as proposed by Bancroft (1906a), the relative ratio of Ca 2 + to other cations appeared to play a crucial role in determination of both the sign and time dependence of the galvanotactic response. This phenomena of temporally oriented movement or ciliary activity of Paramecium in an applied d.c. field and its antagonism or augmentation by cations can be taken as early indicators of the role of ionic gradients in the control of ciliary activity. However, as correctly noted by early workers (Bancroft, 1906b; Kamada, 1931a, b), the laws governing electrical stimulation and response in Paramecium did not conform with the all-ornothing laws already established in nerve. Early workers attributed the phenomena of galvanotaxis to polar effects and vectors in the cell cytoplasm (Vervorn, 1889); to elimination of acid and alkali at the poles of Paramecium (Mouton, 1899) [Mayeda (1928) and Mayeda and Date (1929) note preferential anodal galvanotaxis in alkali solution]; to general electrophoretic effects (Coehn and Barratt, 1905); or to local changes in the calcium ion content of the cell with respect to other cations in the cytoplasm of the stimulated cell (Bancroft, 1906a). Despite excellent contemporary evaluation (Jahn, 1961, 1962b), the mechanisms underlying and controlling the phenomena of galvanotaxis in Paramecium still remain somewhat of an enigma. Such early studies however, without question, serve to demonstrate the depth of thought of early workers. Jahn's (1962a).

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interpretation of the phenomena on the basis of a membrane sited Ca2+/cation exchanger are in accord with some aspects of current thought (see later). In complete accord with proposals of differential surface and cytoplasmic polarizability of the Paramecium as a means to explain galvanotaxis (Vervorn, 1889), we can now consider that the occurrence of such electrically induced phenomena and their perturbation by cations, reflects the presence of either a surface (0 zeta potential or membrane (A@) potential in Paramecium---the latter being verified by Kamada (1934). The presence of a zeta potential as a means to control ciliary activity was proposed by Andrejewa (1931) and tested by studying the effects of various salts on the swimming speed of the cell. A close correlation was noted between the profiles for independently measured zeta potentials of colloidal particles as a function of the concentrations of M +, M 2 + and M 3 + cations and the effect of the same on the swimming speed of Paramecium. Direct correlations between ciliary activity and the plasma membrane potential in Paramecium did not come until the 1960s, although related work on another ciliated protozoan, Opalina, had shown correlation between change in ciliary orientation (ciliary reversal) and depolarizing currents delivered via an intracellularly located microelectrode (Okajima, 1953; Kinosita, 1954; Naitoh, 1958). Yamaguchi (1960a~ showed that external electrical stimulation (galvanic stimulation) of a Paramecium cell not only effected ciliary reversal, but that concommitant depolarization (recorded by use of an intracellularly located KCl-filled microelectrode) of the cell membrane also occurred. In agreement with the earlier results of Kamada (1934), Yamaguchi (1960a) also noted depolarization of the cell membrane by application of inorganic cations to the extra,cellular solutions and showed both short-lived depolarizations following addition of the salts (correlated with the occurrence of ciliary reversal onset) and shifts in the steady-state (resting) potential across the surface membrane. Some correlations between the resting potential, ciliary activity (beating frequency) and the swimming speed of Paramecium were provided by Kinosita et al. (1964b), by recordingthese three parameters for cells bathed in a variety of salt solutions (Fig. 5). Substantive evidence for a correlation between the depolarization of the plasma membrane and changes in ciliary reversal came from the same authors (Kinosita et aL, 1965a, c, 1965) who found that, on bathing Paramecium in Ba2+/Ca 2+ solutions, regular all-or-nothing spike depolarizations occurred coincident with transient ciliary reversal events that lasted the duration of the spike depolarization event (Fig. 6). Subsequent notable studies of workers at the University of California, Los Angeles (Naitoh and Eckert, 1968a, b; Naitoh et aL, 1972; Friedman and Eckert, 1973; Machemer and Eckert, 1973) provided the links between electrical stimulation, ege~icat activity, ciliary activity and both calcium ions and other (monovalent) cations. Their collective studies estabti!th~! that the surface membrane of Paramecium shows graded, Ca 2 +-deIgndent electrogenesis (of a type similar to that found in invertebrate smooth muscle fibers

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

11

1 sec FIG. 6. Active electrogenesis and ciliary activity in Paramecium. Spontaneous, all-or-nothing spike potentials observed in Paramecium caudatum bathed in solution of 2 mM BaCI2 and 1 m u " CaCI 2. pH 7.2. 19-21°C. Ciliary reversal is coincident with the spike potentials. From Kinosita, Dryl and Naitoh, 1964a.

(Werman et al., 1961; Werman and Grundfest, 1961; Hagiwara, 1976)) in response to outwardly directed injected current pulses. This Ca 2 +-dependent regenerative depolarization appeared concommitant with the onset of ciliary reversal, and this correlation was later studied in further detail (Machemer and Eckert, 1973; Machemer, 1974a). The electrophysiological analysis demonstrates the presence of surface membrane located, voltage-dependent Ca 2 +-channels as a primary element in the control of ciliary activity (the mechanochemical basis of which is known to be Ca2+-regulated: Naitoh and Kaneko, 1972). Recent studies (Dunlap, 1977; Ogura and Takahashi, 1976; Machemer and Ogura, 1979) suggests that these Ca 2 + channels are predominantly, if not exclusively, located on the ciliary membrane rather than distributed over the entire plasma membrane (ciliary and somatic membranes). An isolated cilium of Paramecium, thus, in addition to the contractile protein-enzyme components responsible for ciliary motion and its control per se (Doughty, t979b), appears to contain the Ca 2 + channels responsible for regulation of the ciliary activity at the membrane level (Dunlap, 1977; Machemer and Ogura, 1979). The isolated cilia, which can be fairly easily removed from the cell in pure state free of cellular components, thus provide an enriched starting material for biochemical studies on a sensory transduction pathway in addition to containing, as a key interest component from an electrophysiological and neurobiological standpoint, the components of a gated, voltagedependent Ca 2 + channel suitable for molecular dissection and reconstitution studies. To facilitate understanding of the following analysis of the components of the chemosensory transduction pathway in Paramecium and our current knowledge and understanding of its operation, a schematic presentation of its functional components and their localization is given below (Fig. 7). 2.2. STRUCTURE, PROTEIN COMPOSITION AND ACTIVITY OF THE CILIARY MEMBRANE

Conventional sectional electronmicroscopy of fixed specimens of Paramecium cilia and their domain (Watson and Hopkins, 1962; Kennedy and Brittingham, 1968; Jurrand and Selamn, 1969; Ehret and McArdle, 1974) shows that the cilia are inserted through a series of three membranes (see also Allen, 1967 for a three-dimensional schematic presentation of the ciliary domain) (Figs 7 and 8). Each cilium has a terminal "root'--the basal body or kinetesome which is thought to serve as a kind of anchor for the cilium (Pitelka, 1974). An apparently continuous membrane covers both the surface of the cell (the interciliary or somatic membrane) and the cilium (the ciliary membrane). At this time, little is known about the structure or molecular components of the ciliary membrane. The entire plasma membrane of Paramecium is covered with a diffuse, ruthenium red-stainable, surface coat (Wyroba and Przelecka, 1973). The same authors reported that the surface coat density and organization was significantly reduced following treatment of Paramecium with commercially available preparations of either pronase, trypsin or neuraminidase enzymes. Whilst the sensitivity of the coat to proteolytic enzymes indicates the proteinaceous nature of the coat, the presence of substrates for the neuraminidase enzyme is uncertain since, as subsequently reported, the surface coat is labile in buffer solutions alone (Wyroba, 1980). Since Luft (1971) proposed that ruthenium red shows a certain degree of specificity for sialic acid (although will also bind to other

12

M . J . DOUGHTY and S. DRYL

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somatic membrane

\ ciliary necklace ---. terminal/basal plate k inetesome/basal body

FIG. 7. The cilium and its d o m a i n in Paramecium. Schematic illustration to show the basic structures believed to be relevant to sensory transduction.

species such as lipids with a high negative charge density), it was thought that the surface coat contains glycoproteins, in particular sialic acid. Another polycationic dye, Alcian Blue, also recognized as having a certain affinity for glycoproteins (Luft, ¿97•), has been used to visualize a surface coat in a related ciliated protozoan, Tetrahymena (Nilsson and Behnke, 1971). For both ruthenium red and Alcian Blue adsorption, the quality of the surface coat revealed was found to vary with factors such as the cell culture age and culture medium and also with the solutions in which the cells were suspended prior to fixation (Wyroba, 1980). The lectin, concanavalin A, has been shown, in a qualitative manner, to bind to the plasma membrane of Paramecium fWyroba, 1975a, b), with the cilia (i.e. ciliary membranes) of Tetrahymena (Csaba and Madarasz, 1979) and with SDS-denatured ciliary membrane proteins of Paramecium separated by high-resolution sodium dodecylsulphate-polyacrylamide gels (Merkel et al., 1980). At this time, the role of either the surface coat or the lectin-binding proteins is uncertain. Wyroba (1977a) reported that immobilization of Paremecium cells by homologous antiserum (through the i-antigen system--see below) was altered by preincubation of the cells with commercial preparations of either pronase or typsin, prior to exposure to the antiserum. Such treatment, in common with high concentrations of salt and ethanol (which solubilize the i-antigen of the cell membrane; see Preer, 1959; Wyroba, 1977b), also remove the surface coat (Wyroba, 1977b, 1980). The immobilization of Paramecium by homologous antiserum was reported to be unaffected by preincubation of the cells with concanavalin A (Capdeville, 1979). Another surface related phenomena in Paramecium concerns the initial reactions between cells that are normally prerequisite to sexual pairing and conjugation (Butzel, 1974). Mating reactivity is destroyed by mild treatment of Paramecium cells with prote01ytic enzymes (Metz and Butterfield, 1951; Cohen and Siegel, 1963; Kitamura and Hiwatashi, 1976; Watanabe, 1977a) but not neuraminidase (Kitamura and Hiwatashi, 1978). Frisch et al., (1976) reported that concanavalin A only mildly affected Tetrahymena but specifically altered mating reactivity through an as yet unidentified gtycoprotein(s) (Frisch et al., 1977a, b). Several reports (Wiese, 1974; Wiese et al., 1975, 1978; Jamieson et al., 1978; Ray et al., 1978) indicate a glycoprotein nature to the mating type substances in the flagellated alga, Chlamydomonas. However, current evidence indicates that this is not necessarily so in Paramecium (Kitamura and Hiwatashi, 1976, 1978, 1980), although further experimentation appears necessary before definite conclusions can be drawn since the mating type substances have not yet been isolated, The mating reactivity however appears to be ciliary membrane located (Miyake, 1964; Kitamura and Hiwa-

CONTROL OF CILIARY ACTIVITY IN PARA.'~IECIUM

FIG. 12. Protein composition of the cilium, ciliary membranes and demembranated ciliary axoneme of Paramecium aurelia. Gradient polyacrylamide gel containing sodium dodecyl sulphate of, from left, purified i-antigen, molecular weight markers (220, 120, 90. 68 and 43 Kd), isolated cilia, ciliary membranes, heavy membrane fraction,,'incompletely demembranated axonemes and demembranated ciliary axonemes. Doughty and Kaneshiro, unpublished.

13

14

M..1. Dot GH]~ and S. DR~I.

iary necklace

00 w

"--

-

~I00

I

nm

~~

/ ~ "

I

FIG. 9.

FIG. 10.

FIG. 11.

FIGS. 9 1 l. Calcium-dependent deposits in the cilia of Paramecium. Figure 9, (top). Schematic illustration of the arrangement of the ciliary membrane plaques in Paramecium caudatum. Figure (provided courtesy of the author) from Plattner, 1975. Figure 10, Ca 2 + electron-dense deposits seen in cross section of a cilium of Paramecium aurelia (125,000). Figure (provided courtesy of Dr. Kaneshiro) from Fisher, Kaneshiro and Peters, 1976. Figure 11, Electron-dense deposits seen in EGTA-treated cilia of Paramecium caudatum ( × 43,000). Figure (provided courtesy of Dr. Takahashi) from Tsuchiya and Takahashi, 1976 and reproduced with permission J. Protozoology/ Society Protozoologists.

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

15

tashi, 1976, 1980; Fukushi and Hiwatashi, 1970; Watanabe, 1977b; Adoutte et al., 1978, 1980), in common with the i-antigen proteins (Hansma and Kung, 1975; Doughty, 1978a; Adoutte et al., 1978, 1980; Merkel et al., 1980) although the i-antigens at least may cover the entire surface membrane of the cell (Mott, 1965; Klimetzek, 1977; Reisner et al., 1968). While both surface (ciliary membrane) phenomena detailed above lead to loss or alteration of ciliary activity (usually resulting in immobilization of the cells), the interactions of the proteins or glycoproteins involved in the recognition processes associated with these phenomena with those proteins controlling ciliary activity remains unknown. However, a Ca 2+-dependence to mating interaction (leading to loss of ciliary activity) has been detailed (Cronkite, 1976, 1979). Sectional electron-microscopic analysis of either fixed or ruthenium red stained cilia does not reveal any obvious structural specialization of the ciliary membrane or the surface coat. However, Dute and Kung (1978) report discrete localization of cationic ferritin binding to proximal portions of the ciliary membrane in Paramecium. An ultrastructural specialization within a ciliary membrane was first reported by Satir and Gilula (1970). Freeze-etch analysis of metazoan gill cilia revealed a ring of membrane particles close to the junction of the cilium with the cell. This "ciliary necklace" was subsequently demonstrated in Tetrahymena cilia (Satir et al., 1972) and Paramecium cilia (Plattner, 1975; Ogura, 1976; Dute and Kung, 1978). Whilst the function of this necklace array is unknown, it would not appear to simply be mere decoration since the cilia shear from the cell at, or just proximal to, this point when the cells are treated with chloral hydrate (Kennedy and Brittingham, 1968); dibucaine and calcium (Satir et al., 1976); ethanol and calcium (Ogura, 1976) or sucrose and calcium (Adoutte et al., 1980), providing a clean, relatively simple method for obtaining isolated cilia and thus ciliary membranes. This ciliary necklace structure, although differing in detail in different tissue types has been found in protozoan cilia, metazoan cilia, oviduct cilia and occurs sparsely in sperm flagella membranes (Gilula and Satir, 1972; Bergstrom and Henley, 1973; BoisvieuxUlrich et al., 1977). A second structural array, originally termed "intermembranous particle aggregates" (Plattner et al., 1973) but now generally referred to as the "ciliary plaques" (Plattner, 1975; Dute and Kung, 1978) or "patch particles" (Satir et al., 1976; Baugh et al., 1976; Byrne and Byrne, 1978a) has received considerable attention in the last few years. Plattner (1975) noted a close correlation between the presence of the "plagues" in freeze-etch replicas of Paramecium cilia and the occurrence of Ca 2 +-dependent electrondense deposits in ultrathin sections of cilia. Both structures (found in both single and double cilia and both somatic and oral cilia) occurred at the same distance from the ciliary base, had comparable dimensions and periodicity in their distribution around the ciliary shaft (Fig. 9), usually being observed in a position opposite to the peripheral doublet microtubules of the axoneme (see Section 2.4 and Figs 9 and 10). Such particle arrays were also found in Tetrahymena cilia (Satir et al., 1976); these authors additionally noting the absence of these arrays in newly synthesized cilia and their appearance during later stages of ciliary growth. Three independent subsequent studies confirm the presence of these "plaques" in Paramecium ciliary membranes (Ogura, 1976; Dute and Kung, 1978; Byrne and Byrne, 1978b). Dute and Kung (1978) note the coincidence of ferritin binding sites on the ciliary membrane with the disposition of the plaques. Plattner (1975) suggested a Ca2+-binding/deposition (associated with enzyme activity) role for the ciliary membrane plaques~Ca 2+-dependent electron-dense deposits. Ca 2 + (and other divalent cation)-dependent electron-dense deposits in similar positions were independently reported for Paramecium cilia (Fig. 9; see Fisher et al., 1976: Tsuchiya and Takahashi, 1976; Tsuchiya, 1976a) and Tetrahymena cilia (Przelecka et al., 1977). In ciliary cross-sections, and to a lesser extent in longitudinal sections, these Ca 2 +-dependent deposits appear to be sited on the inner surface of the ciliary membrane. Electron-microprobe analysis for the elemental composition of the deposits confirms the presence of calcium for cells fixed in the presence of CaCI2 (Plattner, 1976; Fisher et al., 1976; Tsuchiya, 1976a), although other divalent cations can substitute for Ca 2 ÷ and are revealed in subsequent microprobe analysis (Fisher et al., 1976). Of these other divalent

16

M.J. DOUGHTY and S. DRYL

cations, Mn 2 + and Ba 2+ alter the Ca2+-dependent graded electrogenesis (Naitoh et al., 1972). Ba 2+ and Sr 2+ induce all-or-nothing spike electrogenesis (Kinosita et al., 1964bc, 1965; Dryl, 1964; Naitoh and Eckert, 1968b), whilst Ni 2+ both stimulate the Ca 2÷dependent electrogenesis (Naitoh and Eckert, 1970) and effect ciliary immobilization (Geleii, 1935; Kuznicki, 1963b; Andrivon, 1968). Such reports lend support to current hypotheses that the plaque structures represent an activity relevant to the control of ciliary activity, and it is of further interest to note that the divalent cation dependent deposits were only noted in cells that were actively swimming at the time of fixation (Fisher et al., 1976). Plattner (1976) reports definite enrichment of the plaque deposits with P and S, contrary to Fisher et al., (1976). The latter authors, finding CI but unable to unambiguously identify P (i.e. indicative of phosphate salt presence) enrichment, considered that the deposits were unlikely to have resulted from Ca 2+ interacting with inorganic phosphate released at hydrolytic sites (ATPase/phosphatase activity) (although in the following paragraph I discuss the possibility that the S presence could indicate the presence of functionally important - S H groups). The several reports on the plaques note the detection of Ca, P or S only in electron-micrograph visible plaques suggesting a relation between enrichment and physiological activity. The variability in both occurrence and composition of the deposits may be in part attributed to the glutaraldehyde fixation during which enzyme activity may be inhibited or altered--Doughty and Dodd (1978) present evidence suggesting general perturbation of excitability in Paramecium by extremely low concentrations of glutaraldehyde. Divalent cation-dependent phosphohydrolase activity (phosphatase or ATPase) on ciliary membranes of Tetrahymena has been reported using histochemical studies (Burnasheva and Jurzina, 1968; Dentler, 1977). The activity was lost by non-ionic detergent treatment of the cilia (Dentler, 1977) and Baugh et al. (1976) report both the presence of Ca 2+-ATPase activity in detergent extracts of cilia from Tetrahymena and the marked reduction of the same activity in a mutant strain of the cell found to lack the ciliary membrane plaque arrays. Andrivon et al. (1977) report enrichment of a ciliary membrane fraction (obtained by Tris-EDTA lysis of cilia; see Hansma and Kung, 1975) with Ca z÷ATPase activity, whilst Doughty (1978a) reported a Triton solubilized Ca2+-ATPase from cilia of the same organism. The plaques, regardless of their activity, would appear to reflect some specialized function attributable to the control of ciliary activity in protozoa since, to date, plaques have only been found in the ciliary membranes of holotrich ciliated protozoa (Satir et al., 1976; Dute and Kung, 1978). Plaques do not appear to be present in either flagella membranes, metazoan gill cilia or oviduct cilia (Bergstrom and Henley, 1973; Gilula and Satir, 1972; Boisvieux-Ulrich et al., 1977). However, it should be noted that in these other ciliary types and in olfactory membrane cilia, the membrane particles appear to be randomly organized and far more prominent than in protozoan cilia (Gilula and Satir, 1972; Boisvieux-Ulrich et al., 1977; Menco et al., 1976). Plaque morphology has recently been reported to be altered in a behavioral mutant of Paramecium (Byrne and Byrne, 1978a, b). As a final comment on Ca 2 ÷-dependent deposits in protozoan cilia, it should be noted that the ciliary membranes are not the only site of such deposits. Tsuchiya and Takahashi (1976) report both the presence of membrane-associated Ca 2 ÷-dependent electrondense deposits and the presence of less dense deposits within the ciliary axoneme treated with Ca 2+-free solutions prior to fixation (Fig. 11). Similar histochemical localization (i.e. at the vicinity of the spoke heads--see Section 2.4) of phosphohydrolase activity has been reported for both Tetrahymena cilia (Burnasheva and Jurzina, 1968) and in flagella axonemes (Anderson et al., 1968; Burton, 1973; Nagano, 1965). There have been few studies on either the composition (protein or lipid) or activity (enzymes and ligand binding activity) of ciliary membranes, from either protozoan or metazoan cilia, primarily because techniques for isolation of both cilia and their membranes have only recently been developed to a satisfactory state suitable for quantitative

CONTROl. OF CII.IARY ACTIVITY IN P.~,~4 ~.1t:(1( ~1

FIG. 8. The cilium and its domain in Paramecium. Electron micrograph of longitudinal section through a somatic cilium of Paramecium caudatum. × 100,000. The ciliary membrane, the outer doublet microtubules, the inner microtubule pair, the axosome and the basal plate are clearly visible. Of the central pair of microtubules, only one appears attached to the axosome. Photograph (provided courtesy of Dr. H a u s m a n n t from H a u s m a n and Fisher-Defoy, 1978 and reproduced with permission Cell Biol. Int. Rep./Academic Press.

17

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

19

analysis. Early workers on cilia from Tetrahymena generally agreed that the cilium was rich in protein (Child, 1959; Watson et al., 1961; Watson and Hopkins, 1962; Gibbons, 1963; Watson et al., 1964, contained 0.6-3% carbohydrate (hexose equivalents) (Watson et al., 1961; Watson and Hopkins, 1962; Culbertson, 1966a, b; Watanabe, 1973) and contained significant nucleotide material (Child, 1959). As a result of differences in techniques, it is uncertain what percentage of the isolated cilium is ciliary membrane-estimates ranging from 10 to 40% have been reported or can be estimated (Gibbons, 1963, 1965; Watanabe, 1977b; Kitamura and Hiwatashi, 1976; Doughty, 1978a; Adoutte et al., 1980; Merkel et al., 1980). Ciliary membrane vesicles appear to contain some 12-30 polypeptides with molecular weights ranging from 15 to 250 Kd (Kitamura and Hiwatashi, 1976; Hansma and Kung, 1975; Butzel and Decapiro, 1978; Adoutte et al., 1980; Merkel et al., 1980) (Fig. 12). The ciliary membrane is principally composed (60-80% of total composition) of the i-antigen. This protein (or its precursor; see Hansma and Kung, 1975) has a subunit molecular weight in the region of 250-300 Kd (Hansma and Kung, 1975; Hansma, 1975; Adoutte et al., 1980; Merkel et al., 1980; Steers and Davis, 1977; Davis and Steers, 1978). There are a variety of types of i-antigen--a property which confers serotypic identification to the Paramecium. Only antibodies raised against a particular serotype will immobilize the cell containing that serotype (Capdeville, 1979; Finger, 1974; Preer, 1959), although, within a cell population, more than one serotype (i-antigen) can be expressed at any one time (Finger, 19741. A sulfhydryl agent-stimulated protease activity is associated with isolated i-antigen protein(s) (Hansma, 1975; Steers and Davis, 1977; Davis and Steers, 1978)--an activity which can result in degradation of the i-antigen (Reisner et al., 1969; Hansma, 1975; Steers and Davis, 1977; Davis and Steers, 1978) and other proteins. This i-antigen associated protease activity may represent a physiological function, since recovery of ciliates from immobilization by homologous antiserum appears to involve proteolitic degradation of the immunoglobulins (Eisen and Tallan, 1977). Variations in ciliary membrane polypeptide composition have been discussed in terms of proteolytic degradation (Dentler, 1980; Adoutte et al., 1980). The polypeptide composition of the ciliary membrane has not been unequivocably determined. Some workers consider the presence of "tubulin" (the major structural protein of the ciliary axoneme--see Section 2.4) or tubulin-like proteins in Paramecium ciliary membrane preparations as being due to contamination (Hansma and Kung, 1975; Adoutte et al., 1980; Merkel et al., 1980), whilst other reports suggest the presence of a tubulin-like protein as a normal component of ciliary membranes, especially those from Tetrahymena and gill cilia (Otter, 1978; Dentler, 1980; Dentler et al., 1980; Stephens, 1977b). The presence of tubulin as a normal component has been proposed for flagellar membranes (Adair and Goodenough, 1978; Stephens, 1977b) and also synaptosomal membranes (Blitz and Fine, 1974; Feit and Barondes, 1970; Korngluth and Sunderland, 1975; Gozes and Littauer, 1979). It remains to be established in any of these systems whether tubulin or tubulin-like proteins are integral membrane proteins or simply tightly associated with the~membrane. In Paramecium and Tetrahymena cilia, fine structures are seen in crosssections of cilia to bridge between the membrane and the peripheral doublet microtubules--in Tetrahymena, this structure has been proposed to be at least in part composed of a tubulin-like protein (Dentler et al., 1980). The presence or absence of tubulin in protozoan ciliary membrane preparations might thus simply reflect differences in the selectivity of the preparative procedures (Hansma and Kung, 1975; Kitamura and Hiwatashi, 1976; Stephens, 1977a; Butzel and Decaprio, 1978; Doughty, 1979b; Adoutte et al., 1980). Stephens (1977b) reported that ciliary membrane "tubulin" could be labelled with various impermenant covalent reagents including 125I-lactoperoxidase. This type of radiolabelling technique suggests that at least half the ciliary membrane proteins are accessible to the extracellular environment (due to their being labelled with ~25I) (Merkel et al., 1980). A few studies have been carried out on the activity of ciliary membrane preparations. Gibbons (1965) noted that the ciliary membrane fraction of Tetrahymena cilia contained a labile Mg 2+-stimulated (but not Ca 2+-stimulated) ATPase activity, whilst Baugh et al.

20

M.J. DOUGHTY and S. DRYL

(1976) reported a Ca 2~ > Mg2+-stimulated ATPase in ciliary membranes of the same cell. Using a similar procedure (solubilization of cilia in Triton X-100) to the latter authors, Doughty (1978a) measured, and subsequently purified, a Triton solublized C a 2 + > Mg2+-stimulated ATPase from Paramecium cilia. Neither Mg2+-dependent, Na-K stimulated ATPase or K-phosphatase activity has been so far detected in Paramecium cilia (Doughty, 1978a; Doughty and Kaneshiro, unpublished results). The role of the Ca 2 +-ATPase, which requires further characterization, is unknown, but may serve as the energy-dependent "Ca 2+pump'' proposed by various authors (Eckert, 1972; Andrivon, 1974b; Machemer, 1974a; Browning and Nelson, 1976a; Doughty, 1978a, b) to serve a role in maintaining intraciliary free calcium levels at less than micromolar concentrations in the absence of stimulation (see Section 4.6). Triton treatment of isolated Paramecium cilia also solubilizes an enzyme activity hydrolysing acetylthiocholine--an activity sensitive to neostigmine and eserine (Doughty, 1978c). Acetylcholinesterase activity has also been reported in isolated pellicles of Tetrahymena (a preparation including the interciliary membrane) (Seaman and Houlihan, 1951 ; Seaman, 1951), histochemically demonstrated in cilia of Tetrahymena (Schuster and Hershenov, 1969) and in Paramecium cell homogenates (Andrivon, 1975). The role of the enzyme, if subsequent studies confirm its existence, would presumably be as part of the cholinergic control mechanism for ciliary activity (Doughty, 1978c, 1979a). Triton treatment of isolated Tetrahymena cilia solubilizes several cyclic nucleotide dependent protein kinase enzymes (Murofushi, 1973). Nelson and Kung (1978), in a preliminary communication, report the presence of several phosphoproteins in Paramecium cilia. Their presence might be anticipated if protein phosphorylation is involved in control of membrane Ca 2 + permeability and function as in other excitable membranes (e.g. synaptosomes; see Delorenzo, 1976; Krueger et al., 1977; Michaelson and Avissar, 1979; Berman et al., 1980). Two recent major studies have provided information on the phospholipids of Paramecium cilia. In the related ciliate, Tetrahymena, where the major sterol is Tetrahymenol, the ratio of total phospholipid to sterol in isolated ciliary membranes is approximately 2:1 (molar ratios) (Conner et al., 1971). In Paramecium cilia, of the phospholipids, only the glycerophospholipids have so far been fully characterized. Seventy to eighty-one percent of the phospholipids are ethanolamine lipids (PnE, PsE and CAEP); 11-14~o are choline lipids (principally PC); and the remainder is found to be composed of minor sphingolipids and some lysoderivatives of all phospholipid classes (Rhoads and Kaneshiro, 1979; Andrews and Nelson, 1979). In the total glycerophospholipids, the predominant fatty acid is arachidonate (20:4 aS's'l 1.14); other major fatty acids include palmitate (16:0), oleate (18:1 a9) linoleate (18:2A9"12), linolenate (18:3 a6'9"12) and notable minor classes include stearate (18:0) and 20:5 a5"8'~1'14"17 (Kaneshiro et al., 1979). Analysis to date, on Paramecium cilia, indicates that, in common with Tetrahymena cilia (Smith et al., 1970), the cilia are enriched in phosphonyl lipids (PnE and CAEP) and polyunsaturated fatty acids (Kaneshiro et al., 1979). Further analyses of both protein, enzyme and phospholipids of Paramecium cilia are currently in progress in several laboratories and a substantial increase in our knowledge in these areas can be anticipated in the next few years. 2.3.

FUNCTIONAL ROLE OF THE CILIARY MEMBRANE

The ciliary membrane of Paramecium is now generally accepted as being of fundamental importance in the control of ciliary activity (Eckert, 1972; Machemer, 1977; Eckert and Brehm, 1979). However, the mechanisms underlying this control, at the membrane level, are still largely unknown and the subject of current research. Several early studies recognized the existence and role of a membrane separating the ciliary machinery from the extracellular environment and proposed various theories as to how the surface membrane of Paramecium could play a role in the regulation of cell behavior. Early studies on the galvanotaxis of Paramecium were interpreted in terms of an external electric currentinduced formation of localized areas of either acid or alkali at the surface membrane

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

21

(Dale, 1901 ; Vieweger, 1912). Woodruff and Bunzell (1909) noted a correlation between the ionic potential (redox potential) of various salts and their relative toxicity towards Paramecium and suggested that their penetration, and resultant cytotoxicity, was somehow related to this chemical characteristic of the ions. The intervention of the "cell wall" in the control of penetration of toxic ions into Paramecium was discussed by Collett (1919). Gray (1922) noted correlations between the effects exerted by ions on the cell membrane and ciliary activity of metazoan ciliated epithelia and their action on the theoretical electrical conductivity of these cells. Both Andrejewa (1931) and Wense (1935, 1939a), whilst hypothesizing that the motor reactions (ciliary activity) of Paramecium were controlled through alterations in the colloidal state of the cells' cytoplasm, proposed that the electrical charge at the surface membrane of the cell was linked to the activity of a neuromotor system underlying it. Early concepts of the role of the surface (ciliary) membrane in the control of ciliary activity and behavior in Paramecium are perhaps best summed up by the conclusions of Oliphant (1938): "Precisely how environmental changes which have been observed to induce reversal in the action of cilia effect reversal and the physiological factors involved cannot be completely explained on the basis of our present knowledge. Factors such as changes in the electrical potential at the surface, or in permeability and consistency of the surface layer of the organism, or chemical changes in the receptors or elsewhere in the organism may be involved as has been suggested. It is believed, however, that there is considerable evidence to indicate that reversal is associated with increase in viscosity of the cytoplasm."

Both Kamada (1940) and Wense (1939a, b) acknowledged the role of C a 2+ ions in promoting ciliary reversal in Paramecium but explained the mode of action in different ways. Kamada (1940), in the light of the data of Akita (1941) concerning the concentrations of Na +, K + and Ca 2+ in Paramecium suspended in solutions of varying ionic composition, proposed that a reduction in the free Ca 2 + ion concentration within cytoplasm (which in turn effected ciliary reversal) was effected as a result of a K+42a 2+ exchange at the surface membrane. Wense (1939a, b) proposed that the surface membrane regulated the intracellular Ca 2 + ion concentrations--an increase in which altered the colloidal properties of the cell cytoplasm and thus induced ciliary reversal. Mast and Nadler (1926) proposed that extracellular calcium ions directly entered the ciliary apparatus (axoneme) to induce ciliary reversal. However, their concept is inconsistent with the several reports that sudden elevation in extracellular CaC12, in the absence of any other cations, does not induce ciliary reversal (except at cytotoxic concentrations) (Jennings, 1897, 1899b; Oliphant, 1938, 1942; Grebecki, 1964; Kuznicki, 1966a; Dale, 1913). However, extracellular calcium ions are required for ciliary reversal behavior (Bancroft, 1906a, b; Grebecki, 1965; Kunznicki, 1966a; Hildebrand and Dryl, 1976; Dryl and Kurdybacha, 1978) and have been widely reported to modify K+-induced ciliary reversal (see, e.g., Kamada and Kinosita, 1940; Naitoh, 1968), galvanotaxis (Bancroft, 1906a, b) and geotaxis (Vieweger, 1912; Grebecki and Nowakowska, 1977). Jahn (1962a) reanalyzed the data of Kamada and Kinosita (1940) and noted that, in agreement with the GibbsDonnan principle, constant behavioral responses were observed where the ratio of [M+]o/[M 2+]~ was constant. Furthermore, for maximum duration ciliary reversal behavior for Paramecium adapted to various solutions containing fixed concentrations of K + and Ca 2+ ions, the Gibbs-Donnan ratio, for each series of conditions, was the same regardless of the dilution of the salts. This discovery resulted in renewed interest in a theory that ciliary reversal was effected as a result of removal of bound Ca 2 + from the surface membrane of the cell. However, earlier studies indicated that recognized calciumion chelating agents (oxalate and citrate), whether applied extracellularly (Oliphant, 1938) or intracellularly (Kamada, 1938), could induce ciliary reversal behavior and suppressed galvanotaxis when applied extracellularly (Bancroft, 1906a; Fabre, 1947). Bancroft (1906a, b) also reported that no galvanotaxis occurred in Paramecium when the extracellular medium [salt]/[CaC12] concentration was very large; when the ratio was slightly smaller, annodal movement occurred and at higher CaC12 concentrations, cathodally

22

M . J . DOUGHTY and S. DRYL

directed movement of the cells occurred. Naitoh and Yasumasu (1967) proposed, on the basis of their measurements of the association of 45Ca2+ with Paramecium, that ciliary reversal was effected by removal of Ca 2÷ from the surface membrane since ions that induced reversal (K +, Na +, Rb ÷ and Ba 2÷) inhibited the absorption of 45Ca2 + onto the cell. They furthermore showed that the amount of 45Ca2 ÷ "bound" to the cells, following equilibration of the cells for l min in K-Ca solutions, was constant for a constant Gibbs-Donnan ratio for these cations. The authors concluded that, because of the rapid apparent equilibration, the Ca 2 ÷-ion binding sites were either on, or at least close to, the surface membrane. However, in an earlier study, Yamaguchi (1960b) reported differences in 45Ca2+ "uptake" by Paramecium following adaptation of the cells to different K-Ca solutions. Yamaguchi (1960b) found that, as the ratio [K+]o/[Ca2+]0 or I-K+]0/[Ca2+]~ increased, so the kinetics of 45Ca "uptake" were increased. These two sets of data would therefore appear contradictory, but for the fact that neither study corrects for Ca 2+ efflux from the cell, although Yamaguchi (1960b) makes a study of the "efflux" under different conditions following loading/binding 45Ca2+ to the cells. Yamaguchi (1960b) showed that higher 45Ca 2 + efflux/desorption occurs into solutions with higher Gibbs-Donnan ratios. Therefore, whilst both this and other behavioral data support a hypothesis that ciliary reversal is effected as a result of alteration in the calcium occupancy of either surface or intracellular binding sites, we can conclude that several mechanisms are operative--those regulating Ca 2+ binding to the membrane, Ca 2 + uptake into the cell (intraciliary space) and Ca 2+ efflux from the cell. Naitoh and Yasumasu (1967) note that, in glycerol-treated Paramecium, ciliary reversal could only be induced, in the absence of an intact surface membrane, by Ca z +--i.e. the Ca 2 + at intraciliary sites, and not membrane sites, appeared to be responsible for induction of ciliary reorientation (see Section 4.5). Kamada (1934) first noted that a potential difference was maintained across the surface membranes of Paramecium and that this potential difference was dependent upon the extracellular cation concentrations. These results were confirmed by Yamaguchi (1960a), were studied in further detail later (Kinosita et al., 1964a, b) and quantitatively evaluated by Naitoh and Eckert (1968a). These electrophysiological studies show that increasing concentrations of monovalent and divalent cation salts in the extracellular medium shifted the resting potential in a depolarizing direction. Earlier studies on the galvanotaxis of Paramecium had already showed that the strength of the response (net migration towards either the cathode or the anode) of free-swimming cells was dependent upon the ration of [K +]o/[Ca 2+]~ (Bancroft, 1906a, b). Yamaguchi (1960a) showed that external d.c. stimulation of a microelectrode-impaled specimen of Paramecium not only effected ciliary reversal (in a solution of 5 mM KC1 and 3.3 mM CaC12) but that a concommitant depolarization of the surface membrane also occurred. Kinosita et al. (1964a) also studied the effects of externally applied current on the resting potential of Paramecium and on the ciliary activity of the cells. Using brief (10-100msec) low intensity external galvanic stimulation, they noted occurrence of both spike depolarization (which they attributed to "an artifact due to membrane capacity") and marked alterations in ciliary activity (unspecified). Higher current strengths evoked a hyperpolarizing spike that was coincident with body contraction. Yamaguchi (1960a) noted the occurrence of similar hyperpolarization events that appeared to be associated with the contraction of the contractile vacuole (the osmoregulatory organelle of the cell). Such studies provide a link between the phenomenon of galvanotaxis and that of ciliary reversal and their collective control both by Ca ~+ ions and the activity of the surface membrane. Both involve membrane depolarization and the extent of the depolarization is dependent, in some uncertain manner, on the ratio of K + to Ca 2+ in the extracellular medium. Naitoh and Eckert (1968a) showed that the resting potential in solutions containing various concentrations of K + depended on the Ca 2 ÷ concentration (see Fig. 34). Increases in ionic strength were accompanied by depolarization even if a constant Gibbs-Donnan ration was maintained. Subsequent studies, in which Paramecium were stimulated with brief (200 msec), low intensity (i> 10 -11 A) outward current pulses delivered via an intraceUularly located microelectrode, showed that outwardly directed current could also induce ciliary reversal

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

23

and depolarization of the membrane. The depolarization was found to be graded with respect to the applied current and this regenerative depolarization was found to be Ca 2+ dependent (Naitoh and Eckert, 1968a; Naitoh et al., 1972). Ciliary reversal occurred coincident with the depolarization and did not occur unless the Ca 2+ regenerative response occurred, i.e. simple electrotonic shifts in the resting potential did not elicit ciliary reversal (Naitoh and Eckert, 1968a Machemer and Eckert, 1973; Machemer, 1974a, 1975). The surface membrane of Paramecium thus regulates the galvanotactic response, regulates the cation induced ciliary reversal response in free swimming cells and regulates the current-induced ciliary reversal behavior observed in microelectrode-impaled cells. The surface membrane appears to be the site of interaction between extracellular K ÷ ions and Ca 2÷ ions and both these cations play a dominant role in the control of both ciliary activity and the membrane depolarization events. The relative importance of Ca 2 ÷ ion displacement at membrane K " - C a 2÷ binding sites compared to changes in Ca z÷ at intracellular (intraciliary) loci is uncertain at this time, although we can be reasonably certain that changes in intraciliary Ca 2÷ ion activity actually effect ciliary reorientation. Although accepted by many, it is questionable at this time whether the conservation of the Gibbs-Donnan relationship has any significance in the control of ciliary activity or indicate any particular phenomenon occurrent at the surface membrane. Constancy of this relationship with respect to any particular response profile may reflect the sum of all Ca 2+ events at or through the membrane and include both changes in binding characteristics as well as changes in membrane permeability (both influx and outflux of Ca 2÷ ions). Recent studies (Machemer, 1976) fai_led t O d e t e c t a n y consistent correlation between [K+]o/[Ca2+]~ and the characteristics of ciliary responses (beat frequency and orientation), although earlier reports show relationships between the Gibbs-Donnan ratio and ciliary reversal behavior induced by K ÷ ions (Kamada and Kinosita, 1940; Naitoh, 1968)and the amplitude of the Ca 2÷ regnerative response (Naitoh and Eckert, 1968a). Grebecki (1965) first attempted to reconcile the various theories proposed as to how Ca 2+ ions either effected or controlled ciliary activity in Paramecium: these being the direct action theory (Mast and Nadler, 1926), by which extracellular Ca 2 + directly acted on the cell cytoplasm; Ca 2+-uptake theories (Seravin, 1961; Kuznicki, 1966a), in which increased Ca 2÷ uptake effected ciliary reversal in analogous manner to which Ca 2÷ effected muscle contraction; Ca 2÷ loss theories---either at the surface membrane--displacement of bound Ca 2+ effected ciliary reversal (Jahn, 1962); or at intracellular sites-removal of Ca 2÷ ions from intracellular sites effected ciliary reversal (Kamada, 1938, 1940): the combination resulted in the Ca 2+ content theory (Grebecki, 1965), by which ciliary activity depended not upon either the loss or uptake of liberated Ca 2+ ions but was related to the amount of Ca 2+ ions resting on absorption sites on the cell membrane which in turn influenced the properties of the cell membrane. The degree of excitation depended on gradual changes in the amount of calcium ions bound to the membrane. Grebecki (1965) notes that decalcification (nominally by the use of EDTA) of the cells resulted in loss of sensitivity to K ÷ ions and additionally notes that elevation of the Ca 2÷ concentration in the extracellular medium during a period of K÷-induced ciliary reversal behavior, shortened or even interrupted the response indicating that extracellular Ca 2 ÷ ions are required for control of ciliary reversal as well as its induction. It should be clear to the reader by now that no single factor or mechanism underlies either the induction or control of ciliary reversal. Akita (1941) noted that in intracellular Ca 2 ÷ ion concentration of the Paramecium cell was maintained below that of the extracellular medium. Naitoh and Kaneko (1972) demonstrated that in Paramecium in which the surface membrane had been disrupted by treatment with detergents (Triton X-100), ciliary reversal could be effected if the free-Ca 2+ ion concentration in the bathing solutions was raised above 10-6M. If the calcium sensitivity of these "model" Paramecium, in which membrane control of ciliary activity has been removed, is the same as that of intact cells, then it can be expected that LP.N. 16'1 B

24

M . J . DOUGHTY and S. DRYL

the normal intraciliary free calcium concentrations are maintained below I0-6 M (Naitoh and Kaneko, 1972; Eckert, 1972). Most behavioral and electrophysiological studies on Paramecium have been carried out in solutions containing 10-4-10-3M CaCI2. A large concentration gradient for calcium is thus maintained between the intraciliary space and the extracellular medium. Eckert (1972) proposed that the ciliary membrane thus additionally served to maintain the intraciliary free calcium below that threshold level required for onset of reversal. A transmembrane flux of calcium ions was thus responsible for induction of ciliary reversal and was associated with the Ca-regenerative depolarization of the ciliary membrane (Naitoh and Eckert, 1968a; Naitoh et al., 1972; Naitoh and Kaneko, 1972) (see Sections 4.1-4.5 for full details). By this theory, Ca 2+ ions will enter the intraciliary space, across the ciliary membrane through specific voltage-sensitive channels and that the driving force for the calcium influx will be the relative magnitude of the diffusion gradient (electrochemical potential) between the external medium and the intraciliary space. Renormalization (return to forward beating mode) of ciliary activity was proposed (Eckert, 1972) to be effected either by active removal (pumping out) or sequestration of the calcium ions that entered during the active depolarization. These two basic functions, assigned to the ciliary membrane (influx and outflux) by most hypotheses, could be independently operating. Andrivon (1970b, 1972) studied the kinetics of 63Ni2 ÷ uptake by Paramecium cells. In the absence of extracellular potassium ions, the uptake showed a distinctive Michaelis character which was interpreted as indicating an active transport system for Ni 2+. In the presence of potassium ions, however, the kinetics became sigmoidai: the kinetics were further influenced by the relative concentrations of K ÷ and Ca 2÷, in common with other membrane phenomena. Andrivon, in a series of experiments (1970b, 1972, 1974a) showed that the immobilization of ciliary activity (which occurs as a consequence of treatment with Ni 2÷ ions) was effected by a constant concentration of Ni 2 ÷ ions, and concluded that it was the rate of transport of Ni 2 + ions into the cell that determined the time course of immobilization which was inversely proportional to the Ni 2÷ concentration (see Section 3.4 for details). Like K ÷ induced ciliary reversal, this uptake of Ni 2 + was found to be dependent on pH, temperature and the monovalent and divalent cations present in the extracellular medium. At constant Gibbs-Donnan ratios for K ÷ :Ca 2 ÷, regardless of the absolute concentrations of these two ions, the rate of immobilization was not constant for any particular Ni 2 ÷ ion concentration (Andrivon, 1972). In view of the nature of the sensitivity of this uptake system to conditions that acted antagonistically towards K÷-induced ciliary reversal phenomena, Andrivon (1972, 1974a, b) proposed that there were common characteristics for both molecular components controlling Ni2+-induced ciliary immobilization and K ÷-induced ciliary reversal and its duration. The processes that control these two activities might thus act synergistically. Browning and Nelson (1976a) demonstrated that at 4°C, 45Ca2+ efflux from Paramecium was not detectable, i.e. was inhibited. Under these conditions, they measured a high rate of passive influx of 45Ca2 ÷ into the cells in the absence of recognized stimuli, suggesting that there is either a high steady-state influx of Ca 2 + into the cells or that, upon inactivation of Ca 2+ efttux, influx is enhanced. Browning and Nelson (1976a) additionally showed that this Ca 2~- influx, at 4°C, was enhanced by stimuli known to induce ciliary reversal and thus the fluxes were attributed to activity of the voltage sensitive channels through which calcium normally flows to induce ciliary reversal. Andrivon (1974a) proposed that, in view of the kinetics of 63Ni2 + uptake under various antagonistic conditions, the molecular component(s) responsible for this Ni 2 ÷ permeability were oligomeric in nature, and, being regulated by allosteric control, showed an activity dependent upon the relative occupancy of unidentified sites by K ÷ and Ca 2 +--i.e. similar to the activity of the ciliary reversal induction mechanisms. Andrivon (1974a) further proposed that, for a given relationship between the K + and Ca z+ concentrations in the medium and for those cations bound to the surface membrane, there existed a corresponding overall conformational state of those molecular components responsible for determining both the net excitability and divalent cation transport activity across the ciliary membrane. In the light of the above data and the pro-

CONTROL OF CIL1ARY ACTIVITY IN PARAMECIUM

25

posals of Andrivon, Doughty (1978b, c) further proposed that, on activation of the calcium conductance of the ciliary membrane by K + ions (to effect transmembrane calcium influx; see Eckert, 1972), concommitant reduction in calcium efflux (transport) would occur as a result of an allosteric coupling between the two activities. If alteration in calcium efflux does occur concommitant with activation of calcium influx, it might be anticipated both that the efflux mechanism would be voltage sensitive and that the overall excitability of the membrane would be determined by the resting potential, V.,. Van Houten (1978, 1979) has indeed proposed a model for Paramecium which is indirectly related to the above hypothesis: i.e. the excitability and thus the overt cell behavior to stimuli (kineses) is dependent on V,.. By measurement of the relative changes in V,. following change of the extracellular bathing solutions (in either the cation or anion character), a relationship between V,, and the type of behavior shown by the cell has been found. The results indicate that the overt behavioral response is both controlled by the membrane potential, and more importantly that the strength of the membrane response and thus the relative behavioral response and its time course is determined by the relative change in V,. rather than its absolute value. By use of a mutant Paramecium (Van Houten, 1977), dissection of the role of V,, in determining the nature of the behavioral response has been initiated. Other recent work with mutant Paramecium provides further convincing evidence for, and highlights the role of, the ciliary membrane in the control of ciliary activity and thus cell behavior. Normal (wild-type) Paramecium respond to injected current or K +-stimulation by showing ciliary reversal (Naitoh, 1968; Naitoh and Eckert, 1968a; Machemer, 1974a, 1975, 1976; Machemer and Eckert, 1975; Brehm and Eckert, 1978b). Wild-type cells respond to intracellular injection of CaC12:EGTA mixtures by showing ciliary reversal as well (Saiki and Hiramoto, 1975; Brehm and Eckert, 1978a). Wild-type Paramecium "models", in which the functional integrity of the ciliary membrane has been destroyed by careful treatment of the cells with a non-ionic detergent (Naitoh and Kaneko, 1973), swim forward on transfer to reactivating solutions containing MgATP z-, but swim backwards in similar solutions containing greater than 10-6M free Ca 2+ (Naitoh and Kaneko, 1972). A mutant of Paramecium, derived from wild-type by controlled mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (Kung, 1971a, b), has been isolated on the basis of failing to show a ciliary reversal response to K + stimulation. This "pawn" phenotype, whilst showing normal forward swimming behavior, fails to show cation-induced ciliary reversal and also fails to show a Ca 2 + regenerative response following current injection (Kung and Eckert, 1972; Brehm and Eckert, 1978b) although passive membrane properties appear normal. However, "models" of this phenotype swim forwards in MgATP 2- solutions and show ciliary reversal (and thus backward swimming) in MgATP 2- solutions containing greater than 10-6M Ca 2+ (Kung and Naitoh, 1973), and also can reverse their cilia following intracellular injection of CaC12:EGTA solutions (Brehm and Eckert, 1978b). The mutant is thus apparently defective only in that mechanism determining activation of membrane conductance to Ca 2+ ions and without this activation fails to show ciliary reversal. In conclusion therefore, the ciliary membrane, by our current understanding, is the site of at least two descriptive operative functions, although neither of these have been completely experimentally analyzed. The ciliary membrane both regulates the time course and magnitude of Ca 2 + fluxes into the intraciliary space through the operation of an as yet uncharacterized voltage-sensitive gating mechanism and in addition regulates the time course (and perhaps the magnitude) of Ca 2 + efflux (transport?) from the intraciliary space. The rate and magnitude of the influx appears to determine both the frequency of beating and the relative orientation of ciliary activity (Machemer, 1974a; Machemer and Eckert, 1975). The active removal of Ca 2+ ions from the intraciliary space both serves to maintain intraciliary free calcium concentrations at a level below that required for induction of ciliary reversal in the absence of active influx and also serves to regulate the time course of reversed beating of the cilia following stimulation by removing the calcium inflowing during active electrogenesis (Machemer, 1974a;

26

M.J. DOUGHTYand S. DRYL

Doughty, 1978b; Browning and Nelson, 1976a; Hildebrand and Dryl, 1976; Andrivon, 1974a; Eckert, 1972). These two basic functions may or may not operate independently of one another, but, as would seem possible in the light of currently available data, could operate in a synergetic manner through an, as yet uncharacterized, aUosteric linkage. 2.4. STRUCTURE, PROTEIN COMPOSITION AND ENZYME ACTIVITY OF THE CILIARY AXONEME

Several major reports on the morphology and structural organization of Paramecium appeared in the first 50 years of this century (Schuberg, 1905; Rees, 1922; Klein, 1926; Gelei, 1926, 1937a, b). Further details of the structure came from extensive electron microscope studies (Pitelka, 1963; Jurrand and Selman, 1969; Ehret and McArdle, 1974). Unfortunately, however, there have been few studies on either the structure of whole cilia or the ciliary axonemes (cilia without the ciliary membrane) from Paramecium. Cilia from the related holotrich protozoan, Tetrahymena, have been the choice material for most recent studies on the structure, biochemistry and biomechanics of cilia supplemented by studies on cilia from metazoan sources. Thus, what little knowledge we do have of ciliary structure and ciliary mechanochemistry comes largely from studies on cilia from sources other than Paramecium. The current status of our understanding of the molecular mechanisms underlying ciliary activity is largely the result of extensive recent studies made on the sperm tails (flagella) of sea-urchins although a few correlation studies have been carried out. In view of common structural components and "contractile proteins" (as we currently understand them), the mechanisms underlying ciliary activity and its control at the axonemal level appear to been largely assumed to be sufficiently similar to those in sperm flagella as to negate the need to study similar processes in protozoan cilia. Although cilia and flagella undoubtedly have some common structural and enzymic components, the very nature of the motion of the respective organelles argues against there being common or identical molecular mechanisms controlling the frequency, beat profile and relative "orientation" of motion. Likewise, whereas the nature of ciliary activity and membrane responses of Tetrahymena have not been studied in any detail, the majority of biochemistry has been carried out on cilia from this cell. Since this review is on Paramecium, we will define what knowledge we currently have of Paramecium ciliary mechanochemistry in the context of studies carried on on Tetrahymena cilia and draw attention to studies on flagella mechanochemistry where these either support or clarify certain concepts. The readers attention is drawn to several recent major reviews on flagella motion (Blum and Lubliner, 1973; Summers, 1975; Mohri, 1976; Blum and Hines, 1979). The first electron micrographs of either cilia or ciliary axonemes from Paramecium (Jakus and Hall, 1946; Anderson, 1951) showed that the ciliary axoneme was composed of 11 fibrils enclosed in a membranous sheath. These observations were confirmed by Child (1959) and Watson and Hopkins (1962), who further noted that, in common with the flagellar axoneme, cilia contained a central pair of fibers or tubules (microtubules) surrounded by a ring of nine peripheral, doublet microtubules. Many cilia and flagella have this common morphological arrangement--the 9 + 2 (Blum and Lubliner, 1973; Blum and Hines, 1979; Warner, 1974; Summers, 1975). Cilia and flagella normally originate from "basal bodies" (or kinetosomes) (Watson and Hopkins, 1962; Pitelka, 1963, 1974; Blum and Lubliner, 1973) (Fig. 7). The basal body appears to serve as an anchor for the cilium (Pitelka, 1974) and contains nine triplet sets of microtubules. At the transition region between the kinetesome and the ciliary shaft proper (the axonemel, one subfiber of each of the triplets terminates and the other two continue as the axonemal outer (peripheral) ring of doublet microtubules (Fig. 13). The transition region is demarcated by a "terminal" or "basal" plate. Just above the basal plate originate the central pair of microtubules from the "axial granule" or "axosome". Several reports (Ehret and McArdle, 1974; Hausman and Fischer-Defoy, 1978; Dute and Kung, 1978) indicate that only one of the central singlet microtubules is actually linked to the axosome (Fig. 8). In

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

a

27

cb

FI

FIG. 13. Structure of the motile axoneme. Schematic illustration of a cross-section of a typical "9 + 2" axonene, a--A-tubule; b--B-tubule; c---central tubules; cb---central bridge; cs--central sheath; ia--inner arm; oa--outer arm; m--membrane; n--nexin link; s--spoke; sh--spoke head. Figure (provided courtesy of the author) modified from Fig. 1 in Mohri, H. (1976) Zool. Mag. 85, l 16 (In Japanese) and reproduced with permission of The Zoological Magazine/Zoological Society of Japan.

Tetrahymena ciliary axonemes, the individual doublet microtubules can be identified from a difference in length--one doublet is shorter than the rest and is assigned number 5 (Sale and Satir, 1976) in accordance with the nomenclature for metazoan cila where a permanent bridge appears to exist between doublets 5 and 6 (Warner and Satir, 1974). We will not concern ourselves in this review with the fine structure of the microtubules since the role of this fine structure in the control of ciliary activity is completely unknown. Details may be found elsewhere (Mohri, 1976; Amos et al., 1976). In Tetrahymena cilia, the central pair of singlet microtubules are enclosed in a central sheath. The central sheath is surrounded by a ring (some 1600 A in diameter; see Blum and Lubliner, 1973) of nine pairs of doublet microtubules composed of one complete microtubule (subfiber a) and an incomplete microtubule fused to the complete one (subfiber b) (Figs 10 and 13) (Watson and Hopkins, 1962; Gibbons, 1963, 1965; Allen, 1968; Dentler and Cunningham, 1977). The 9 + 2 structure extends along the length of the axoneme thus forming the structural "skeleton" of the axoneme. The peripheral terminate short of the tip while the central pair extends right into the tip. The outer nine doublet array is maintained by the presence of proteinaceous linkages between each of the a-subfibers along the whole length of the axoneme. These links are made of a protein termed "nexin" with a subunit molecular weight of 165,000 daltons (Stephens, 1970). These links are permanent and have been shown to hold the flagellar structure together even after removal of all other linkages within the axoneme (Summers and Gibbons, 1971, 1973). On trypsin digestion of axonemes, the nexin links are disrupted and the cilia or flagella axonemes can then, under controlled conditions, be induced to break apart in a characteristic manner (Summers and Gibbons, 1973; Sale and Satir, 1976). The peripheral doublet microtubules are linked to the central sheath in cilia (and flagella) by "secondary links" (Watson and Hopkins, 1962; Gibbons, 1965), "radial links" (Warner, 1970) or "radial spoke" (Allen, 1968) (Fig. 13). All these terms describe a common heterogeneous structural array for which the term radial spokes appears to be now generally accepted. In mussel gill cilia, the radial spokes occur in groups of three and distributed along the length of the a subfiber of the outer doublets (Warner and Satir, 1974). These observations were later confirmed in studies on Tetrahymena cilia (Sale and Satir, 1976). Within each group of three, the spokes are arranged at nonequidistant intervals--the most basal spoke of the group, in Tetrahymena cilia, being 280 ~, from the next spoke and the third spoke is 200-210 A from spoke number two, and then there is a gap of 360--400 A before the next spoke triplet (Sale and Satir, .1976; Dentler and

28

M . J . DOUGHTY and S. DRYL

Cunningham, 1977). The spokes appear to be aligned with two rows of projections along the central sheath. The relationship between the horizontal alignment of the spoke groups on one doublet compared to an adjacent one is uncertain (Sale and Satir, 1976). Overall, however, it would appear that the radial spokes form some sort of helical arrangement around the central sheath (Warner and Satir, 1974; Chasey, 1974; Sale and Satir, 1976; Dentler and Cunningham, 1977). A segment of the radial spokes in Tetrahymena cilia were observed to be thicker than adjacent regions. These regions were termed "spoke heads", although there have been differences of opinion as to whether the spoke heads are located along the length of the spoke (Gibbons, 1965; Allen, 1968) or on the central sheath end of the spoke (Summers and Gibbons, 1973; Summers~ 1975: Mohri, 1976; Dentler and Cunningham, 1977). Warner (1970) describes the presence, in fixed sections, of a radial link, a link head and a transitional link in flagella axonemes, while Dentler and Cunningham (1977) estimate that the spoke head is sited 40-I 00/~ from the central sheath. Using the more recently developed technique of critical point drying-- a method in which the three-dimensional form of the cilium is believed to be more accurately preserved (Sale and Satir, 1976}--it would appear that, in Tetrahymena cilia at least, the radial spokes terminate in a club-like head. Longitudinal sections and crosssections of mussel gill cilia reveal similar morphological arrangements without the presence of an intermediate transitional link between the spoke head and the central sheath (Warner and Satir, 1974). An open mind is perhaps advisable concerning some of the finer details of the axonemal structure, due to inherent problems with specimen contraction or expansion during preparation for electron microscopy and the obvious dependence of at least cross-bridge (dynein-microtubule interaction) activity on fixation conditions (see later). Details, as currently understood, are given so as to indicate the structural complexity of the 9 + 2 axoneme. We chose to present the organization of the cilium, as seen in cross-section, as schemetically illustrated in Fig. 13. Several authors have either noted or detailed the presence of electron-dense deposits or even specific structures "linking" the ciliary membrane to a site between the peripheral doublet microtubules (Gibbons, 1965; Allen, 1968; Satir, 1974a; Dute and Kung, 1978; Dentler et al., 1980). Dentler et al. (1980) provide evidence indicating that these "wine glass" or "champagne glass" structures (alternatively called "linker" structures) are composed of a high molecular weight ATPase protein and three smaller molecular weight proteins and suggest that the linker structures serve a functional role to either transduce events from the membrane to axonemal structures or to regulate membrane-axoneme disposition (see also Dute and Kung, 1978). A schematic illustration of the organization of the ciliary membrane and its morphological detail in relation to the axonemal skeleton is given in Fig. 14. As shown in Fig. 13, two "arm-like" projections are observed in ciliary (and flagellar) cross-sections, to extend from the a-subfiber of each of the peripheral doublet microtubules towards the adjacent b-subfiber on the next peripheral doublet. These structures were first characterized by Gibbons (1963), who subsequently demonstrated (Gibbons, 1963, 1965) that these arms could be extracted; that in Tetrahymena cilia, the extract contained two types of protein complexes with characteristic sedimentation coefficients of 30S and 14S (on continuous sucrose gradients), and that on incubation of extracted axonemes with the 30S complex resulted in reappearance of the arms predominantly in their original sites, although some other minor structures, extending outwards from the doublets, were also noted. The extracted protein complexes were named "dyneins" (forceproteins) (Gibbons and Rowe, 1965) since it was also found that the protein complexes possessed ATPase activity (see later). While it has been claimed that only 30S dynein from Tetrahymena cilia will combine significantly with extracted axonemes (Gibbons, 1963, 1965; Shimizu, 1975), caution should perhaps be advised in the following respects prior to assessment of the disposition of the dyneins within the axoneme and their role in axonemal motion. Due to further resolution in techniques, it has been shown that one type of dynein (thought not yet proved homogeneous), dynein 1 or dynein A-band, can be extracted from and recombined with either Tetrahymena cilia (Warner et al., 1977) or

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

29

FIG. 14. The cilium: 3-D schematic illustration of the structure of the plaque and necklace regions of a cilium of Paramecium aurelia. Figure (provided courtestyof Dr. Kung)from Dute and Kung, 1978. sea-urchin sperm tails (Kincaid et al., 1973; Gibbons and Gibbons, 1976; Ogawa et al., 1977b) and appear to form the outer arms. 30S dynein, as extracted by dialysis from T e t r a h y m e n a cilia and then fractionated on sucrose gradients, is not dynein 1, although this fraction appears to contain a major polypeptide which may be dynein 1 (dynein A-band; see Mabuchi and Shimizu, 1974; Mabuchi et al., 1976). Recombination studies have been carried out with either salt-extracted axonemes and salt extracts (an alternative method of obtaining dyneins, especially dynein 1) or with low ionic strength dialysis techniques and with the dialyzate. In view of differences in both the kinetics of extraction of flagellar dynein ATPase at least (Mohri et al., 1969; Gibbons and Fronk, 1972) and in residual structures after the extraction procedures for both cilia and flagella (Gibbons, 1963, 1965; Renaud et al., 1968; Kincaid et al., 1973; Gibbons and Gibbons, 1972, 1973; Shimizu, 1975), some cross correlation studies would appear desirable if not essential. It is unknown, e.g., if, during the different types of extraction procedure, some protein components conferring binding capacity to the dyneins are lost or retained. The most important observation made by Gibbons (1963, 1965), from the point of view of stimulating research in this area, was that the arm-like projections possessed magnesium ion-dependent adenosine triphosphatase (ATPase) activity (Gibbons, 1965: 1966: Gibbons and Rowe, 1965). 14S dynein was also found to possess similar activity. Direct histochemical demonstration of such enzymic activity at the arm sites in situ has not yet been demonstrated in P a r a m e c i u m cilia. Burnasheva and Jurzina (1968) note the presence of unspecified ATPase activity only "in basal areas of the peripheral doublets" in Tetrahymena cilia. Cytochemical work on flagellar axonemes has revealed the presence of either Mg 2÷ ATPase activity (Anderson et al., 1968; Burton, 1973) or ATPase activity (Nagano, 1965) on or near the peripheral doublet microtubules amongst other sites. A

30

M.J. DouotTVand S. DRYL

trypsin fragment of dynein 1, still possessing ATPase activity in vitro, has been shown by immunocytochemical methods to be located at the distal end of the outer arms (Ogawa et al., 1977a, b). Worley (1934) and later Seravin (1961) proposed that each individual cilium had its own mechanism responsible for conferring motion to the organelle. Hoffman-Berling (1955) had independently reported ATP-dependent motion in isolated fragments of glycerol-treated flagella. Seravin (1961)demonstrated that in two ciliated protozoa (Euplotes and Spirostomum), following treatment of the cells with saponin-EDTA mixtures, the cells could be reactivated (with restoration of motility) by transfer to solutions of ATP and MgCI2. Wincur (1967) reported similar reactivation of isolated, glycerol treated Tetrahymena cilia and his results were further elaborated by Saavedra and Renaud (1975). Burnasheva et al. (1963, 1965) separated cilia from broken Tetrahymena by an ethanolKC1 procedure and reported Mg 2÷ and Ca 2 ÷ stimulated ATPase activity in salt extracts from the cilia fractions. Gibbons (1965, 1966) showed that ATPase proteins could be extracted from isolated, demembranated Tetrahymena cilia by either high ionic strength salt extraction (0.5 M KC1 or KI) or dialysis at low ionic strength in the presence of a chelating agent (EDTA). Both techniques, with minor modifications (e.g. substitution of NaC1 for KCI), are still widely used for extraction of dynein proteins from both cilia and flagella. Gibbons (1965, 1966; Gibbons and Rowe, 1965) showed that the extracted dynein proteins from Tetrahymena cilia could be resolved into two major fractions on sucrose gradients (30S and 14S fractions), and many authors (Raft and Blum, 1969; Otokawa, 1972; Hoshino, 1974; Mabuchi and Shimizu, 1974; Shimizu and Kimura, 1974; Nishino and Watanabe, 1977a; Hayashi and Takahashi, 1979) have since confirmed this separation. As recently suggested by Gibbons (Gibbons et al., 1976), the term dynein proteins should be applied to all high molecular weight (greater than 30,000 daltons subunit size) ATPase proteins found within the ciliary or flagellar axoneme. To date there have been no definitive studies on the enzyme activity of the dyneins primarily because complete purification of any one dynein protein has not yet been achieved. Crude dynein extracts (salt-extracts) from Paramecium cilia contain several polypeptides of high molecular weight (Doughty, 1979b). The estimate of greater than 500,000 daltons subunit size for these proteins appears to be incorrect and a more recent estimate (Hayashi and Takahashi, 1979) of 340--380,000 daltons appears more suitable in view of similar molecular subunit sizes reported for flagellar axoneme dynein proteins. Similar results, i.e. subunit size of 350-550,000 daltons, have been reported for dynein proteins of Tetrahymena cilia (Gibbons and Rowe, 1965; Daiya et al., 1972; Hoshino, 1974, 1975; Mabuchi and Shimizu, 1974; Nishino and Watanabe, 1977a, b), in flagella axonemes from various sources (Gibbons et al., 1976; Witman et al., 1978; Ogawa and Gibbons, 1976; Link, 1973; Bell et al., 1980) and metazoan ciliary axonemes (Linck, 1973; Warner et al., 1977). Studies on the enzyme activity of the dyneins from Tetrahymena cilia have been on either crude extracts (Raft and Blum, 1969; Blum, 1973; Hoshino, 1974) or sucrose gradient purified dyneins (30S and 14S) fractions (Gibbons, 1966; Raft and Blum, 1969; Otokawa, 1972, 1973; Hoshino, 1974, 1975, 1977a, b). However, it should be noted that neither of the sucrose gradient "purified" fractions are homogeneous with respect to dynein composition as revealed by SDS-polyacrylamide gel electrophoresis (Mabuchi and Shimizu, 1974; Mabuchi et al., 1976; Hoshino, 1974, 1975, 1977b; Nishino and Watanabe, 1977a, b; Warner et al., 1977; Hayashi and Takahashi, 1979). The possible presence of lower molecular weight regulatory proteins associated with dynein fractions is indicated both from differences in the calcium ion sensitivity of dynein fractions (Blum and Hayes, 1977; Doughty, 1979b)and by the presence of such proteins associated with dynein fractions as revealed by high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bell et al., 1980). Sepharose chromatography of crude salt fractions from Paramecium ciliary axonemes, whilst yielding one apparently homogeneous dynein fraction, failed to resolve others (Doughty, 1979b). Thus, few of the enzymic properties of the dyneins so far reported can be confidently ascribed to the dyneins themselves or to any particular class of dyneins, but rather represents the activity of a heterogeneous

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

31

mixture of dynein proteins and associated proteins although the predominant activity is due to the dyneins. Slight differences in the extraction procedures can additionally apparently alter the relative quantities of different dynein proteins in the extract and thus alter the properties of the extract (Mabuchi and Shimizu, 1974; Mabuchi et al., 1976; Doughty, 1979b; Kincaid et al., 1973). The presence of axonemal proteins (principally tubulin) has been reported to considerably influence the enzymic activity of the dyneins (Otokawa, 1972; Ogawa, 1973; Hoshino, 1977a). In reviewing the reported properties of the dynein activities, we must additionally question whether many of the properties have any physiological significance. At this time, neither the pH or ionic milieu of the intraaxonemal space is known. The K ÷ concentration of the cell cytoplasm has been reported to be around 20mM (Yamaguchi, 1963) whilst intra-axonemal ATP concentrations appear to be around l mu (Naitoh, 1969; Browning and Nelson, 1976b). Due to the presence of ATPase (Burnasheva and Jurzina, 1968) or Na-K stimulated phosphatase activity (Dentler, 1977) in the vicinity of the basal plate region in Tetrahymena cilia, we consider that the intra-axonemal monovalent cation concentrations are regulated and are perhaps close to that of the cell cytoplasm proper. Since both ciliary activity of reactivated axonemes (Naitoh and Kaneko, 1973) and the activity of crude dynein extracts (Doughty, 1979b) from Paramecium cilia show an optimum at stochiometric ratios of ATP:Mg 2÷ (1:1) such stoichiometry presumably indicates the normal intra-axonemal environment. The origin of intra-axonemal ATP, required for ciliary motion, is unknown but is generally assumed to originate from intracellular sites and is thus presumably controlled and maintained at a reasonably constant level. From what data is available, the preferred dynein substrate would appear to be MgATP 2- (Doughty, 1979b; Gibbons, 1966; Mohri et al., 1969; Daiya et al., 1972; Blum, 1973; Hoshino, 1974, 1975, 1977b; Shimizu and Kimura, 1974; Gibbons and Fronk, 1972; Ogawa and Mohri, 1971; Hayashi, 1974). All other nucleosides are poorly used by Tetrahymena ciliary dyneins in the presence of MgClz (Gibbons, 1966; Blum, 1973), and are largely ineffective (except for ADP--see later) in activating "model" Paramecium (Naitoh, 1969) or restoring motion to isolated, glycerol-treated cilia of Tetrahymena (Saavedra and Renaud, 1975). Dynein extracts from Paramecium cilia were found to poorly utilize either Zn 2÷ or Ni 2÷ (with ATP) (Doughty, 1979b) and similar results have been reported for Tetrahymena ciliary dyneins (Gibbons, 1966) and flagellar dyneins (Ogawa and Mohri, 1971). The utilization of CaATP 2- by dynein proteins is uncertain. Ca 2÷ ions cannot substitute for Mg 2÷ ions in reactivation experiments with "model" Paramecium (Naitoh and Kaneko, 1972, 1973; Kung and Naitoh, 1973); i.e. CaATP 2- does not support motility. The mechanochemical cycle underlying ciliary activity would therefore appear to be magnesium dependent. However, various authors have reported Ca 2+-stimulated ATPase activity of dyneins from both Paramecium cilia (Doughty, 1979b), Tetrahymena cilia (Gibbons, 1966; Raft and Blum, 1969; Daiya et al., 1972; Shimizu and Kimura, 1974; Hoshino, 1974, 1975, 1977b), Chlamydomonas flagella (Watanabe and Flavin, 1976) and sea-urchin sperm flagella (Mohri et al., 1969; Gibbons and Fronk, 1972; Ogawa and Mohri, 1971; Hayashi, 1974). However, in most cases, the ATPase activity was less than that reported in the presence of an equal concentration of magnesium ions and significant activity was only obtained at millimolar concentrations of calcium ions. Calcium ions can effect a marked alteration of both ciliary beat frequency and also the swimming speed of reactivated "model" Paramecium in the presence of MgATP 2-, as well as effecting reorientation (reversal) of the ciliary power stroke at concentrations above 10-~M (Naitoh and Kaneko, 1972). A maximum frequency for cilia in reverse beating occurs at around 5 × 1 0 - 6 M free Ca 2+ ions in model Paramecium, while higher concentrations effect apparent immobilization of the cilia, in the presence of MgATP 2- (Naitoh and Kaneko, 1972). The isolated ciliary dyneins do not, however, appear to have lost a sensitivity to physiological (micromolar) concentrations of calcium ions (Doughty, 1979b) and thus the calcium ion concentrations hitherto used in most dynein activity assays (millimolar) have questionable significance with respect to the role of calcium ions in the ciliary mechanochemical cycle. Similarly, the reported alteration in magnesium-dependent dynein

32

M.J. DOUGHTY and S. DRYL

ATPase activity by high concentrations of KCI (up to 1 M) (Gibbons, 1966; Raft and Blum, 1969; Blum, 1973; Hoshino, 1974, 1975) is unlikely to have any physiological significance for fresh water species of Paramecium (all known species except Paramecium calkinski are fresh water protozoans), Tetrahymena or Chlamydomonas. A K ÷ ion sensitivity for sea-urchin sperm flagella dyneins (Gibbons and Fronk, 1972) or for the dyneins that appear to be present in brain tissue (Burns and Pollard, 1974) may, however, be physiologically relevant. The dynein ATPase activity may additionally be sensitive to high NaC1 concentrations, and since the dyneins can be extracted with 0.5 M NaC! instead of KCI (Mohri et al., 1969; Gibbons and Gibbons, 1973), concentrations of such salts in any assay procedure for dyneins should be carefully considered. Any salt effect, on either dynein ATPase activity or dynein recombining activity or interaction with axonemal proteins in situ may however simply reflect an ionic strength effect, since the dynein activities generally appear to be inhibited by high concentrations of any monovalent or divalent cation salt. The major protein component of both ciliary and flagellar axonemes is tubulin and forms the structural basis of the microtubules. A full description of either the physical, chemical or ultrastructural properties of the tubulins is outside the scope of this review. Other reviews are available (Mohri, 1976; Amos et al., 1976). Both the a- and b-subfibers of the peripheral doublet microtubules and the central pair of microtubules contain two species of tubulin with subunit molecular weights of 54-58,000 and 46-54,000daltons (Mohri, 1976) as determined from analytical sodium dodecy sulfate polyacrylamide gel electrophoresis of Paramecium (Adoutte et al., 1980; Merkel et al., 1980), or Tetrahymena (Adoutte et al., 1980) ciliary axonemes or flagellar axonemes (Olmsted et al., 1971; Linck. 1976). The two tubulins, which are non-uniformly distributed between the a- and b-subfibers of the peripheral doublets (Linck, 1976)appear to be related but distinct proteins (Luduena and Woodward, 1973; Piperno and Luck, 1975). Polyacrylamide gel analysis reveals a large number of protein components in both ciliary and flagellar axonemes. The activity and function of most of these is unknown, although by use of mutants, tentative localization of some of these proteins has been achieved (Piperno et al., 1977) and chemical and thermal fractionation methods have indicated which proteins are associated with the microtubule structures (Linck, 1976) in flagellar axonemes. Some other proteins and enzymes have been tentatively identified in axonemes. Early workers (Seravin, 1961) considered that the contractile biochemistry of cilia and flagella would be different from that found in skeletal or smooth muscle. However, there have been many attempts to draw parallels between the two systems, and a substantial proportion of dynein studies have been directed towards either showing the existence of or disproving common activities between dyneins and muscle myosins and the dyneintubulin and myosin-actin contractile systems. Consequently, in addition to a search for enzymes common to both ciliary/flagellar axonemes and muscle, considerable attention has been paid to the characteristics of the axomeme contractile protein pair and those in muscle. Ogawa et al. (1975) showed that flagellar dyneins had a different amino acid composition to muscle myosin; that dynein did not interact with muscle actin and that no common immunological cross-reactivity existed between either dynein and myosin or their primary tryptic fragments. Finch and Holtzer (1961) reported there were no common antigenic properties between skeletal muscle and avian ciliary proteins. From a report by Burnasheva et al. (1965), that actin-like proteins were not present in Tetrahymena cilia, a series of studies between 1967 and 1976 (for details see Mohri, 1976) compare the physical, chemical, biochemical and ultrastructural properties of tubulin and actin. These studies show that the two proteins differ in both subunit molecular size, in amino acid composition, in peptic digest maps and overall antigenic character. The ultrastructure of the units that the two proteins form on self-polymerization are very different and most importantly, from the point of view of the role which tubulin plays in axonemal motion, is the observation that the bound nucleosides are different--being guanosine nucleosides for tubulin and adenine nucleosides for actin. More recently, it has also been reported that tubulin, unlike actin, does not inhibit DNase 1 (Castle et al.,

CONTROL OF CILIARY ACTIVITYIN PARAMECIUM

33

1976). Thus it would appear that dynein and tubulin are distinct from their counterpart principal proteins in muscle. However, the presence of a different contractile protein pair in motile axonemes does not necessarily rule out the presence of an actin-myosin system as well. However, since it would appear that actin, if present, is there in exceedingly small quantities compared to tubulin at least in Tetrahymena cilia (Rubin and Everhart, 1973), it would appear reasonable to assume that the two muscle type proteins are unlikely to play any significant role in the mechanochemical cycle responsible for axonemal motion per se. Some muscle type enzymes have been reported to be present in cilia and flagella. Adenylate kinase activity has been reported in Tetrahymena cilia (Culbertson, 1966a, b; Otokawa, 1973, 1974), and Chlamydomonas flagella (Watanabe and Flavin, 1976). Arginine kinase activity was detected in Tetrahymena cilia (Watts and Banister, 1970) but was not detected in Chlamydomonas flagella (Watanabe and Flavin, 1976). Isolated cilia from Tetrahymena (Saavedra and Renaud, 1975) or "model" Paramecium (Naitoh, 1969) can be reactivated for longer time periods by nucleotide triphosphates other than ATP in the presence of ADP than in the presence of ATP alone. These reports indicate the presence of phosphotransferase enzyme(s) in cilia which could conceivably serve the purpose to maintain intraciliary ATP levels at concentrations optimum for motion under conditions of demanding energy supply. Murofushi (1973) reported the presence of an ATP-dependent protein kinase activity in Tetrahymena cilia that was found to selectively phosphorylate tubulin. Phosphorylation of tubulin in Chlamydomonas flagella has also been reported (Piperno and Luck, 1976). Murofushi (1974) also reported the presence of both cAMP- and cGMP-dependent protein kinases in detergent (Triton) extracts of Tetrahymena cilia. As a final note, soluble Ca 2+ ATPase enzymes have been found associated with ciliary preparations (Andrivon et al., 1977; Doughty and Kaneshiro, unpublished results; Nelson, personal communication to MJD) which are distinct from the membrane Ca 2+ ATPase (Doughty and Kaneshiro, unpublished results) and could be from ciliary axonemal origin. A soluble, cytoplasmic Ca2+-ATPase, different again from the above enzymes, has also been reported for Tetrahymena (Chua et al., 1977). At present, it should be acknowledged that uncertainties still exist concerning the purity of both ciliary and flagella preparations, and in several instances contamination of ciliary fractions by broken cells or their inclusions have been reported (Child, 1959; Watson and Hopkins, 1962; Gibbons, 1965; Subbiah and Thompson, 1974). More recently, efforts have been made to characterize isolated ciliary preparations of Paramecium both by microscopy and by enzyme analysis (Doughty, 1979b; Kaneshiro et al., 1979; Adoutte et al., 1980). Further development of such characterization methods is necessary, such that we can be sure that activities reported in ciliary preparations are indeed ciliary membrane or ciliary axoneme enzymes or receptor proteins, for example.

3. Passive Behavior Ciliary Activity and Electrical Properties of Paramecium 3.1. SWIMMING CHARACTERISTICS OF PARAMECIUM IN DIFFERENT CHEMICAL ENVIRONMENTS

Paramecium swims through the water as a result of the coordinated activity of the many cilia that cover its entire body surface. An analysis of the motion of the individual cilium shows that the activity of the organelle consists of two different sequences: an effective (power) stroke, followed by an ineffective (recovery) stroke. In the first, the cilium is relatively straight and can be observed clearly in profile against the body surface, and in the second phase of the beating cycle, the cilium, now bent, returns to its original position at the start of the power stroke by following a trajectory close to the body surface (see Section 3.2 for full details).

34

M.J. DOUGHTY and S. DRVL

( \\

-

.~

FIG. 15. The swimming path of Paramecium. Schematic representation of the 3-D nature of the swimming path of a Paramecium cell in forward left spiralling mode. Measures of the length of complete rotation (spiral pitch = p) and the radius of the spiral (r) can be made from the projection in 2-D (heavy dashed line). Based on Sears and Elvebank, 1961.

During forward motion of the cell, the ciliary power stroke follows a direction from the anterior end of the cell towards the posterior end of the cell and thus drives the cell forward in an analogous manner to the action of the oars on a boat. However, as noted in early studies (Jennings, 1897, 1901 ; Alverdes, 1922; Bullington, 1925), the power stroke is somewhat oblique with respect to the long axis of the cell. As a result of the combined action of all of the body cilia beating in an oblique direction, the cell rotates on its long axis (Jennings, 1901). As a result of a screw-like conformation of the body, the rotation on the long axis is developed into a precession of the cell around a rotational axis of progression such that a spiral path is prescribed (Jennings, 1901 ; Alverdes, 1922; Bullington, 1925; Ludwig, 1929; Haiderer et al., 1949; Grebecki et al., 1967b). As noted by these authors, and confirmed by Sears and Elvebank (1961) in a quantitative study of the spiral swimming in Paramecium, one rotation of the body axis is completed in one revolution of the spiral "orbit". Figure 15 shows a schematic representation of the spiral swimming path of Paramecium. For the "aurelia" group of Paramecium (P. aurelia, P. caudatum and P. multimicronucleatum), the spiral is left handed (anticlockwise as viewed from the side or from the posterior end looking towards the anterior end) with respect to the direction of revolution of the cell on its long axis and the direction of progression of the spiral (Alverdes, 1922; Ballington, 1925, 1930). The resultant forward swimming behavior is termed "forward left spiralling" (FLS) (Grebecki et al., 1967a, b). The pitch of the spiral is proportional to the width of the spiral regardless of the overall swimming speed of the cell (Sears and Elvebank, 1961). A wealth of studies since the turn of the century have noted changes in the swimming behavior of Paramecium following changes in either the chemical or physical characteristics of the cells' environment. We will concern ourselves only with those reports that are relevant to the review title. The reader is referred to monographs or reviews on Parame-

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

35

cium for a more exhaustive listing (Kalmus, 1931; Wichterman, 1953; Jahn and Bovee,

1968; Dryl, 1974). In considering the effects of extracellular environment on the "resting state" behavior of Paramecium several factors need to be considered. Firstly, we define resting state behavior as that in which cells are not undergoing a short-term, stimulus-induced response (phobic response) from which they subsequently recover (adapt) in the presence of continued presence of the stimulus. Such responses are typically observed in Paramecium as either reversed swimming behavior for finite periods or temporary acceleration of forward motion. For resting state behavior, two aspects of the cells' motion need to be considered. Firstly, as recorded in some detail by Bullington (1925), Paramecium swim in FLS for a finite time period only. The cell can spontaneously change its direction of motion from anything from a few degrees to 90 °, and then continues in FLS (usually). The resultant motion of Paramecium under "constant environmental conditions" (if such a condition is ever realized) is therefore essentially random in three-dimensional time and space and has been described as a random walk (Often and Roberts, 1973). The rate at which such, apparently random, directional changes occur is affected by a variety of extracellular conditions to thus produce a biased random walk behavior. A note on terminology is essential here. The recommendations of a recent committee proceedings on terminology for description of the behavior of motile unicells are adopted here (see Diehn et al., 1977). An increase in the rate of forward motion that is maintained is a positive orthokinesis (a change in the linear velocity of the cell) whilst an increase in the rate of directional changes in the forward motion of the cell is termed a positive klinokinesis (a change in the rate/frequency of directional changes). Other descriptions can be derived accordingly for reductions in the rates of these two basic behavioral phenomena which are generally sufficient to define the translational motion of motile unicells in space and time. Only the effects of electrolytes, hydrogen ion concentration (pH) and temperature on the swimming behavior of Paramecium have been studied in any detail, so we shall confine ourselves to these reports. As a general point, however, since the swimming behavior of Paramecium is markedly influenced by these three factors in particular, it is not possible to directly correlate the various studies carried out over the last 70-90 years simply because of differences in media (culture or buffer solutions of different pH, ionic strength and electrolyte composition) and physical conditions (principally temperature) under which studies have been carried out. The relative changes in the behavior of Paramecium with respect to these three principal variables do, however, appear to show remarkable correlation. Several authors (Andrejewa, 1931; Sears and Elvebank, 1961; Kinosita et al., 1964b; Nakatani, 1970; Fujui and Asai, 1976) have reported alterations in the swimming velocity of Paramecium with respect to the concentrations of various inorganic cation salts in the experimental solution. These results are summarized in Fig. 16, and show that an optimal swimming velocity occurs for most cations within fairly narrow concentration ranges. It is pertinent here to introduce the behavior phenomena often ascribed to "chemotaxis" (directed movement towards or away from a stimulus source) of Paramecium--i.e., the relative accumulation of cells in a defined area containing either certain electrolytes, other chemicals or having a different pH to the surrounding medium. Positive "chemotaxis" has never been demonstrated in Paramecium--the cells accumulate or disperse from defined areas through a combination of orthokinetic and klinokinetic responses (Van Houten, 1977, 1978, 1979). Experimentally however, various workers have noted accumulation of Paramecium (and other ciliated and flagellated protozoa) into areas of different chemical compositions (Jennings, 1897; Pearl, 1901; Massart, 1889; Fox, 1925; Dryl, 1959, 1961b); relative migration of cells between two environments (two chambers or drops of solution) (Massart, 1891; Vieweger, 1912; Abderhalden and Schiffman, 1922; Nowikoff, 1908; Johnson, 1929; Rostowska, 1964); between three drops of solution (a T-maze principle) (Nowikoff, 1908; Wense, 1935); into capilary tubes (Massart, 1891; Garrey, 1903; Nakatani, 1968) or distribution between the three arms of a glass T-maze

36

M.J. DOUGHTYand S. DRYL

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FIG. 16. Swimming behaviour of Paramecium and inorganic cation salts: swimming speed. Effects of various inorganic cation salts on the speed of forward motion of Paramecium caudatum. Left pannel---CaC12, middle panneI--KCI, rioht panneI--NaCI. Data obtained from various sources as indicated by the following symbols. Conditions given in brackets. Q---Andrejewa, 1931 (15°C, pH 6.8); O--Nakatani, 1970 (20-22°C, pH 7.2); II--Fukui and Asai, 1976 (20-25°C, pH 6.8); E]--Dryl, 1961b (21-22°C, pH 7.1); A--Doughty, 1979a (19°C, pH 7.1); &--Kinosita, Dryl & Naitoh, 1964b (19-20°C, pH 7.2).

(Van Houten et al., 1975; Doughty and Dodd, 1978; Doughty, 1979a). Dryl (1959, 1961) studied migration of Paramecium across a glass plate covered with a thin film of solution with areas of different electrolyte composition and showed relative migration of cells between different concentrations of inorganic cation salts. Nakatani (1968) showed migration of Paramecium into capillary tubes containing various salt solutions and Doughty and Dodd (1978) reported migration of Paramecium into arms of a T-maze ;tOO 4

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FIG. 17. Swimming bchaviour of Paramecium and inorganic cation salts: chemoaccumulationchemorepulsion (chcmokinesis, "chemotaxis') responses. Figure shows published data on chemoaccumulation where a range of concentrations of inorganic cation salts were tested. - . - O - . K C I ; - - O - - NaCI; - - A - - CaCi2. Data from sources indicated by numerical superscripts by data points. Details of control solutions and cells given after authors. ~Dryl, 1959: P. caudatum. Dryl+s solution (2raM NazHPO+, 1 mM NaHzPO+, 2mM Na-citrat¢, 1.5mM CaCl2, pH 7.1), 21-22°C. 2Dryl, 1961b: P. caudatum. Dryl's solution, pH 7.1. 21-22°C. 3Nakatani, 1970: P. caudatum. 3.3 mM NaCl, 0.4mM CaCI2, l mM Tris, pH 7.2. 21-23°C. +Doughty and Dodd, 1978: P. aurelia. 0.5 mM CaCI2, - 6 mM Na (OH), 5 mM Hepes-Pipes, pH 7.1.23-24°C.

CONTROL OF CILIARY ACTIVITY IN

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pH FIG. 18. Swimming behaviour of Paramecium and pH : swimming speed. Effects of pH on speed of forward motion of Paramecium caudatum. Q--from Dryl, 1961a: Cells in Na2HPOd-NaH2PO, buffers containing either citric acid or boric acid-NaOH to give desired pH. 18 + I°C. O---from Kinosita, Dryl and Naitoh, 1964b: Cells in unspecified salt solutions. 19 20°C.

containing certain concentrations of salt solutions. These results are presented in Fig. 17. The similarity between plots of the effects of cations on the swimming velocity of the cell and the occurrence of chemoaccumulation indicates that chemoaccumulation can be mediated through a change in the linear velocity of the cells (as a principal contributory mechanism); i.e. chemoaccumulation is the result of positive orthokinesis rather than a chemotaxis. There would also appear to be a similar correlation between changes in the swimming speed of Paramecium and changes in the extracellular medium (Dryl, 1961a, Kinosita et 160

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FIG. 19. Swimming behaviour of Paramecium and pH: Chemoaccumulation-chemorcpuision (chemokinesis, "'chemotaxis") responses to solutions of different hydrogen ion concentration. Left pannel: O--Dryl, 1959: Plate assay. Control solution (100% response), pH 6.4 obtained by combination of Na2HPO4 and NaH2POd. P. caudatum. Other solutions by addition of either citric acid or boric acid-NaOH. 21-22 C. O---Johnson, 1929. 2-way maze principle-~listribution of Paramecium between 2 linked drops of solution of a glass slide. Arbitrarily normalized to pH 6.4 (100%). P. eaudatum. Cells in culture medium (hay infusion) pH 7.7 with additions of various inorganic acids to alter pH 21-28°C. Right panel: Unpublished data of Dr. Van Houten. Taken from Ph.D. thesis 1976 with permission of author. P. aurelia 2-way glass T-maze. pH responses obtained by testing cells against formic acid (test arm) relative to K-formate (control solution) for low pH values or relative to KOH (control) for high pH values. 100% arbitrarily chosen at 6.4 and other responses calculated from relative indices of "chemotaxis" (Ich,: see Van Houten, Hansma and Kung, 1975 for full details). Room temperature.

38

M.J. DOUGHTY and S. DRYL 120

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FIG. 20. Swimming behaviour of Paramecium and pH : Klinokinesis. Changes in the frequency of directional alterations in the swimming paths of Paramecium (unspecified sp.) as a function of extracellular pH. Data from Gunn and Walshe, 1941. Room temperature. Cells transferred from culture medium at pH 8,0 to culture medium containing various quantities of either acetic acid or sodium carbonate and responses measured 6-10 sec. (0) or 1-2 minutes (O) after transfer to these solutions.

al., 1964b; Chase and Glaser, 1930) (Fig. 18) and the occurrence of accumulation of Paramecium into either areas of different pH (Vieweger, 1912; Dryl, 1959); into one side of a two-way T-maze system (Johnson, 1929); or into capillary tubes containing solutions of different pH (Nakatani, 1968, 1970) (Fig. 19). An inverse relationship, with respect to pH of the medium, is shown for klinokinesis in Paramecium. Gunn and Walshe (1941) reported dynamic changes in the frequency of directional change of the cells' swimming

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showing effect of temperature on the forward swimming speed of cells. @----data from Glaser, 1924. Paramecium (sp. u n ~ ) in culture medium (hay infusion; probably slightly alkaline). O--from Nakaoka and Oouwa, 1977 (with permission Dr. Oosawa/.l. Protozootogy and Society Protozoologists). Paramecium caudatum in culture medium (lettuce infusion supplemented with Tris-maleate, pH 7.0).

39

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

path in solutions of different pH (Fig. 20). In addition, there have been several qualitative observations of the migration of various protozoa into or away from capillary tubes containing various acids or alkalis (Massart, 1891; Garrey, 1903; Frank, 1904). Glaser (1924) made a quantitative study of the effect of temperature on the swimming velocity of Paramecium. Later studies were carried out by Tawada and Oosawa (1972) and Nakaoka and Oosawa (1977). The results of these studies are presented in Fig. 21. We chose to plot the data on Arrhenius axes to emphasize the effect of temperature on swimming velocity, and thus ciliary activity, in Paramecium. In both cases, within a fairly narrow temperature range, there is an abrupt change in swimming velocity observed. The actual "transition temperature" seems to depend on the temperature at which the cells are grown (Tawada and Oosawa, 1972). It is uncertain whether this transition reflects changes in ciliary axoneme activity or ciliary membrane activity, or both. Evidence to support the former case is given by Toyotama and Nakoaka (1979), who reported that demembranated ("model") Paramecium responded to temperature change in a similar manner to electrically excitable, membranated cells. In contrast, the temperature sensitivity of cell chemosensory behavior (K÷-induced ciliary reversal) (Doughty, 1978b; Hildbrand, 1978) of cell responses to current injection (Machemer, 1974a) and cell thermosensory behavior (Hennessey and Nelson, 1979) has been interpreted in terms of alteration of membrane activity. Differences in thermosensory behavior have been reported for a membrane excitation mutant (Hennessey and Nelson, 1979). Since this mutant has been found to possess normal axonemal responses ("model" cells swim backwards in the presence of greater than 10-6M Ca2÷; see Kung and Naitoh, 1973), and since thermosensory behavior is strongly influenced by agents effecting chemosensory behavior of the cells and vice versa (Mendelssohn, 1902a; Hennessey and Nelson, 1979; Oliphant, 1938; Doughty, 1978b; Andrivon, 1970b), temperature induced alteration of membrane activity seems a reasonable explanation for thermosensory behavior. Paramecium are very sensitive to temperature. Mendelssohn (1902b) estimated that Paramecium could detect a temperature difference of 3-4°C. Later studies (Tawada and Oosawa, 1972) indicate similar sensitivity of the calls to temperature change. Bullington (1925) studied the characteristics of the swimming paths of Paramecium at different temperatures and observed (Table 1) that the length of a "straight run" (i.e. a straight swimming path without any spontaneous directional change) varied with temperature since the frequency of directional change varied with temperature. Similar observations were made by other workers (Jennings, 1901; Mendelssohn, 1902a, b; Yapp, 1941; Doughty, 1978b; Hennessey and Nelson, 1979). The effect of temperature on the steadystate frequency of directional change in the swimming path of Paramecium was carefully evaluated by Nakaoka and Oosawa (1977) (Fig. 22). Current data shows a reasonable correlation between the swimming speed of Paramecium and the frequency of ciliary beating (e.g. see Fig. 5). Machemer (1972a) showed that the beat frequency of Paramecium oral cilia was influenced by temperature and found similar alteration, as a function of temperature, in beat frequency as seen on cell swimming speed. The strict relationship between ciliary beat frequency and cell swimming speed reflects the power of the axonemal processes per se since this relationship is TABLE 1. EFFECTOF TEMPERATUREON THE SPIRALSWIMMINGPATH OF PARAMECIUM

11~C Number of paths Total length of paths (cm) Average length of paths Longest single path Longest single distance in a relatively straight line in one path Total distance in straight lines

20~C

Room temp.

7 124 17.5 35

10 247 24.7 46

47 1060 22.5 51

3 3

25 165

31 640

22~C 23 802 35.5 63 25 333

25°C 21 1130 54.5 183 20 312

29°C

Total

7 251 33 78

115 3600

28.5 35

1493

From Bullington (1925). Reproduced with permission from Archives Protistenkunde, Gustav Fisher Verlag. ~,P.~. 16/1~c

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FIG. 22. Swimming behavior of Paramecium and temperature: Klinokinesis. Effect of temperature on the frequency of directional changes in the swimming path of Paramecium caudatum. Cells in culture medium (lettuce infusion supplemented with Tris maleate, pH 7.0). From Nakaoka and Oosawa, 1977. Reproduced with permission J. Protozoology/Society Protozooiogists.

preserved in demembranated "model" cells (Naitoh and Kaneko, 1972, 1973; Toyotama and Nakaoka, 1979) despite the overall reduction in the rate of both parameters (see Section 4.5 for details). In conclusion, the rate of locomotion of Paramecium cells and the frequency of ciliary beating change in an approximately parallel manner under a variety of external perturbations (chemicals, pH and temperature). The mechanisms that control ciliary beating in forward mode are thus sensitive to temperature, pH and cations. 3.2. CHARACTERISTICS OF CILIARY MOTION Paramecium propel themselves through water by means of the cilia distributed over the entire body surface. The cilia can be seen to execute a power stroke followed by a recovery stroke. In the first, the ciliary beat profile is relatively straight and is directed towards the rear of the cell to thus drive the cell forward. The cilia then "relax" (the recovery stroke) to return to their position at the start of the power stroke. The individual cilia beat in an organized relationship to their immediate neighbors such that the cilium in front is just starting the particular part of the beat cycle that its immediate posterior neighbor has just completed. Ciliary beating is thus synchronized. In addition, over the entire field of cilia covering the body surface, the cilia beat in groups--the cycle of timing of which and the direction of progression of which is termed metachrony. By casual observation of the ciliary beat of Paramecium in profile, one can only readily see the power (effective) stroke which is seen essentially perpendicularly aligned to the body surface. Jennings (1897, 1899a, b, 1901, 1906) noted that the cilia did strike perpendicularly backwards and to the right and thought that this contributed to the rotation of the cell on its long body-axis. Early workers studying ciliary beat in profile (Metzner, 1923; Gelei, 1926; Gray, 1928), considered that the individual cilia beat in a pendular manner such that, after the power stroke perpendicular to the body surface, the return stroke was in the same plane with the cilium simply relaxed. They reasoned that the return stroke for some reason was ineffective. Ludwig (1929) adopted the view of Metzner in his classic study of the mechanics and hydrodynamics of ciliary (and flagellar) activity and the forms of locomotion in ciliated and flagellated protozoa. Ludwig (1929) analyzed ciliary beating in Paramecium in terms of an effective power stroke that could be generated with the cilium beating perpendicular to its site of attachment. He considered that

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

41

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the individual cilium moved in an ellipsoidal trajectory and that the rearward swimming of the cell was effected simply as a result of a reversal in the direction of the effective stroke. From early studies by Gelei (1926, 1937b), detailed studies by Parducz (1954a, b, 1967) and later studies by Machemer (1969) and Tamm (1970), using different methods, it has been established that the cilia actually beat in a three dimensional profile. Observation of Paramecium, in profile, under dark field microscopy, shows that the cilia beat in groups and that waves of activity pass from the posterior to the anterior end of the cell body with each cilium successively assuming the position of its immediate posterior (rather than anterior) neighbor (Parducz, 1954a, b, 1967; Tamm, 1970) (see Fig. 1). The sequence of motion of a cilium within one of the waves of activity (a metachronal wave) has been studied either by treatment of swimming cells with chloroform vapor (Parducz, 1967); by instantaneous fixation of swimming cells with osmium tetroxide and a fixative for staining (Parducz, 1954a, b, 1967; Machemer, 1969, 1972b); by scanning electron microscopy (Tamm, 1970); or by high speed cinematography (Machemer, 1972c). In all cases, it would appear that both the beating profile of the individual cilia and the metachronal waves are preserved. As noted by all these authors, cilia at the end of the power stroke point at a position approximately parallel to the wavefronts. Cilia in the successive stages of the recovery stroke are observed (from above) to be progressively curved and aligned more perpendicular to the wave front as they return to the original start of the power stroke following a trajectory close to the body surface. At the end of the recovery stroke and preparatory to the next effective stroke, the cilia assume an S-shaped profile directed distinctly obliquely to the NW (the anterior of the cell being designated N). From above, the cilia in the power stroke are in an errect position and sticking out from the body surface. The effective stroke takes place in a N W - , SE direction. The three dimensional profile of the ciliary beat is thus in the form of a rotary motion that can be likened to the periphery of a tilted cone with respect to the body surface (Fig. 24). This is the current viewpoint of the ciliary beating profile in Paramecium (Sleigh, 1974; Rikmenspoel, 1976)--there having been no subsequent studies. We adopt this description in preference to either the true helical three-dimensional form described by Kuznicki et al. (1970), or the simple asymmetrical profile proposed by Tamm (1970). The recovery stroke is not in the same plane as the effective stroke and progresses in a counterclockwise direction close to the body surface (as viewed from above). This threedimensional profile has taken the place of earlier models in which it was considered that the cilium simply collapsed at the end of the power stroke and returned in a bent form in the same plane as the power stroke (Sleigh, 1962; Gray, 1928; Ludwig, 1929). In addition, the relative velocities of the two aspects of the beat cycle are noticeably different, at least

42

M.J. DOUGHTY and S. DRYL

,:-SI-.

k

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FIG. 24. Ciliary beating cycle in Paramecium: schematic illustration of the 3-D nature of the normal beat cycle of cilia. From Sleigh, 1974. Reproduced with permission Academic Press.

for forward swimming cells, with the recovery stroke being the faster (Rikmenspoel, 1976). The nature of the beating profile of Paramecium cilia in "reversed" orientation has not been studied in much detail partly because, in reversed mode, the cilia beat with 2-4 times the frequency observed in forward mode (Machemer, 1974a). It would appear unlikely, in view of what we currently know about the structure of the ciliary axoneme and the characteristics of ciliary beating in forward mode, that a true "reversal" in the direction occurs in which, at the onset of reversal, a cilium that has just completed a power stroke merely reverses the sequence of activity. At this time there is little firm evidence that the axoneme twists--a requirement, it would seem, for a simple "reversal" of activity. The possibility of such an event has been discussed (Sale and Satir, 1976; e m o t e and Kung, 1979) and is supported by observations that the local direction of ciliary beat is often nearly perpendicular to the central microtubules of the axoneme. This has been observed as differences in the orientation of the central pair in both ciliary and flagellar axonemes (Gibbons, 1961 ; Satir, 1968; Tamm and Horridge, 1970). There is no evidence that the basal body rotates, but recently e m o t e and Kung (1979) have presented evidence indicating that the central pair in Paramecium cilia rotates in a continuous or discontinuous manner. Perturbation of this potential control element, dependent upon a discontinuous linkage of the central pair of microtubules at the axesome (Hausman and Fischer-Defoy, 1978; Dute and Kung, 1978) could serve to switch the cilia into reverse mode. It is conceivable, in view of the discontinuous linkage, that reversal of the effective power stroke orientation is achieved as a result of an angular shift in the disposition of the envelope of beating (see Fig. 24) through 90 ° in an analogous manner to that observed for envelope profiles in viscous solutions (Maehener, 1972b, 1974b). The position previously occupied by the recovery stroke would now be perpendicular to the body surface and for hydrodynamic reasons, now assume the role of an effective stroke. Such an concept is supported by observations that, in viscous media, the cone shaped envelope of ciliary beating becomes widened and symmetrical to the body surface (Kuznicki et al., 1970). In forward swimming Paramecium, metachronal waves pass from the posterior end of the cell towards the anterior end, The orientation of the wave fronts with respect to the long axis of the: cell is not perpendicular but in a NW --->SE orientation with the wave fronts progr¢~ing in a SW--, NE direction (Fig. 25a). Fixation of Paramecium, with osmium tetroxide, during rearward swimming behavior induced by either mechanical (Parducz, 1967), chemical (BaCI2; see Machemer, 1969) or electrical (galvanic; see Par-

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

43

I ! FIG. 25. Ciliary metachronal waves and direction of swimming in Paramecium. Direction of swimming indicated by heavy arrows. Lines represent the metachronal wave fronts while small arrows show direction of progression of the wave fronts. Taken from Machemer, 1972b with permission. Copyright J. experimental Biology/Cambridge University Press.

ducz, 1963) stimulation permits observation of metachronal patterns during rearward swimming. The metachronal wave fronts now lie parallel to the long axis of the body and the wave fronts travel in an E--* W direction (Fig. 25b). As demonstrated by both Parducz (1967) and Machemer (1969, 1974b), at the onset of ciliary reversal and thus rearward swimming behavior of the cell, ciliary metachrony is completely lost. The reversed swimming metachronal pattern develops rapidly and towards the end of reversed swimming behavior, normal ciliary metachrony reappears at the anterior end of the cell and is observed to slowly progress over the entire body surface as gradual renormalization occurs and forward swimming is eventually restored (Fig. 26) (see Section 4.1 for complete descriptions). Full details of the character of ciliary metachrony during a cycle of rearward swimming induced by K ÷ ion stimulation of Paramecium (see Section 4.1) have not been published but are probably close to that sequence observed following mechanical stimulation of the anterior end of the cell (Fig. 26).

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FIG. 26. Ciliary metachronal waves during cycle of depolarization induced ciliary reversal behaviour of Paramecium. 1,9--forward swimming; 2--contact depolarization (loss of metachrony~; 3--5)--rearward swimming (continuous ciliary reversall: 6-8--cell reorientation under partial ciliary reversal. From Machemer, 1969. Reproduced with permisssion of author and J. Protozoology/Society Protozoologists.

44

M.J. DOUGHTYand S. DRYL

The individual cilia thus beat in a polarized manner with each cilium working first in "power stroke mode'--the action of which can be likened to an oar working with a large angular amplitude and in a spherical plane which appears to be more or less perpendicular to the body surface. In the following "recovery stroke mode", the cilia are bent close to the body surface and are turning in a counterclockwise direction back towards the starting point for another power stroke (Machemer, 1972b, c). Metachronal coordination of the activity of the body cilia or Paramecium is not a requirement per se for translational motion of the cell. Metachrony should perhaps be considered as working to effect an averaging of the mechanical output of all of the individual cilia to thus maximize their efficiency. The power output can thus be maximized under conditions where high speed (escape) motor behavior is required and relies as much on the beating of the individual cilia as upon the hydrodynamic effects imposed upon the motion by the nonsymmetrical shape of the cell body (Ludwig, 1929; Grebecki et al., 1967b). Thus, continuous ciliary reversal activity and the rearward swimming behavior associated with this form of ciliary beat is considered to be the most hydrodynamically favorable of the motion types of Paramecium (Grebecki et al., 1967b). 3.3. MEMBRANE RESTING POTENTIAL IN DIFFERENT ENVIRONMENTS

Andrejewa (1931) appears to have been the first to propose that a correlation could be drawn between the surface potential of Paramecium (altered by extracellular cations) and the swimming speed of the cell. Later studies (Kinosita et al., 1964b), indicated that there did exist a relationship between the forward swimming speed of Paramecium, the frequency of ciliary beating in forward mode and the steady state (resting) potential across the surface (plasma) membrane of the cell with respect to the extracellular concentrations of various inorganic cation salts. Subsequent work (Naitoh and Eckert, 1968a; Naitoh et al., 1972) showed that increasing extracellular concentrations of both monovalent and divalent cation salts, shifted the resting potential in a depolarizing direction. They (Naitoh and Eckert, 1968a) also showed that a marked antagonism existed between K + and Ca 2 + with respect to their effects on the resting potential. The resting potential measured was that found in different salt solutions after the membrane had showla the Ca 2 ÷ action potential in response to injected current (see Sections 4.3 and 4.4). Since the resting potential appears to be related to the forward swimming speed of the cell the resting potential measured by Naitoh and Eckert (1968a) could be regarded as the resting potential of the cell once it has adapted, at the membrane level, to a new ionic environment. On sudden transfer of free-swimming Paramecium into solutions of monovalent cation salts, the cells show a variety of temporary rearward swimming behaviors after which forward swimming is resumed, usually permanently (Jennings, 1897, 1899b; Mast and Nadler, 1926; Oliphant, 1938, 1942; Grebecki, 1964, 1965; Kuznicki, 1966a; Naitoh, 1968; Doughty and Dodd, 1978). All available data indicates that any inorganic cation salt and a variety of organic cation salts, unless excessively toxic, when applied extracellularly, will effect a depolarization of the cell membrane of Paramecium (as measured by intracellularly located microelectrodes) (Kamada, 1934; Yamaguchi, 1960a; Kinosita et al., 1964a, b; Naitoh and Eckert, 1968a, b; Naitoh et al., 1972; Friedman and Eckert, 1973). Doughty and Dodd (1976) reported changes in the spectral properties of a potentiometric dye, added to suspensions of Paramecium, following changes in the extracellular concentrations of a variety of inorganic and organic cation salts. The relative amplitude of these spectral changes (fluorescence emission intensity or absorbance) was found to be related to the expected resting potential across the cell membrane of Paramecium, i.e. showed relative changes over a 10 fold change of cation concentration similar to those found in microelectrode impaled cells for changes in resting potential. Both the behavioral sensitivity of Paramecium to extracellular stimuli (see Sections 4.1 and 4.2) and also the membrane resting potential in different salt solutions, are very dependent on the nature of the solutions in which the cells were prepared prior to experimentation (the adaptation or equilibration medium: Kamada and Kinosita, 1940; Naitoh, 1968;

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Doughty, 1978b), and the ionic composition of the test solutions (Kamada and Kinosita, 1940; Jahn, 1962a; Naitoh, 1968; Naitoh and Eckert, 1968a). Unfortunately, at the present time, there are at least 20 different buffer-salt combinations in use for studies on Paramecium, and adaptation periods range from a few minutes to over 24 hours. The same variation holds for electrophysiological studies. Therefore, only qualitative correlations can be drawn between most behavioral, electrophysiological and biochemical studies. Several studies (Yamaguchi, 1960a; Kinosita et al., 1964b; Naitoh and Eckert, 1968a; Doughty and Dodd, 1976) have shown that increasing extracellular concentrations of both monovalent (Na t, K ÷ : Fig. 27; Rb + and Li +) and divalent cations (Ca 2+, Ba 2+ : Fig. 28; Mn 2+, Co 2÷ and Mg 2÷) shift the resting potential towards zero from an initial value of - 2 0 to - 4 0 m V (inside negative) depending on the equilibration solution. In addition, increasing concentrations of alkyl-quaternary ammonium ion salts (Naitoh et al., 1972; Friedman and Eckert, 1973; Doughty and Dodd, 1976); choline salts (Doughty and Dodd, 1976" Fig. 29) and NH~ salts also effect depolarization of the cell membrane. Using the potentiometric carbocyanine dye, diS-C3(5), to monitor membrane potential changes following cationic stimulation of free-swimming Paramecium, spectral measurements were made 30 sec after addition of the stimulus solution. Observation of the cells, in the same buffered solutions, under identical conditions, following such cationic stimulation, for all salts tested by the potentiometric dye method, shows the Paramecium have all completed initial stages of ciliary responses to stimulation, i.e. continuous rearward swimming of the cells has been completed. However, the cells have not necessarily readopted forward swimming behavior under these conditions, i.e. within 30 sec after stimulation (Doughty and Dodd, 1978; Doughty, 1978b, c and unpublished). Several cations (Ca :÷, TEA +, TMA + and choline+), in the presence of 0.5 mM CaC12, were found to induce ciliary resting potential shifts but fail to induce ciliary reversal. However, TEA + have been reported to induce alterations in cell swimming behavior, analogous to those observed with K+-stimulation of the cells, in solutions containing lower concentrations of free calcium (Chang and Kung, 1976). With increasing extracellular CaCI: concentrations, the membrane resting potential becomes smaller and smaller (Fig. 28). Thus, subsequent addition of K ÷ effects a smaller depolarization than at lower Ca 2 ÷ concentrations (Fig. 30) (Naitoh and Eckert, 1968a; Doughty,.

46

M.J. DOUGHTYand S. DRYL 10

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unpublished). Such a response of the Paramecium membrane is given here in this form since it serves to emphasize the close linkage between K + ions and Ca 2+ ions in determining the overall chemical and electrical excitability of the membrane and thus the cell behavior. The steady state resting potential across the surface membrane of Paramecium is adopted immediately after the active conductance increase to Ca 2 + ions (the Ca 2+ regenerative response, see Sections 4.3 and 4.4) (Naitoh and Eckert, 1968a; Naitoh et aL, 1972). This resting potential thus represents adaptation of the excitable membrane to the new ionic environment and is distinct from the adaptation of the motile machinery (the

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47

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

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FIG. 30. M e m b r a n e resting potential in Paramecium: Interactions between K ÷ and Ca 2 ÷. Effect of KCI on the absorbance signal of the potentiometric dye, diS-C3(5) bound to free-swimming Paramecium caudatum in solution of 5 mM Hepes-Pipes buffer, pH 7.2 plus 0.5-4.0 mM CaC12. Cells adapted to the CaC12 solutions for 4 hr prior to experiments 19-21°C. Doughty and Dodd, unpublished results.

axonemal mechanochemical cycle) that determines the duration of the behavioral response of the cell (Doughty and Dodd, 1976). This resting potential is adopted in both microelectrode-impaled cells (Yamaguchi, 1960a) and free-swimming cells (Doughty and Dodd, 1976) in the new salt concentration and is generally maintained, with only slight fluctuations of a spontaneous type (Kinosita et al., 1964a; Satow and Kung, 1974), for as long as the cell(s) remain bathed in that solution (except Ba2+/Ca 2÷ mixtures, see Section 4.3). Temperature shift from 5-27°C (Yamaguchi, 1960a) or extracellular pH shift from 4.6 to 8.6 (Kinosita et al., 1964b) results in an increase (hyperpolarization) of the resting potential. The data presented in this section and Section 3.1 clearly indicates that there is no simple relationship between cell swimming speed and resting potential. The alternative factor determining behavior would therefore appear to be the frequency and character of ciliary activity and to date, no clear relationship between resting potential and ciliary frequency has been established. Small (5-15mV deviation from resting potential) depolarizations or hyperpolarizations appear to generally increase ciliary beat frequency. However, according to the nature of the perturbing agent or condition, the hyperpolarization, for example, can either increase or decrease the frequency of changes in the cells swimming path direction despite generally increasing ciliary frequency and cell swimming speed. Large depolarizations generally decrease cell swimming speed, can increase the frequency of directional changes and can result in ciliary reorientation of longer duration (ciliary reversal). However, the depolarization per se is not requisite for ciliary reversal since, for example, both CaC12 and choline chloride depolarize the membrane but do not induce ciliary reversal. Van Houten (1977, 1978, 1979) provides data to show relationships between resultant cell behavior (attraction or repulsion from chemicals) and the resting potential--a relationship which generally holds for both hyperpolarizing and depolarizing stimuli. The change in resting potential per se however is not the sole determinant. Neither is behavior determined by the absolute magnitude of the resting potential. The chemical nature of the perturbant appears to be a critical factor although the nature of the interacting sites remains to be elucidated (see Sections 4.1 and 4.2). Under normal conditions, the membrane behaves as a mixed type of electrode, since plots of V, versus extracellular cation concentration never attain the values predicted by the Nernst equation for either K ÷, Na ÷, or even Ca 2÷ (Naitoh and Eckert, 1968a; Naitoh et al., 1972; Doughty and Dodd, 1976). Ciliary activity and thus cell behavior is determined by the nature of the perturbant altering the resting potential and would

48

M.J. DOUGHTYand S. DRVL

appear to be controlled by the relative magnitude of the resting potential change for stimuli of a common type (Van Houten, 1978, 1979). 3.4. CATION FLUXES ACROSS THE SURFACE MEMBRANE OF P A R A M E C I U M

Both behavioral and electrophysiological studies have provided evidence that both the membrane resting potential and the characteristics of active electrogenesis of the excitable membrane of Paramecium under various conditions, is strongly dependent on the relative permeability of the membrane to calcium and potassium ions. It would therefore be most useful to be able to determine both the magnitude and kinetics of these ion fluxes under different conditions. The duration of active electrogenesis (the Ca 2 ÷ regenerative response) in response to cationic stimulation of the membrane of Paramecium is unknown, but would appear to have a maximum duration of between half and at the very most a few seconds (Kinosita et al., 1964a; Yamaguchi, 1960a; see also Machemer, 1975; Machemer and Eckert, 1975). Spectroscopic monitoring of membrane potential indicates that, following cationic stimulation, the active membrane potential changes (spike potentials) last for 1-2 sec (Doughty, unpublished, Fig. 31). Measurement of active potential changes following changes in extracellular solutions flowing past microelectrode impaled cells, indicates that the duration of graded (with respect to stimulus strength) spike potentials can range from around 0.25 sec (quinidine-HCl: Van Houten, 1979) to around 0.5-1 sec (Na+: Satow and Kung, 1974) in normal, wild-type Paramecium. All-or-nothing electrogenesis can be induced under some conditions and the spike potentials have a duration of around 1 sec (Kinosita et al., 1964b, c, 1965; Van Houten, 1979). The total duration of monovalent cation induced continuous ciliary reversal behavior however lasts many seconds, dependent on the concentration of cation used (see Sections 4.1 and 4.2).

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F,G. 31. Active electrogenesis in Paramecium following KC! stimulation. Spike potentials recorded from free-swimming Paramecium aurelia by monitoring the absorbance signal of the potentiometric dye, diS-Ca(5) bound to the cells in solution of Hepes-Pipes buffer 0.5 m~ CaCI~, pH 7.1 23°C. 1cells plus diS-Ca(5) and given KC! stimulus at s; 2cells plus dye and given sham stimulus (buffer solution) at s, 3.%ells in absence of dye and given KCI and sham stimuli respectively at s. Lower section shows relationship between amplitude of the spectral transient and the KCI concentration (+ s.d.). Doughty unpublished.

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

49

There have been several reports of measurements of both monovalent and divalent cation fluxes in and out of Paramecium cells which have been interpreted to represent fluxes across the ciliary membrane (Yamaguchi, 1960b; Naitoh and Yasumasu, 1967; Browning, 1976; Browning and Nelson, 1976a, b; Browning et al., 1976; Hansma and Kung, 1976; Satow et al., 1976; Hansma, 1979; Ling and Kung, 1980). Recent reports show that the ciliary membrane is differentially specialized in both structure and function from the rest of the surface (plasma) membrane (Plattner, 1975; Satir et al., 1976; Ogura and Takahashi, 1976; Dunlap, 1977; Brehm et al., 1978; Machemer and Ogura, 1979). There are however at least three types of membrane in Paramecium in direct communication with the extracellular environment: those of the cilia (the ciliary membranes), the rest of the surface membrane between the cilia (the interciliary or somatic membranes), and that membrane which delineates the contractile vacuoles (osmoregulatory organelles) of the cell. In addition, Patterson (1978) reports the presence of membranous sacs invaginating from the somatic membrane into the cell cytoplasm in the vicinity of the mitochondria. Thus we have to acknowledge the many reports of ion fluxes in Paramecium and other protozoa that have been interpreted as being associated with osmoregulatory capacity and environmental adaptation (Akita, 1941; Dunham and Child, 1961 ; Andrus and Giese, 1963; Dunham, 1964, 1969; Jones, 1966). Protozoa have no osmotically regulated cell fluids and therefore all fresh water protozoa have to cope with the problem of continuous water influx (Jahn and Boggs, 1971). Significant water flux apparently occurs across the ciliary membranes of Paramecium since deciliation results in significant reduction in contractile vacoule activity (Ciccolello and Gibor, 1978). We do not yet know how osmoregulation in fresh water protozoa is achieved. However, the importance of at least monovalent (and possibly divalent) cations in the control of this process appears important, as judged from the effects of cations on the contractile vacuole activity of the cell (Stempell, 1924; Czarska, 1964; Dunham and Child, 1961). Direct and indirect coupling between cation and amino acid fluxes have been reported (Hoffman and Kramhoft, 1969; Stoner and Dunham, 1970) and changes in monovalent cation flux activity has been reported following changes in medium osmolarity (Dunham, 1964; Kramhoft, 1970). Failure of osmoregulation mechanisms probably accounts for the uncertain ability of fresh water protozoa to adapt to sea water or concentrated salt solutions (Massart, 1889, 1891; Frisch, 1939 and ref. cit.; Dunham, 1964). The rate of contractile vacuole activity in Paramecium has been reported to be markedly affected by temperature (Cole, 1925), by both monovalent and divalent cations (Kamada, 1935; Frisch, 1939, Gelei, 1935, 1937b; Seravin, 1958) and pH (EisenbergHamburg, 1929). Andrus and Giese (1963) report that cooling of Tetrahymena led to an entry of Na + and loss of K + - - a process that was reversible. Jones (1966) found that the kinetics of 45Ca2+ uptake into the ciliated protozoan, Spirostomum, were markedly influenced by temperature. Cation exchange and/or water flux activity can thus be expected to occur across ciliary, somatic and contractile vacuole membranes. Thus, although data on 45Ca2+, K + and Na + distributions, in both wild-type and mutant Paramecium defective in active electrogenesis, under conditions of external cation perturbation, conveniently supports a hypothesis that the mutant behavior is the result of large differences in the permeability of the excitable membrane to cations compared to wild-type cells, the topographical location of the flux activity should perhaps be considered. It should be noted that the methodology used to determine these latter flux activities is very similar to that used to determine flux activities assigned to osmoregulatory activity. The kinetics of all ion fluxes are very slow with many minutes, rather than seconds, required for uptake to reach saturation or show discontinuous characteristics (Yamaguchi, 1960b; Naitoh and Yasumasu, 1967; Browning and Nelson, 1976a, b; Ling and Kung, 1980). Similar kinetics have been reported for the osmoregulatory studies (Akita, 1941; Dunham and Child, 1961). Large differences in Na + uptake have been reported for a mutant showing prolonged active electrogenesis (Hansma and Kung, 1976; Satow et al., 1976). The same mutant (d4-90) was also found to be defective in Na ÷dependent K + efflux (Hansma and Kung, 1976; Satow et al., 1976), in Na~ dependent

50

M.J. DOUGHTY and S. DRYL

Ba 2+ influx (Ling and Kung, 1980) and Na~-dependent Ca 2+ uptake (Browning et al., 1976). The kinetics of 22Na+ uptake in Paramecium have recently been studied in detail (Hansma, 1979)---fluxes assigned to a voltage-dependent Na + influx through (ciliary membrane) Ca 2+ channels. At this time there is no strong evidence for a role for Na + in the voltage-dependent activation of Ca 2+ channels or in their inactivation although voltage clamp experiments will need to be carried out to analyze this more carefully. Na ÷ ions do not alter the characteristics or amplitude of the Ca 2+ spike (Naitoh et al., 1972) and recognized Na + conductance antagonists (procaine-HCl; Naitoh et al., 1972; tetrodotoxin; Satow and Kung, 1974) appear to be without significant effect on the Ca 2 + spike potentials. From a membrane transport, rather than conductance, aspect, no enzyme activity of a type that might nominally be expected to indicate the presence of an active N a / K transport system (Mg 2 +-dependent, N a - K stimulated ATPase activity or K-phosphatase activity: Skou, 1975) has so far been detected in Paramecium ciliary membranes (Doughty, 1978a, and unpublished), or Tetrahymena (Baugh et al., 1976). The presence of Na +, K + stimulated ATPase activity (phosphatase) has been detected by histochemical methods in basal body regions of the cilia in Tetrahymena (Dentler, 1977). A role for Na + ions in regulation of either Ca 2 + influx or efflux or the occurence of Na ÷ fluxes across the ciliary membrane relevant to changes in ciliary activity remains to be established. It is, for example, conceivable that somatic membrane late Na + currents could in part control Ca 2÷ conductance activation or inactivation at the ciliary membrane or that Ca 2 + efflux from the intraciliary space might be dependent upon a sodium electrochemical gradient (see Sections 4.4 and 4.6). In conclusion, since, in part, current hypotheses predict the presence of voltage dependent, gated Ca 2 + conductance channels as a principal component of the ciliary membrane related to control of ciliary activity, one might anticipate being able to record Ca 2+ influx associated with the activation of these channels. Experiments, at 4°C for 45Ca z÷ or at room temperature for 131Ba2+ (Browning and Nelson, 1976a; Ling and Kung, 1980), indicate that, in response to stimulation, there is an increase in divalent cation flux activity (calcium or barium) across the Paramecium surface membranes above a background flux activity measured in the absence of stimulation. Such fluxes are thought to represent the divalent cation influx associated with voltage dependent activation of the ciliary membrane calcium channels through which calcium ions flow, down their electrochemical gradient, to activate the ciliary reversal mechanism at axonemal sites. In a series of unrelated studies, indirect information has been sought on the cation fluxes associated with, or required for, induction of ciliary reversal. Such indirect evidence comes from studies on the kinetics of immobilization of Paramecium cells by low concentrations of nickel ions. Gelei (1935) reported that Ni 2+ ions, at low concentrations, effected non-lethal immobilization of Paramecium and his results were confirmed and extended by subsequent studies (Thomas, 1953; Bovee, 1958; De Puytorac et al., 1963; Andrivon, 1968,-1974; Grebecki et al., 1967a, b). The immobilization of a ciliated protozoan by a heavy metal ion is hardly of interest at first glance since it is a well documented phenomena (see Section 4.2). However, while Ni 2 + induce immobilization of ciliary activity, the immobilized cilia can still be reorientated (shifted into reverse mode) by the application of depolarizing stimuli to the cell (Naitoh, 1966). This indicates that the mechanisms underlying the reorientation process at the axonemal level are not affected. The membrane resting potential appears unaffected by Ni 2 + ions (Naitoh, 1966). However, in the presence of Ni 2 ÷, the amplitude of the regenerative calcium response is increased from 22-32 mV (solutions contain either I mM CaC12 and 2 mM KCI, or the same plus 1 mM NiC12) and the kinetics of activation are increased (Naitoh and Eckert, 1970). Following current injection, the cilia are permanently placed in reverse mode (although they are nonbeating) indicating that the mechanism(s) responsible for renormalization of ciliary activity following stimulation are inactivated and thus forward beating orientation of the cilia is not regained. The immobilization, by Ni 2 ÷ ions, is effected at nonlethal concentrations (Gelei, 1935; Kuznicki, 1963b; De Puytorac et al.,

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

51

300 1 1112

z001"//

lift+

1

/

/

/o

3

/

1- NiCl2.6H20

O'O@&2 mM

2-

~0szs m.

3' /

.. ..

0

0

~

0 60

120 min~es

180

2/0

FIG. 32. Ni 2+ uptake in Paramecium: kinetics of 63Ni2+ uptake into Paramecium caudatum as a function of [Ni 2+ ]o. Cells in 2% proteose peptone, pH 6.0 25°C. Redrawn from Andrivon, 1970b with permission of the author and Protistologica/Centre Nationale de la Recherche Scientifique (C.N.R.S.).

1963; Andrivon, 1968). The immobilization of ciliary activity is reversible. Motility can be restored by washing the cells in CaCI2 containing solutions or adding excess CaC12 to the solution of Ni 2÷ immobilized cells (Kuznicki, 1963b; Grebecki et al., 1967a). Nicomplexing agents (dimethyglyoxyme) also reverse immobilization (Andrivon, 1968). Low concentrations of Ni 2 + ions also change the swimming behavior of Paramecium from the normal forward left spiralling pattern to forward right spiralling, i.e. the cells, although still forward swimming, now rotate clockwise on their long axis rather than counterclockwise (Grebecki et al., 1967a). Ni 2÷ ions do not inhibit the activity of crude dynein extracts from Paramecium cilia (Doughty, 1979b) nor the activity of semi-purified dynein proteins from Tetrahymena cilia (Gibbons, 1966). Andrivon (1974c), however, does report inactivation of swimming in "model" Paramecium by concentrations of Ni 2 + that are probably sufficient to complex ATP. It is known that Ni 2÷ ions affect activities in Paramecium other than ciliary motion and its control--these activities including contractile vacuole activity, food vacuole activity and overall metabolism (Gelei, 1935, 1937b; Brutkowska, 1967). However, since after recovery from immobilization, active motility and growth is regained (Andrivon, 1968, 1970a, b), the actions of Ni 2 ÷ on the cell and the mechanism(s) by which they effect reversible alteration in ciliary activity and its control is thought to provide an indirect approach to furthering understanding of the cation fluxes associated with ciliary activity and orientation and the control of these fluxes for the following reasons. Andrivon (1968, 1970a, b) studies the rates of cell immobilization 8O 70

o

i/. 0 s0

2o 10 ,"

/ o ~ .

•

~'~'o.* "°" L

0

1

I

I

2

3

I

/

l~Ni2+]o

FIG. 33. Ni 2. uptake in Paramecium: Competitive inhibition of 6aNi2+ uptake by Paramecium caudatum by Co 2 ÷ ions, (Lineweaver-Burke plot). Conditions etc as in Fig. 32. Redrawn from Andrivon, 1970b with permission of the author and Protistologica/C.N.R.S.

52

M.J. DOUGHTYand S. DRYL

TABLE 2.

Ni 2+

I N H I B I T I O N OF C I L I A R Y M O T I O N IN

PARAMECIUM. D E T E R M I N A T I O N OF THE Q U A N T I T Y OF N i 2+

NECESSARY T O S T O P C I L I A R Y M O V E M E N T IN PARAMECIUM CAL'DATUM 1N THE PRESENCE OF D I F F E R E N T C O N C E N T R A T I O N S OF K + A N D C a 2 +

63NIC12 concentration mM

0.0105

Other cation Concentration mM

None

c.p.m. 63Ni

178.4

0.0105 Ca 2 ÷ 0.0425 171.6

0.0105 Ca 2 ÷ 0.17 187.2

0.0105 Ca 2 ÷ 0.34 169.0

0.0105 K* 0.084 174.6

0.0105 K÷ 0.34 180.2

0.0105 K~ 1.35 167.6

Data taken from similar Table in paper by Andrivon (1972), with permission of the author and Acta

Protozoologica.

b y N i 2 ÷ in the presence of a large number of metabolic inhibitors and other inhibitors of enzyme activity and concluded that Ni 2 ÷ have to cross the cell (plasma) membrane to effect ciliary immobilization but that the immobilization was not simply the result of poisoning of the cell. Treatment of Paramecium with a variety of inhibitors of oxidative metabolism, but not of mitochondrial respiratory chain activity, resulted in an enhancement of the time required for a constant concentration of Ni 2 ÷ ions to effect immobilization of the cell (147-153 sec to 387-2216 sec). The resistance of Paramecium to Ni 2 ÷ ions had thus been increased (Andrivon, 1970a). Andrivon (1970b) subsequently investigated the kinetics of 63Ni2 ÷ uptake into Paramecium and found that, not only could the cells concentrate Ni 2 ÷ ions from the medium, but that the uptake showed distinct Michaelis type kinetics. The kinetics were found to be concentration dependent (Fig. 32). On a double reciprocal plot, the rates of uptake versus Ni 2 ÷ concentration give a straight line plot (Fig. 33). Cobalt ions act as a competitive inhibitor of the uptake (Fig. 33). The time taken to immobilize Paramecium is in inverse proportion to the Ni 2 ÷ concentration (De Puytorac et al., 1963; Andrivon, 1970b, 1972; Doughty and Dodd, 1978; Doughty, unpublished). The immobilization of the cells always occurred during early stages of the Ni 2÷ uptake where linear kinetics were observed (Andrivon, 1970b) and the quantity of Ni 2 + required to effect immobilization remained approximately constant under different conditions (Andrivon, 1972; Table 2). The rate of immobilization of the cells by Ni 2÷ therefore appears to reflect the rate of uptake of Ni 2 ÷ ions into the cell (Andrivon, 1970b, 1972). Thus, the reciprocal of the time taken to effect immobilization of the cells is taken as an indicator of the rate of Ni 2 ÷ influx into the cell and presumably into the intraciliary space---since ciliary immobilization is effected. Therefore, as proposed by Andrivon

8oo[ 700 [

Jio-ii

600 I"

O0 300 2 O0 100 0

i

0.0031

t

I

00125

i

I

1

0.05 0.1

|

I

I

0-2 0./+ 0.8

t

•

1.6

3-2

raM. NiCl 2 FIG. 34. Ni 2÷ in Paramecium: Evidence for allosteric control by Ca 2+ and K ÷ ions. Rate of Ni z * uptake in Paramecium caudatum (105/tN~ where tN~ is the time for ciliary immobilization to be effected: see Andrivon, 1970b for derivation of relationship) as a function of extracellular NiCI2 concentration in the presence of CaClz or KCI. 10.085 mM CaCI2; 20.0425 mM CaCI 2 plus 4 mM KCI; 30.085mM CaC12 + 5.7ram KCI; 40.17raM CaCI 2 + 8.05raM KCI; ~l.3mM CaCI2 + 6.1mM KCI; 61 mM CaCI2. pH 6.8 0 - 5 ) and 7.1 (6). 25°C (t-5) and 23°C (6). 1-5 taken/replotted from Andrivon, 1974a with permission of the author and Prostistologica/C.N.R.S.

6~Doughty, unpublished.

CONTROL

OF CILIARY

ACTIVITY

1 0oo

1000

9OO

900

BOO

800

700

53

PARAMECIUM

IN

'XO\o\\

o

7oo

O

500

/\

400 300

100 0

o/

/

20°

500 400 30O

\

200

O

1 00 t

i

5

10

m

15

i

20 25

0

|

30 35 40

I

I

i

i

i

4

5

6

7

8

°C

pH

FIG. 35. Ni 2+ in Paramecium: Effect of temperature and pH. Rate of Ni 2÷ uptake in Paramecium caudatum (= 105/ts~--see Fig. 34) as a function of temperature (left) (pH 6.0) and pH (right) (25°C). Cells in 2% proteose peptone. Replotted from Andrivon, 1970b with permission of the author and Protistologica/C.N.R.S.

(1972, 1974a, b), alteration in the rate of immobilization of the cells by various cations and pharmacological reagents, reflects an alteration in those mechanisms determining the uptake of Ni 2 ÷ into the cell. The rate of immobilization increases with increasing Ni 2+ concentration (Fig. 34). The rate of immobilization is dependent on both temperature and extracellular pH (Fig. 35) (Andrivon, 1970b). In the presence of monovalent cations, the rates of immobilization, as a function of Ni 2÷ concentration, change (Fig. 34). Extra° cellular Ca 2 + reduce the rate of Ni 2+ uptake in a competitive manner (Fig. 36) (Andrivon, 1972). In contrast, the rates of uptake, determined from rates of immobilization, are markedly reduced by extracellular KC1 or NaC1 (Andrivon, 1972; Doughty and Dodd, 1978; Doughty, unpublished) (Fig. 37). The Ca 2+ antagonist, ruthenium red, reduces the rate of uptake at low concentrations (Fig. 38) and increases the rate at higher concentrations. Acetylcholine, at micromolar concentrations, has the opposite action (Doughty, !979a). Concentrations of K ÷ and ruthenium red that reduce the rate of Ni 2+ uptake effect inhibition of the detergent solubilized Ca 2 ÷-ATPase of Paramecium cilia (Doughty, 600

o/

500

4OO

300

200

1 O0

L

t

i

i

i

|

m

0-2 04. 0"6 OB 1"0 1-2 1-/. 1.6 , ~ N i~ lo x 105

FIG. 36. Ni 2÷ uptake in Paramecium: Competitive inhibition by Ca 2+. Rate of Ni 2+ uptake into Paramecium caudatum (= 105/tN0 in the presence of different extracellular CaC12 concentrations. Lineweaver-Burke plot. pH 6.0 25°C. Redrawn from Andrivon, 1972 with permission of the author and Acta Protozoologica.

M.J. DOUGHTYand S. DRYL

54 700

,oo t \ 50O

~00

30O

20C 10C 0

i

.

0

.

I

i

0.1250-2506

.

.

1

2

raM.

|

/,

I

S 16

I

32

KCl

FIG. 37. Ni 2+ uptake in Paramecium: Inhibition by KCI and NaCI. Rate of Ni 2+ uptake in Paramecium (105/tsl) in the presence of different extracellular concentrations of NaCI (El) and KCI (O, O). [], O--P. aurelia, 0.SmM NiCI2, pH 7.1, 0.SmM CaCI2 present, 19-21°C. From Doughty and Dodd, 1978. q~--P, caudatum, 1.6 mM NiCI2, pH 7.2, 1 mM CaCI2 present, t8°C. Doughty, unpublished.

, : -,.: "k~)st Ni 2 + alone also inhibit the enzyme (Doughty, unpublished). Treatment of Paramecium with low concentrations of a recognized cross-linking agent ~lutaraldehyde) effects marked alterations in ciliary reversal behavior of the cell to monovalent cations and also alters the action of Na + ions on the rate of Ni 2+ uptake (Doughty and Dodd, 1978). Ni 2 + enhance the duration of K + induced ciliary reversal behavior of Paramecium (Doughty, unpublished). The characteristics of Ni 2+ induced ciliary immobilization under different chemical and physical conditions have similarities to the effects of these conditions on cell motility

13ooF 12ooI 11ooI lOOOI 9oo I 105 80O .~q~Ni700 600 500 L,O0 300 200 I O0 0

t

t

i

I

t

I

0 ~,25 1,25 2.s s 1 2 .la 8- --1~7 --lg L Molar concentration R R FIo. 38. Ni 2+ uptake in Paramecium. Effect of ruthenium red. Rate of Ni 2+ uptake (10s/tN0 in Paramecium caudatum in the presence of different concentrations of ruthenium red. 1.6 m~ NiCI2, 1 mM CaCI2, pH 7.2 20~C. Doughty, unpublished.

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

55

(see Section 3.1), membrane resting potential (Section 3.3), the membrane C a 2 +-ATPase (Section 2.2) and the duration of K ÷-induced ciliary reversal (Sections 4.1 and 4.2). The rates of Ni 2 ÷ ion uptake are modified in Paramecium mutants defective in active electrogenesis (Andrivon, 1974a; Nelson, D. L., personal communication). Modification in Ni 2 ÷ uptake appears to be related to changes in both active electrogenesis and membrane adaptation. As a result of these common characteristics, there has been discussion that the molecular components controlling active electrogenesis and those controlling membrane adaptation and Ca 2 ÷ efflux are closely functionally linked and that the vectorial direction of activity is determined through the operation and activity of an as yet uncharacterized allosteric control (Andrivon, 1974a, b; Andrivon et al., 1977b; Doughty, 1978b).

4. Active Behavioral Responses, Ciliary Activity and Electrical Responses Following Depolarizing Stimuli under Different Physical and Chemical Conditions 4.1. CILIARY REVERSAL BEHAVIOR IN PARAMECIUM TO INORGANIC CATION SALTS "Chemotactic and tonotactic movements (of ciliate infusoria) have been the subject of scattering notices by various authors" (Jennings, 1897, p. 259).

Jennings, in a series of studies around the turn of this century, initiated systematic studies on the behavior of Paramecium towards chemicals, His studies on a variety of unicellular and multicellular organisms are summarized in his book, The Behavior of Lower Organisms (1906). Prior to the studies of Jennings, Massart (1891) noted that "Tousles Hypotriches protozoa que j'ai examin6s se sont montr6s tr6s sensibles h la concentrations: ils restent h une plus grand6 distance des fragments salins que des Infusories appartenant ~ d'autres ordes. Le Paramecium aurelia notamment se permet assez souvent une petite incursion dans les zones etr~mes de la solution saline, dans lesquelles, par suite de la diffusion, la concentration est moindre. Les Hypotriches 6vitent m~me ces liquids dilu6s". Such studies indicate a sensitivity of ciliate protozoa to chemicals. Massart (1891) presented lists of various infusoria (a collective term used to include any usually unicellular organism found in decaying infusions rich in organic material or in standing water rich in the same) as to whether they were sensitive or insensitive to drops of salt solutions. He notes that two species of Paramecium were sensitive and additionally notes that P. aurelia readily adapted to solutions of monovalent cation salts. The behavior reaction (response) that Paramecium show on contact with salt solutions is a phobic response. This is to say that the organisms show a behavioral response to a test chemical for a finite time period but adapt, within a period varying from a few seconds to a few minutes at the extreme, to the new ionic environment and resume forward swimming of a type prior to stimulation, unless the composition of the new environment is either extreme or toxic to the cells. In this case, the behavioral responses are repeated until escape from that environment is successfully achieved through a series of trials (Jennings, 1897, 1899a, b). Unfortunately, over the last 15 years in particular, perhaps undue emphasis has been placed upon this "avoiding reaction" behavior of Paramecium. This response, originally described by Jennings and named "the avoiding reaction" (1899a), that Paramecium show in culture medium or pond water following collision, at their anterior end, with any mechanical obstruction (debris or rotting vegetation). The cell backs off from the object for a few body lengths (as a result of ciliary reversal), stops translational motion momentarily, turns through a small angle and then swims forward again, hopefully to avoid the obstruction (see Fig. 26). The process can be repeated over and over again until the obstacle, e.g., a drop of concentrated salt solution, is successfully bypassed. However, such a behavioral response, is by no means the only one that the cells can show. Mechanical contact with the posterior end of the cell results J.P.N. 16, I--D

56

M.J. DOUGHTY and S. DRYL

in acceleration of forward swimming motion (Jennings, 1899b, 1906). In addition, the cell can change its swimming velocity, can change the rate at which its swimming path direction changes in unit time and also the overall three-dimensional character of the swimming path in addition to showing a variety of phobic responses to shock stimulation with chemical and physical stimuli (see previous sections). Jennings himself carried out few studies of the "avoiding reaction". This reaction is not typical of the behavioral responses that can be shown to stimulation. Certainly, the behavioral responses, shown by Paramecium following perception of (usually) abrupt increases in the concentrations of common salts in its environment, invariably involve a period of backward swimming of the cell (Jennings, 1897, 1899a, b; Vieweger, 1912; Mast and Nadler, 1926; Oliphant, 1938, 1942; Kamada and Kinosita, 1940; Grebecki, 1964, 1965; Kuznicki, 1966a; Naitoh, 1968; Doughty and Dodd, 1978; Doughty, 1978b). However, the reversed swimming behavior differs remarkably in detail according to the nature of the stimulus and more importantly, to some stimuli a single response is given prior to adaptation and return to forward swimming, and to others, several cycles of response occur prior to return to forward swimming (which does not always occur). Jennings (1906, pp. 47-52) describes several types of behavioral responses of Paramecium observed according to the type and strength of chemical stimuli. These behavioral types and the response sequence following stimulation of Paramecium with inorganic cation salts, were studied in greater detail and photographed in more recent years (Grebecki, 1965; Kuznicki, 1966a; Grebecki et al., 1967a; Nakatani, 1970). Despite this excellent documentation, many contemporary studies (1968-76) have not addressed the detailed nature of the responses and instead have either used ambiguous terminology or have simply noted whether or not the cells swim backwards following stimulation. The contemporary reader is thus often left in doubt as to whether a measurement of the duration of the period of actual reversed swimming behavior has been made or, for example, whether the duration refers to the period during which the cell is perturbed from forward swimming behavior. The finer details of the response sequences have been only occasionally noted. As detailed in previous sections, the sensitivity of Paramecium to external current stimulation (galvanic stimulation with resultant galvanotaxis), the cell swimming speed and the membrane resting potential are very dependent on both the nature of the media in which the cells are suspended and the physical conditions under which the behavioral character is analyzed. Likewise, as can be expected, the sensitivity of Paramecium to chemical stimulation depends both on the nature of the solutions in which the cells are studied (pond water, culture medium, tap water, distilled water, water plus salts, defined buffered solutions containing various salts etc.) and on the conditions under which the cell responses are evaluated (especially temperature). Jennings (1899b) studied the chemical sensitivity of Paramecium by introducing a small drop of a chemical solution beneath the cover slide on a glass slide covered with a suspension of Paramecium (usually in tap water or distilled water). He then observed whether the cells reacted against the drop (by forming a ring around it), whether they swam through the drop without showing a response or whether they swam into the drop and remained there. He termed these three responses "negative chemotaxis", "indifferent behavior" and "positive chemotaxis". He additionally notes, however, that the use of such terminology was for convenience, rather than to imply that a true behavioral "taxis" was the cause of the observed macroscopic phenomena. To our knowledge, true chemotaxis of Paramecium (specific orientation and movement of cells with respect to a stimulus source) has never been documented in the literature. Jennings (1899b) compiled a list of the chemicals to which the cells showed either negative or positive "chemotaxis" (Table 3) as well as noting the survival of cells in various solutions, While noting the occurrence of "avoiding reactions" associated with the negative behavior, Jennings makes few notes of the time that the cells exhibited rearward swimming behavior. Similar results were obtained by both Vieweger (1912) and Mast and Nadler (1926) for many of these

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

57

TABLE 3. SENSITIVITY OF PARAMECIUM TO CHEMICALS

1. Repellent power strong in proportion to injurious effects: chemotaxis protective.

2. Repellent power very weak in proportion to injurious effects: chemotaxis not protective A. Attractive Substances

Sodium fluoride, Potassium biochromate, Ammonium bichromate, Potassium ferricyanide.

(KBr), (KCI), (RuCl), (CsCI)1

HF, HCI, HBr, H1, H2504, HNOa, Acetic Acid, Tannic Acid, Picric Acid, Chromic Acid, Potassium fluoride, Potassium permanganate, Ammonia alum, Ammonio-ferric alum, Chrome alum, Potash alum, C u S O 4 , C u C I 2, C u ( C 2 H 3 O 2 ) 2 ,

ZnC12, HgCI2, AICI3, LiC1, NaC1, (KCI), (RuCI), (CsC])1. LiBr, NaBr, (K Br), RuBr, Lil, Nal, KI, RuI, LizCO3, NazCO3, K2CO3, LiNO3, NaNO3, KNO3, NaOH, KOH; KBrO3, NH4F, NH4C1, NH4Br, NH4I, CaCI2, SrCI2, BaC12, Ca(NO3)2, Sr(NO3}2, Ba(NO3)2.

B. Repellent Substances Cane sugar, Lactose, Maltose, Dextrose, Mannite, Glycerin, Urea.

~In the case of the substances enclosed in parentheses, the reactions of Paramecia from different regions varied. From Jennings (1899b), with permission Amer. J. Physiology/American Physiology Society.

chemicals, with the notable exception of the calcium salts. Mast and Nadler (1926) and Oliphant (1938, 1942) attempted to quantify the ciliary reversal behavior shown to salt solutions by measuring the duration of the reversed swimming behavior. They took single cells and "dropped" them into various test solutions and observed their behavior continuously under a microscope "until the reversed action of the cilia stopped". Oliphant (1938, 1942) reported that strong ciliary reversal action (45-120sec) was given to all salts of K ÷, Ba 1÷, Mn2+; moderate reaction was given to salts of Na +, Li + and NH2, while no actual reversed swimming behavior, of measurable duration, was given to all Ca 2+, Mg 2÷ and Ni 2+ salts tested although shock reactions were shown to higher concentrations. Similar results were obtained by Mast and Nadler (1926) for some of these salts. Jennings (1899b) notes shock reactions given to inorganic (HCI, HNO3, HzSO4, H3PO4) and organic (acetic, lactic, propionic, maleic, citric, tartaric and picric acids) acids. Salts of Zn 2+, Ctl 2+, Sn 2+, Hg 2+, Co 2+, AI 3+, Fe 3+ and Fe 2+ did not induce ciliary reversal behavior except just prior to death of the cells in these salts since, even at low concentrations, most of these salts (metals) proved to be very toxic to Paramecium. A variety of alcohols (methyl, ethyl iso-propyl, n-butyl and benzyl) typically effected an acceleration in the forward motion of Paramecium (Oliphant, 1942). Sugars (glucose, sucrose and lactose) induced shock reactions but no defined ciliary reversal behavior (Mast and Nadler, 1926; Oliphant, 1942). Oliphant (1938) notes, as does Dryl (1959), that adaptation of Paramecium (incubation for several hours; see Kamada and Kinosita, 1940) to fairly concentrated salt solutions (25 mM KCI) results in a marked reduction of their sensitivity to monovalent cation salts, i.e. after adaptation to KC1 solutions, subsequent transfer to KC1 solutions does not effect ciliary reversal of the same magnitude as found in non-adapted cells. The adaptation is however temporary and after 10-20rain in solutions containing low concentrations of salts, the cells regain their sensitivity to salt stimulation (Dryl, 1959; Hildebrand, 1978; Hildebrand and Dryl, 1976; Dryl and Hildebrand, 1979). The de-adaptation is not apparently due to the resting potential being maintained at a depolarizing level (Kinosita et al., 1964a; Dryl and Hildebrand, 1979). Monovalent cations show the following effectiveness at inducing ciliary reversal behavior in Paramecium: K + > Li + > Na + > NH2 (Oliphant, 1938, 1942) with the anions of the salts following the effectiveness series CI- > Br- > I- > NO3 > C O ] - (Oliphant,

58

M. J. DOUGHTYand S. DRYL

CCR gy~tio n~ (sideview) %.,/

,Q 2

spinning (as viewed

fromabove)

tumbling

~?,,') ~,rcting

PaCR

looping FL$ FiG. 39. Swimming behaviour of Paramecium following stimulation with depolarizing stimuli. Diagrams to show sequences of behavioral responses following stimulation of free-swimming cells with either NaCI (periodic ciliary reversal, PCR) or with KCI (continuous ciliary reversal, CCR through partial ciliary reversal, PaCR and the return to forward left spiralling swimming behavior, FLS).

1942). The anion effects are far less pronounced than those for cations. Satow and Kung (1974) report that substitution of extracellular chloride for propionate has no significant effect on the Ca 2÷ regenerative response. From early studies by Mast and Nadler (1926), exhaustive studies by Kamada and Kinosita (1940) (studies later reanalyzed by Jahn (1962a)) and confirmatory studies by Naitoh (1968), it has been established that the duration of the ciliary reversal behavior responses shown by Paramecium in response to KCI stimulation, critically depends on the concentration of calcium ions to which the cells are adapted and the concentration of calcium ions in the stimulation solutions. Jahn (1962a) found that, on reanalysis of the data of Kamada and Kinosita (1940), for any particular K ÷ concentration, the maximal duration of ciliary reversal behavior was shown in those test solutions having a certain fixed ratio between the concentrations of K ÷ and Ca 2÷ for cells adapted to a fixed concentration of these two ions. Paramecium exhibits several distinctive types of behavioral response following stimulation with inorganic cation salts. These are detailed below: Upon transfer to solutions of NaCI (or other sodium salts apart from NaF), Paramecium respond with a behavioral type known as periodic ciliary reversal (PCR) (Dryl, 1961c, 1964---terminology subsequently adopted by other workers; Grebecki, 1965; Kuznicki, 1966a). This behavioral response (see Fig. 39), is t y p i ~ by a, relatively short duration, period of rearward swimming (a few seconds) during which the cell moves backwards for only a few body lengths. The speed of reversed swimming is higher than that for cells in forward swimming (FLS). The rearward swimming ends abruptly as the cells stops translational motion momenta.,ily and then swims off again in FLS (forward left spiralling). At low concentrations of NaC1, a single response is shown and the cells return to FLS although, after return to forward swimming, there is a higher occurrence of spontaneous changes in the direction of the cells' swimming path. With higher concentrations of NaCI, the response is repeated, i.e. the cells show a sequence of rearward

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

59

swimming ~ FLS--~ rearward swimming ~ FLS etc--thus the terminology, periodic ciliary reversal. The duration of each successive rearward swimming event is less and less, and the cells readopt FLS usually within 30 sec regardless of the NaCI concentration. The strength of the response (the duration of each rearward swimming event and the number of times the sequence is repeated) is dependent upon the extracellular calcium concentration (Doughty, unpublished). The occurrence of directional changes in the swimming path of the cells, especially under conditions of stimulation with some salts, has been described as an avoiding reaction by some authors. However, care should be taken to discriminate between a frequency of occurrence of active rearward swimming events of short duration and the frequency of changes in cell swimming path where the cells predominantly simply change the direction of motion through an angle less than 180 °. The use of terminology such as frequency of avoiding reaction (FAR) to describe changes in swimming direction is perhaps open to misinterpretation. Van Houten (1977, 1978) reports that, following transfer to solutions of sodium salts, the FAR declines from an initial high value to close to zero within a few minutes indicating adaptation of the cells to the sodium solutions. A change in the frequency of directional changes in the cells swimming path is a klinokinesis (Fraenkel and Gunn, 1961 ; Diehn et al., 1978). The PCR response is shown by P a r a m e c i u m that have been adapted to various concentrations of Ca 2÷ (Grebecki, 1965; Kuznicki, 1966a; Doughty and Dodd, 1978). In the presence of low concentrations of extracellular Ca 2 ÷ (distilled water or spring water), a longer lasting ciliary reversal behavior is shown, the details of which were not given (Mast and Nadler, 1926). Transfer of P a r a m e c i u m to K ÷ salts, typically KC1, the cells immediately adopt a behavioral response known as continuous ciliary reversal (CCR) (Dryl, 1961c). In CCR, the body cilia are all striking (beating) towards the anterior of the cell (Parducz, 1954a; Machemer, 1969); i.e. are all in reverse beating mode. The cell body now rotates clockwise, instead of counterclockwise, and the rotation is far faster than normal. As a result of the reversal in beating direction and the change in body rotation character, the cell is propelled backwards for many seconds in a straight path. The speed is at least twice that of FLS but no measurements have appeared in the literature. The speed of CCR slows visibly after a few seconds (depending on the KC1 concentration) and the cell eventually ceases translational motion--this marks the end of CCR behavior. The cell then undergoes a series of very characteristic behavioral reactions (Jennings, 1906, pp. 47-52; Doughty and Dodd, 1978). At the end of CCR, ciliary activity is such that, although no translational motion is possible, the cell gyrates like a top (Fig. 39). The gyration gives way to a spinning response in which the cells prescribe a circular locus with the body length being the radius of the circle. Spinning behavior gives way to a circling behavior in which the anterior end of the cells is definitely leading, i.e. ciliary activity is such that partial return of forward beating and forward metachrony patterns has occurred at this stage. However, since the majority of the body cilia are still in an inactive mode (see later), the cell prescribes a circle rather than swimming forward. Initially the diameter of the circles is very small (I-2 body lengths) but it gets progressively larger over a period of a few seconds. During this period, movement is largely confined to a two dimensional plane but gradually the cells start to move in either ascending or descending circles (looping behavior) as the ciliary activity progressively renormalizes. During looping, little or no body rotation occurs. Counterclockwise rotation of the body on its long axis appears, often abruptly, and the cell readopts FLS (forward left spiralling) (Bullington, 1930; Grebecki et al., 1967a, b). Such a sequence of behavioral responses (the sensory response of the cell to KC1) serves to amply illustrate both the complexity of control mechanisms in P a r a m e c i u m and the degree of fine control of ciliary activity. Studies using high speed cinematographic analysis of ciliary activity in microelectrode impaled cells under conditions of maintained depolarizing stimulation (Machemer, 1974a; Machemer and Eckert, 1975; Eckert and Machemer, 1975) show that the maximal frequency of ciliary beating in reversed mode occurs at time when maximal outward current occurs (the late outward K ÷ current; see Sections 4.3 and 4.4). Ciliary activity (frequency of.

60

M.J. DOUGHTY and S. DRYL

beating) declines after reaching a maximum and then transiently stops (Machemer, 1974a). This inactivation is followed by a resumption of forward beating, initially at low frequency and the cilia slowly shift in orientation, in a counterclockwise direction (as viewed from above), back to that normally shown for forward, dexioplectic, metachrony (Machemer, 1974a, b, 1975). Cinematographic analysis of single, microelectrode-impaled cells, shows that the ciliary reversal behavior sequence of the cell, if analyzed in detail, gives an accurate picture of the activity of the cilia following application of depolarizing stimuli. CCR behavior reflects the activation of ciliary reversal mechanisms and is the result of enhanced frequency, reversed beating of the cilia. The slowing of cell rearward swimming and the onset of gyration is the result of a gradual inactivation of the cilia and the onset of spinning marks the onset of forward beating. The gradual change in behavior from spinning, through circling, looping and the resumption of FLS reflects the gradual renormalization of ciliary activity and the gradual return of forward metachrony. The initial phase of the rearward swimming response to KC1 is termed CCR and the period from the start of spinning through to return of FLS is termed partial ciliary reversal behavior (PaCR)(Grebecki, 1965; Kuznicki, 1966a; Doughty and Dodd, 1978). The total duration of the behavioral sequence from the induction of CCR through to return to FLS is usually termed tR. Analysis of membrane potential changes that occur following application of a depolarizing stimuli (injected current) to microelectrodeimpaled cells, shows that reversed beating reaches a maximum at, or shortly after, the end of the active conductance change of the membrane to Ca 2 ÷ (the Ca 2 + regenerative response) as determined from the first derivative of the membrane potential. Activation and inactivation of outward current follows a similar time course to augmentation and subsequent decline in reversed ciliary beating (Machemer, 1974a, 1975; Machemer and Eckert, 1975). Active conductance changes thus occur at the onset of and during the early course of CCR behavior and thus the mechanisms that effect and control the active conductance changes can be in part studied through careful analysis of the CCR behavior. During PaCR, resting potential is constant and thus the membrane mechanisms that determine the duration of activation of the ciliary mechanochemical cycle (reversal activation) at intraciliary sites can be studied through analysis of PaCR behavior. These relationships between ciliary activity and membrane potential changes are shown in Fig. 40. Only a single cycle of CCR, through PaCR to FLS is shown following stimulation of Paramecium with KC1. A third type of behavioral response has been described for Paramecium although is of uncertain physiological significance. Eisenberg-Hamburg (1930, 1932) reported that Paramecium transferred into solutions of strontium salts showed a repeated series of forward and backward swimming. The response is qualitatively similar to that observed following transfer of cells to Na ÷ solutions except that the response is repeated over far longer time periods and the rearward swimming event predominates over forward swimming periods. Dryl (1961c, 1964) reports similar behavior on transfer of Paramecium to either Sr 2 +--Ca 2 + or Ba 2 +-Ca 2 ÷ solutions. Transfer of cells, without mixing, into Ba 2 +-Ca 2 ÷ solutions results in a behavior described as the "barium dance" (Kung, 1971a) in which the cells, being repelled by the Ba 2 + solutions repetitively, form a star like pattern of tracks. In solutions containing relatively equal concentrations of Ba 2 + and Ca 2 +, a PCR type behavior dominates and the cells reverse their direction of swimming many times a minute (Yarbrough and O'Kelley, 1962; Kinosita et al., 1964b, c). Similar behavior is observed in solutions containing equal concentrations of Ca(OH)2 and citric acid (t m i each) and 1.7 - < . 5 triM BaC12 (Ling and Kung, 1980). As the Ba 2+ to Ca 2+ ratio is raised above a certain value (estimated 5:1; Ling and Kung, 1980), the duration of backward swimming events get pregressively longer so that the cells swim backwards for many seconds at a time and in the extreme, for over a minute. The prolonged rearward swimming can however give way to repeated, short (< 2 sec) reversal events at lower B a 2 +:Ca 2 + ratios (Doughty, unpublished). The Ba 2 + induced behavior can last for many minutes (20-30 rain) but the cells, rather than recovering FLS, are frequently immobilized after this time period or continue to show weaker and weaker responses.

61

CONTROL OF CILIARY ACTIVITY IN PARAMECIL, M Hz

4O 20m

a

~

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FIG. 40. The ciliary reversal response in Paramecium and the Ca 2 +-dependent active electrogenesis of the ciliary membrane. Depolarization-induced (injected current) changes in the ciliary beat frequency (Hz), ciliary reversal (hatched areas) and membrane potential in Paramecium caudatum at 18°C and 8°C. Cells in l mM Tris buffer, l mM CaC12, l mM KCI, pH 7. INSETS show membrane potential deflections (middle trace) at expanded time scale. Stimulus current (lower trace) adjusted to provide same peak potential of depolarization at both temperatures. Upper trace: first derivative of membrane response indicating latency and reduced rate of rise of regenerative deflection at the lower temperature. Figure (provided courtesy of the author) from Machemer, 1974a. Copyright J. Comparative Physiology/Springer-Verlag.

The duration of the initial period of PCR-type behavior following NaCI stimulation increases with increasing salt concentration (Oliphant, 1938, 1942; Mast and Nadler, 1926; Hansma and Kung, 1976; Kung, 1971a; Doughty and Dodd, 1978). Depending on the CaCIz concentration, above a certain concentration of NaCI, the response becomes shorter (Doughty and Dodd, 1978; Doughty, unpublished) (Fig. 41) although other reports indicate no such decline in response (Hansma and Kung, 1976). Oliphant (1938) reports that the total duration of Na+-induced rearward swimming behavior shows an inverse relationship to temperature. The duration of K+-induced CCR behavior increases with increasing KC1 concentration (Oliphant, 1938, 1942; Kamada and Kinosita, 1940; Naitoh, 1968: Doughty, 1978b) (Fig. 42b). As with the Na + response, higher concentrations of K + do not effect further enhancement of the response (Naitoh, 1968) and at very high KC1 (80mM at 0.5 mM CaC12, 40 mM at 10- 5 M CaClz; Doughty, unpublished) the CCR response is all but eliminated. The relative response to K +, as well as being dependent on the CaC12 concentration (see Section 4.2) is also very sensitive to temperature (Fig. 42). The duration of the total reversed swimming behavior (tR) shows a different degree of enhancement as a function of KC1 concentration for this reason (Fig. 42a). The temperature sensitivity is very marked however for both CCR (Oliphant, 1938; Doughty, 1978b; Hildebrand, 1978) and both PaCR and thus tR (Doughty, 1978b) (Fig. 43). With increasing temperature, all responses get shorter. As with the effects of temperature on either the cell swimming velocity (Fig. 21) or the frequency of directional changes in the cells' swimming path (Fig. 22), there would appear to be a critical transition temperature for the molecular components controlling these behaviors as evidenced from a sharp discontinuity in the temperature plots. The anion sensitivity of Paramecium has been poorly studied--in part because of the relative insensitivity of the cell to the nature of the anion species in the extracellular solution. However, since the cells do show slight anion sensitivity (different dose response

62

M . J . DOUGHTY and S. DRYL

6

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FIG. 41. Ciliary reversal response in Paramecium to NaCI. Duration individual periodic ciliary reversal responses (PCR) as a function of NaCI concentration. P. aurelia in Hepes-Pipes buffer, 0.5 M CaCI2, pH 7.1 23-24°C. Modified from Doughty and Dodd, 1978.

plots for K ÷ concentration and CCR behavior with different potassium salts; Oliphant, 1938, 1942), this sensitivity perhaps warrants further examination. Van Houten (1977, 1978, 1979) shows significant sensitivity of Paramecium to some organic anions relative to inorganic anions. Doughty (1978c) notes differences in ciliary reversal behavior according to whether ammonium chloride or ammonium acetate is used as the stimulus. Ammonium chloride induces a sequence of behavior like that seen towards KCI, i.e. CCR PaCR FLS. Ammonium acetate, however, at low concentrations induces PCR-typ¢ behavior and the CCR response is only seen at higher ammonium acetate concentrations. The duration of CCR behavior in Paramecium to either KC1 or T12SO~ is additionally sensitive to extracellular pH--the response duration increasing with increasing pH (Fig. 44) (Kuznicki, 1966b).

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FrG. 43. Temperature sensitivity of depolarization-induced ciliary reversal responses in Paramecium to KC1. Duration of total reversed swimming behavior (ta), continuous ciliary reversal (CCR) and partial ciliary reversal (PaCR) in response to 16 mM KCI as a function of temperature. P. aurelia in Hepes-Pipes buffer, 0.5 mM CaCl 2, pH 7.1. From Doughty, 1978b.

4.2. INORGANIC CATION-INDUCED CILIARY REVERSAL BEHAVIOR IN P A R A M E C I U M : A PHARMACOLOGICAL ANALYSIS

The behavioral responses of Paramecium to changes in its chemical environment are the result of the operation of a chemoreceptor system and an associated sensory transduction mechanism. The behavioral responses thus provide a convenient monitor of the cells sensory responses, i.e. the output response of a sensory system. The molecular components and control mechanisms underlying the sensory output of excitable membrane systems are characterized by their physiological and electrophysiological responses under a variety of chemical and physical conditions, most notably conditions of different cation composition of tissue, or organelle bathing solutions, and in the presence of a variety of pharmacologically active agents. The sensory system has thus been dissected through an ionic and pharmacological analysis of the sensory response. A similar rationale can presumably be adopted, with caution, in a study of the sensory, excitable, membrane of Paramecium. Such an analysis, by virtue of its comparatively novel nature for protozoan studies, is necessarily limited at this time since the actual sites of action of even inorganic cations is unknown. The ionic dependence of the regenerative depolarization of the ciliary membrane that underlies the ciliary reversal response, has been reasonably well characterized by electrophysiological methods (see Sections 4.3 and 4.4) and is in part based on an ionic and pharmacological analysis. In applying such an analysis to the sensory behavior of Paramecium, it is obviously important that both the responses induced by the ligands in question and inorganic cations be characterized in detail in addition to studying the effects of these ligands on the inorganic cation induced

64

M.J. DOUGHTY and S. DRYL 200

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pH FIG. 44. Depolarization-induced ciliary reversal in Paramecium. Effect of pH. Duration continuous ciliary reversal responses induced in Paramecium caudatum by 8 mM (O) or 16 m~t (F-t) KCI. Cells in Tris buffer plus either potassium phthalate-NaOH or boric acid-HCl to give desired pH. All solutions contain 0.5 mM CaCt2. 21 + I°C. Redrawn from Kuznicki, 1966b with permission author and Acta Protozoologica.

ciliary reversal response. Current evidence indicates that ciliary reversal is triggered as a result of an activation of the calcium conductance of the ciliary membrane and that the resultant influx of calcium ions into the intraciliary space activates the reversal mechanism (see Sections 4.3-4.5). Stimulants such as ionorganic cations trigger the calcium conductance activation by an unknown mechanism. The ciliary reversal response is thought to persist for as long as the intraciliary free calcium ion concentrations remain above a concentration necessary to either trigger or maintain the reversal mechanism in activated state (see Section 4.6). Therefore, modification of Na ÷ or K ÷ ciliary reversal responses by the presence of pharmacological ligands (that do not themselves induce significant perturbation of the cells' behavior) provides a priori evidence as to the nature of the control mechanisms underlying ciliary reversal activation and its control. As detailed in previous sections, the characteristics of Paramecium "resting state behavior", the cell membrane resting potential and the relative sensitivity of the cells to stimulation are all markedly influenced by the chemical and physical character of the medium in which the cells are acclimatized (adapted) prior to study and the nature of the medium in which the cells are tested. Over the 90 years that Paramecium has interested workers as a cell showing chemical sensitivity, many different solutions have been used in these studies. Even in contemporary years, some 20 different buffered salt solutions have been used. Solutions of note, and widely used still, are Dryl's solution (a calcium containing, phosphate buffered solution with additional citrate to chelate heavy metals) developed for genetic studies on the cell and widely used in behavioral studies as well (Dryl, 1959); Tris buffered-KCl-CaCl2 solutions widely used in electrophysiological studies (Naitoh and Eckert, 1968a) and Tris buffered-Ca(OH)2-citric acid solutions also used in electrophysiological studies (Satow and Kung, 1974). More recently there has been a tendency to use EDTA instead of citrate (Dunlap, 1977; Brchm and Eckert, t978a; Hansma, 1979). Heavy metals are extremely toxic to Paramecium (Woodruff" and Bunzell, 1909; Collett, 1919; Peters, 1919; Wiehterman, 1953; Andrivon, 1968,). The use o f ~ l a t ing agents to remove undesirable traces of heavy metals even from double distilled water would therefore appear desirable. However, it must be noted that chelating agents such as citrate alter the cells' sensitivity to inorganic cation stimulation (Grebecki, 1965; Kuznicki, 1966a); the galvanic sensitivity of the cells (Bancroft, 1 ~ b; Fabre, 1947) and pharmacological sensitivity of the cells (Doughty, 197be, and unpublished). These effects are conceivably simply due to alteration in the calcium concentrations in the membrane microenvironment. In the presence of chelating agents such as citrate or EDTA, although bulk solution-free calcium ion concentration can be calculated with

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

65

reasonable accuracy (see Satow and Kung, 1979; Ling and Kung, 1980), membrane sited calcium, which is surely influenced by the presence of a chelating agent, and the kinetics of membrane calcium-chelating agent-bulk solution equilibria cannot be ascertained with any accuracy. The concentration of calcium ions in bulk solution and membrane bound calcium for "optimum" excitability in Paramecium is uncertain (Jahn, 1962a; Hildebrand and Dryl, 1978; Dryl and Kurdybacha, 1978; Naitoh, 1968; Satow and Kung, 1979) and currently being investigated. Changes in the calcium equilibria at the membrane of Paramecium can be induced by short term changes in bulk solution (test solution)-calcium ion concentrations, either by direct addition of calcium salts (Kamada and Kinosita, 1940; Naitoh, 1968), or by use of chelating agents (Dryl and Kurdybacha, 1978; Grebecki, 1965). Alternatively, calcium equilibria can be changed, by adaptation of the cells over several hours to solutions containing different free calcium ion concentrations (Kamada and Kinosita, 1940; Naitoh, 1968). Any change in calcium equilibria can be expected to change the sensitivity of Paramecium to any kind of stimulation or more importantly, their relative sensitivity in the presence of, for example, agonists or antagonists of sensitivity whether these be other inorganic ions or pharmacologically interesting ligands. The effects of free calcium ion concentrations, the effects of adaptation and the effects of chelating agents on the excitability of Paramecium remain to be characterized in further detail. An alternative, but as yet unproved, solution to this problem is the use of buffering reagents with minimal metal ion chelating capacity (Good et al., 1968) and to deionize all buffer solutions prior to use, to remove both calcium and heavy metals (Doughty and Dodd, 1976). The lack of consistency in choice of experimental solutions, although understandable, makes comparative analysis of the sensory sensitivity of Paramecium difficult. The sensitivities of Paramecium to both salts and their alteration by other ligands (in terms of dose-response relationships) are thus of a relative nature only. The duration of monovalent cation-induced ciliary reversal behavior is markedly dependent on the relative concentrations of monovalent and divalent cations in both test and adaptation buffers. Extracellular calcium ions are a requirement for motility in Paramecium and survival. The cells die shortly after transfer to solutions containing less than 1 0 - v u free calcium but strong motility is supported in double distilled water containing only calcium ions at this concentration (10-~-10-6M) (Doughty, unpublished). Hildebrand and Dryl (1976) reported that for K* ions to induce ciliary reversal, a minimal extracellular free calcium ion concentration of 3 × 1 0 - ~ was required. Dryl and Kurdybacha (1978) found that similar calcium concentrations were necessary for the expression of behavioral responses of Paramecium towards quinine and that the threshold for this response (repulsion away from quinine) decreased linearly parallel to decrease in the free calcium ion concentration. In these experiments, the calcium ion concentrations are those to which the cells were adapted prior to stimulation. Both Kamada and Kinosita (1940), and Naitoh (1968) found that the concentration of calcium ions in the adaptation solutions markedly affected the duration of the ciliary reversal responses of the cells when subsequently tested in solutions containing different concentrations of both K + and C a 2 +. Naitoh (1968) reported that, for cells adapted to solutions containing a fixed concentration of calcium to potassium ions ([K+]o/[CaZ+]~), the duration of the ciliary reversal response in test solutions containing a fixed concentration of Ca 2 + ions and different K + ions (1-6 mu), remained the same. Mast and Nadler (19261 note that additions of Ca 2 + ions to test solutions, containing KC1, reduced the duration of the ciliary reversal behavior of Paramecium. They termed this a neutralizing effect and found that the quantity of C a 2+ ions required to supress K+-induced ciliary reversal behavior increased as the test solution K + ion concentration was raised. It is of interest to note that they found that ferric chloride was considerably more active than calcium chloride in this respect. Grebecki (1964) (Fig. 45) and Naitoh (1968) (Fig. 46) report that higher concentrations of calcium in test solutions containing KCI, effect reduction in the duration of the ciliary reversal response while lower concentrations are apparently stimulatory (Naitoh, 1968; Kamada and Kinosita, 1940). Neutral calcium ion salts do not induce ciliary reversal behavior in Paramecium (Mast and Nadler, 1926; Oliphant, 1942).

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Such behavioral data shows that the ciliary reversal response is initiated as a result of activation of mechanism(s) that requires extracellular calcium ions but that calcium ions alone cannot directly activate these mechanisms. As discussed in the previous section, the ciliary responses to either Na ÷ or K ÷ stimulation are different. The reason is unknown. Since the response to stimulation by potassium ions is generally far longer in duration than that given to sodium ions, the former response has received most attention. It should therefore be noted that the pharmacological sensitivity of the two responses is not necessarily the same and preliminary data suggests that the two are in fact different (see later). K+-induced ciliary reversal can be significantly reduced or abolished by extracellular application of ruthemium red--a reported specific inhibitor of both Ca 2 ÷ ionophore and Ca 2 + ATPase activity of biological membranes (Shamoo et al., 1975; Watson, 1971; Ash and Bygrave, 1977; Caroni et al., 1977). The nature of the inhibitory component is uncertain. Both Onimaru (1976) and Doughty (1978b) report reduction in the duration of 120 100

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OF CILIARY ACTIVITY IN PARAMECIUM

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FIG. 47. Depolarization-induced ciliary reversal in Paramecium: Effect of ruthenium red. Left effect of ruthenium red on the duration of continuous ciliary reversal (CCR)~ partial ciliary reversal (PaCR) and total duration ciliary reversal behavior (tR) induced in Paramecium aurelia by 16 mM KCI. 22-23°C. Right effect of ruthenium red on the duration ciliary reversal responses given to different concentrations of KCI. 20-25°C. All cells in Hepes-Pipes buffer, plus 0.5 mra CaCI2, pH 7.1. From Doughty, 1978b.

K +-induced continuous ciliary reversal (CCR) behavior by micromolar concentrations of ruthenium red (Fig. 47). By stimulating Paramecium with K ÷ ions in the presence of ruthenium red without any preincubation of the cells with the polycationic dye, there is additionally a dramatic enhancement of the duration of the responses following the CCR behavior despite the reduction in CCR response (Fig. 47). Preincubation of Paramecium with ruthenium red for only a few seconds prior to stimulation with K ÷ ions causes complete abolition of the CCR response (Doughty, unpublished; Hildebrand, personal communication; Kaczanowska, 1979). Thus preincubation with ruthenium causes progressive loss of excitability (Kaczanowska, 1979). The inhibition is temperature dependent (Kaczanowska, 1979). The same concentrations of ruthenium red (micromolar or less), very rapidly (15-30 sec) abolish both the graded Ca 2 ÷ regenerative response and the all-or-nothing response of the membrane (see later) (Naitoh, personal communication). The compound additionally alters the kinetics of Ni 2 ÷ uptake by Paramecium and inhibits the ciliary membrane Ca 2+ ATPase enzyme (Doughty, 1978a and unpublished). Ruthenium red is toxic to Paramecium and alters other functions apart from those associated with ciliary activity (Kaczanowska, 1979) in common with Ni 2÷ ions. Other Ca 2 + ion antagonists such as verapamil or methoxy-verapamil (D-600) (Kohlhardt et al., 1972; Baker et al., 1973; Cranefield et al., 1974; Malaise et al., 1977) can effect complete or partial supression of K+-induced ciliary reversal depending on the relative K + to Ca 2+ concentrations and the reagent concentrations (Doughty, unpublished). Similar inhibition, by verapamil, of metazoan gill ciliary activity in response to electrical (but not chemical) stimulation has been reported (Motokawa et al., 1975). Ammonium ion salts (ammonium chloride and ammonium acetate), at low concentrations (0.25 mM) do not effect either the characteristics or duration of K+-induced ciliary reversal behavior while higher concentrations prolong the responses (Doughty, 1978c). Alkyl derivatives of quaternary ammonium ion salts (TEA and TMA) also prolong the duration of K+-induced ciliary reversal behavior at low concentrations but at higher concentrations (>0.5 raM), like both the Ca 2÷ antagonists and ammonium ion salts, induce ciliary reversal themselves (Doughty, unpublished; Satow and Kung, 1976).

68

M . J . DOCGHTV and S. DRYL

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FIG. 48. Chemoaccumulation response of Paramecium to acetylcholine. Relative distribution of Paramecium aurelia in a T-maze as a function of acctylcholine (AcCh) chloride in test arm. Iche is the index of chemoaccumulation ("chemotaxis") = T / T + C where T is the number of cells in test arm and C the number of cells in the control arm of the maze (buffer without AcCh). Cells in Hepes-Pipes buffer plus 0.5 mM CaCl2, pH 7.1 22.5-23°C. From Doughty, 1979a.

Cholinergic ligands appear to have first been tested on infusoria in 1924 by Kritz who noted that low concentrations of nicotine caused aggregation and then looping swimming paths in the hetrotrich ciliate Chilodon. Wense, during studies on the effects of adrenalin on the contractile vacuole cycle in Paramecium (being studied as a model system for the inflammatory actions of adrenalin in tissues of higher phyla) notes (1934, 1935) that both choline and acetylcholine acted as antagonists of the action of adrenalin o n the contractile vacuole cycle. Wense (1935, 1939a) also reported that, unlike adrenalin, choline and acetylcholine increased the swimming velocity of Paramecium. A similar action of acetyleholine on ciliary activity of Paramecium was reported by Oliphant (1943), Browning and Nelson (1976b) and Doughty (1978c). Doughty (1979a) made measurements of this velocity enhancement and suggested that it was, at least in part, responsible for the observed accumulation of Paramecium into solutions containing micromolar concentrations of acetylcholine (as assayed in a T-maze assay) (Fig. 48). Using a T-maze analysis (see Van Houten et al., 1975), Doughty (1979a) reported that Paramecium showed only a slight accumulation response to choline chloride and was repelled by higher concentrations. Similar sensitivity was reported by Wense (1935) using the same T-maze principle. Wense (1935) noted marked repulsion of Paramecium by choline chloride in the absence of CaCI2 while Doughty (1979a) reported that accumulation responses of Paramecium to acetylcholine were suppressed by high concentrations of CaC12. Such preliminary results indicate that Paramecium can detect low concentrations of acetylcholine in its environment and that this perception is sensitive to Ca 2~ ions. This Ca 2+ ion sensitivity is also found for the effects of acetylcholine on K *-induced ciliary reversal behavior. At 0.5 mM CaCI2, low concentrations of acetylcholine enhance the duration of the CCR induced behavior (Fig. 49) while in the presence of equal concentrations of Ca(OH) 2 and citric acid, acetylcholine, at these concentrations, no longer alters the K +-induced CCR behavior (Doughty, 1978c). Acetylcholine additionally markedly prolongs the duration of the K +-induced PaCR behavior and thus the total duration of the ciliary reversal behavior shown to K* ions (Fig. 49). Muller and Toth (1959) also reported that micromolar concentrations of acetylcholine enh~mc,ed the duration of K+-ion induced ciliary reversal behavior in Paramecium. The action of acetylcholine on K+-induced ciliary reversal behavior is altered by very low (10 -s M) concentrations of d-tubocurarine (Doughty, 1978b). This cholinergic antagonist itself prolongs the duration of K+-induced PaCR behavior without apparently effecting the CCR behavior (Fig. 50). A similar effect is seen on stimulation of Paramecium with potassium ions in the presence of gallamine triethiodide (flaxedil)(Doughty, 1978b) and both antagonists block

69

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

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FIG. 49. Depolarization-induced ciliary reversal in Paramecium: Effect of acetylcholine chloride (AcCh). Duration of ciliary reversal responses e---CCR, II--PaCR and O---tR) in Paramecium aurelia to 16 mM KCI in the presence of different concentrations of AcCh. Cells in Hepes Pipes buffer, plus 0.5 mM CaCI2, pH 7.1 18-18.5°C. From Doughty, 1978c.

the accumulation behavior shown to micromolar concentrations of acetylcholine (Doughty, 1979a). Muller and Toth (1959) also reported "stimulation" of K÷-induced ciliary reversal behavior by the anti-acetylcholinesterase drug, eserine (physostigmine) and Koshtoyants and Kokina (1957) report alteration in galvanotactic behavior and threshold by other anti-cholinesterase drugs (proserin and phosphacol) and acetylcholine itself. Enhancement of K +-induced PaCR, but not CCR, behavior by both eserine and neostigmine was also reported by Doughty (1978c) (Fig. 51). Neither acetylcholine or 130 120 I10 too

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FIG. 50. Depolarization-induced ciliary reversal in Paramecium: Effect of d-tubocurarrine. Duration ciliary reversal responses (@---CCR, I - - P a C R and O--tR) in Paramecium aurelia to 16 mM KC1 in the presence of different concentrations of d-tubocurarrine (d-Tc). Cells in Hepes-Pipes buffer plus 0.5mM KCI, pH 71.1 23°C. From Doughty, 1978c.

70

M.J. DOUGHTY and S. DRYL leo RiO

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FIG. 51. Depolarization-induced ciliary reversal in Paramecium: Effect of neostigmine bromide. Duration ciliary reversal responses (O--CCR, i - - P a C R and O---tR) in Paramecium aurelia to 16 mM KCI in the presence of different concentrations of neostigmine. Cells in Hepes-Pipes buffer plus 0.5 mM CaCI2, pH 7.1. 18-19°C. From Doughty, 1978c.

acetylcholinesterase inhibitors induce ciliary reversal themselves (Oliphant, 1943; Browning and Nelson, 1976b; Doughty, 1978c) although Grebecki (1965) reports that high concentrations (1 m~,l) of acetylcholine induce shock responses in Paramecium. Nicotinic, but not muscarinic (nicotine and muscarine respectively) agonists decrease the duration of K+-indueed CCR behavior and the resultant PaCR behavior (Doughty, 1978c). A neurochemical sensitivity (for cholinoeeptive and adrenoceptive drugs) has also been reported for the photobehavior of the dinoflagellate, Gymnodinium (Forward, I977) and a sensitivity to cholinergic compounds was also reported for the slime mould, Polysphondylium (Clark, 1977) indicating that the holotrich ciliated protozoa are not unique, amongst protozoa, in their sensitivity to cholinergic compounds. In support of the pharmacological sensitivity of protozoa, several authors report the presence of acetylcholinesterase activity in these cells. Bayer and Wense (1936a) noted that lytic extracts of Paramecium caused contraction of eserine treated smooth muscle (dorsal muscle of the leech and frog heart). They found that the potency of the extract could be increased by treatment of the extract with acetic anhydride or by the inclusion of eserine in solutions during preparation of the extract. Incubation of the extract for several hours at 37°C also enhanced its activity (Bayer and Wense, 1936a; Wense, 1937). The action of the extract on the eserine treated muscle could be blocked by atropine and pilocarpin (Bayer and Wense, 1936a; Wense, 1937). The authors concluded that acetytcholine and an acetylcholinesterase were present in Paramecium. Seaman (Seaman and Houlihan, 1951; Seaman, 1951) reported the presence of acetylcholinesterase activity in subcellular (pellicle) fractions of Tetrahymena while Andrivon (1975) reported the same activity in cell homogenates of Paramecium. Although successful detection of acetylcholinesterase activity (on the basis of substrate specificity and sensitivity to inhibitors) has been reported, other workers were unable to detect aeetylcholinesterase activity or acetylcholine in holotrich ciliated protozoa (Bullock and Nachmansohn, 1942; Mitropolitanskaia, 1941; Aaronson, 1963). Schuster and Hershenov (1969) report localization of acetylcholinesterase activity in cilia of Tetrahymena using histochemical reaction product techniques in addition to uncertain activity in pellicle regions. While pellicle fractions prepared according to Seaman (1951) have aeetylcholinesterase activity, Tetrahymena pellicles prepared by an alternative techniques (Nozawa, 1975) did not contain detectable

CONTROL OF CILIARY ACTIVITY IN

PARAMECIUM

71

acetylcholinesterase activity (Doughty, unpublished). Isolated intact cilia from both Tetrahymena and Paramecium show acetylcholinesterase activity (neostigmine sensitive turnover of acetylthiocholine) (Doughty, unpublished) although further studies are necessary before assignment of the activity type or the localization of the enzyme can be made with certainty. As noted above, Na+-induced ciliary reversal behavior differs markedly from that shown to potassium ions (PCR versus CCR behavior). The Na +-induced response differs from the K + responses in its sensitivity to agonists and antagonists. Mast and Nadler (1926) reported that the sensitivity of Na+-induced ciliary reversal (PCR behavior) towards changes in extracellular calcium ion concentrations was very much less than that for the K+-induced responses. Far higher concentrations of calcium ions were required to suppress Na+-induced ciliary reversal behavior compared to the K + response. Early reports on the duration of Na +-induced ciliary reversal behavior indicate the response(s) can be of relatively longer duration (10-30sec) although later studies (carried out in buffers containing relatively high concentrations of Ca 2+ instead of distilled water or spring water) report far shorter duration responses (Kung, 1971a; Hansma and Kung, 1976; Doughty and Dodd, 1978). Na+-induced ciliary reversal responses show the opposite sensitivity, towards cholinergic agonists and antagonists, to that shown for K+-induced behavior. Na+-induced PCR type behavior is stimulated by low concentrations of nicotine and markedly reduced by the same concentrations of gallamine that enhance the duration of K +-induced PaCR behavior (Doughty, unpublished). Early studies on the effects of various "irritant" chemicals on the motility and behavior of Paramecium were initiated primarily as a consequence of the development of the idea that unicellular organisms possessed an intricate, intracellularly located nerve net (see Section 4.7). In addition, various workers considered that, through a study of the action of various pharmacologically active natural and synthetic substances on the motility, physiology and growth of unicellular organisms, the mechanisms of action of these substances on the tissues of higher phyla could be elucidated. Thus, in the literature are to be found reports of the effects of endocrine gland extracts on Paramecium (Nowikoff, 1908; Abderhalden and Schiffman, 1922; Bramstedt, 1937), adrenalin (Bauer, 1926; Wense~ 1934, 1935), various toxins (Bokorny, 1896; Putner, 1904; Lohner, 1913; Tuncliff, 1928; Philpott, 1930; Essex and Markowitz, 1930; Chopra and Chowhan, 1931; Oliphant, 1943; Roux and Serre, 1964a, b; Zacks and Sheff, 1966) and a variety of other active compounds (Wense, 1935; Seaman and Houlihan, 1951; Koshtoyants and Kokina, 1957; Koshtoyants, 1960; Van Eyes and Warnock, 1963; Jones and Jahn, 1965). Compounds have been tested for their effects on the motility, "chemotactic responses" and galvanotactic responses in addition to such vital functions as osmoregulation. While it is difficult, in view of our current knowledge, to side with the authors on their conclusions as to the mode of action of these "irritant" chemicals on Paramecium, the authors should perhaps be credited with their foresight for believing that unicells might show forms of control mechanism usually attributed to the presence of differentiated tissue in multicellular organisms (Wense, 1939a, b). 4.3. CILIARY REVERSALAND THE Ca-REGENERATIVERESPONSE As outlined in previous sections, the ciliary reversal behavior in Paramecium is associated with the occurrence of a Ca 2 + ion dependent regenerative depolarization of the ciliary membrane. Yamaguchi (1960a) and Kinosita et al. (1964b, c) provided the first evidence that ciliary reversal behavior was related to the electrical activity of the surface membrane of Paramecium by recording both the ciliary activity and membrane potential changes simultaneously. Kinosita et al. (1964a-c, 1965) observed that, following bathing Paramecium in solutions containing calcium ions and high concentrations of barium ions, a series of spike depolarizations occurred and that each spike depolarization episode was coincident with a short-lived reversal of ciliary activity (Fig. 6). It should be noted however that the J.P.N: 16 l--E

72

M.J. DOUGHTYand S. DRYL

frequency of spike depolarizations and ciliary reversal events in microelectrode-impaled cells was, at 26 cycles/min, far smaller than the ciliary reversal episodes measured in free-swimming cells under the same conditions (83 cycles/min). Machemer and Eckert (1973) analyzed, through use of high speed cinematographic techniques, the ciliary activity in microelectrode imptaed cells following stimulation of the cells by injected current (delivered via a second intracellularly located microelectrode) of constant duration (10 msec) and varying amplitude ( >/10- to A). With increasing strength of injected current, the rate of rise of the regenerative depolarization increased (see Section 4.4). Machemer and Eckert (1973) also found that the duration of the ciliary reversal response was related to the rate of rise of the regenerative response with an approximate exponential relationship being shown between the two. The duration of the reversed beating showed a linear relationship to the amplitude of the regenerative depolarization (millivolt depolarization-resting potential to peak). Ciliary reversal did not occur without the regenerative depolarization. The ciliary response was only seen if the membrane responded to current injection with this typical regenerative spike depolarization and did not occur if the current intensity was only sufficient to induce an electrotonic shift in the resting potential. Doughty and Dodd (1976) report transient changes in the spectral absorbance of the potentiometric dye, diS-C3(5), bound to P a r a m e c i u m following stimulation of the cells with K ÷ ions. The amplitude of these spike transients in the spectral signal increases with increasing KC1 concentration (Doughty, unpublished; Fig. 31). Since similar spectral transients were shown following stimulation of P a r a m e c i u m with Na ÷ or Ba 2 + and not seen following stimulation with Ca 2+, Mg2 ÷ or choline chloride, the spectral changes may represent the regenerative depolarization occurrent in free-swimming cells following stimulation with K + ions, despite the long duration of the absorbance changes. Naitoh et al. (1972) showed that the amplitude of the regenerative depolarization (given to 2 msec, 10-9A current injections) was unaffected by the extracellular sodium ion concentration (Fig. 52), increased linearly as a function of the logarithm of the extracellular potassium ion concentration (Fig. 53a), and was most prominently dependent on the extracellular calcium ion concentration with the slope of the plot being close to that predicted for a membrane showing pure calcium ion conductance (Fig. 53b), The only other inorganic cation salt that was found to significantly affect the regenerative

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Fro. 52. Electrical responses of the Paramecium surface membrane. Effects of ions on the passive electrical properties and active responses to depolarizing stimuli. Resting potential (resting level) and amplitude of regenerative spike potentials (overshoot: induced by current injection) in Paramecium eaudatum in the presence of varying concentrations of different cation chloride salts. Cells in 1 mM Tris, 1 mM CaCI2, pH 7.2 18-21°C. Cells impaled with both stimulating and recording microelectrodes, current clamp. From Naitoh, Eckert and Friedman, 1972. Reproduced with permission J, experimental Biology/Cambridge University Press.

CONTROL OF CILIARY ACTIVITY IN

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FIG. 53. Electrical responses of the Paramecium surface membrane. Effects of KCI (left panel) and CaCI~ (right panel) on the resting potential and amplitude of the regenerative spike potentials [induced by current injection) in Paramecium caudatum. Cells stimulated with 2 msec current pulses of supramaximal intensity. Top of each section shows potential (Vm) and first derivative (Vm) recordings. Cells in 1 mM Tris, pH 7.2 18-21°C. From Naitoh, Eckert and Friedman, 1972. Reproduced with permission J. experimental Biology/Cambridge University Press.

spike amplitude was manganese, while other cations, having little effect, followed a series of Mg 2+ > Cs + > Rb + > Co 2+ > NHZ > Li + = 0 in the presence of 1 mu CaC12 (Fig. 52). Neither the membrane resting potential nor the regenerative spike amplitude was affected by procaine (Naitoh et al., 1972). Following perfusion of Paramecium, held in a chamber by recording microelectrodes, with NaCI solutions, a train of spike potentials are seen shortly afterwards. These depolarization episodes were apparently associated with ciliary reversal episodes. The spike depolarization events were unaffected by the presence of tetrodotoxin (Satow and Kung, 1974). The regenerative depolarization of the Paramecium membrane would therefore appear to be dependent on extracellular calcium ions and that the inward current is carried by calcium ions rather than sodium ions despite the all-or-nothing nature of the depolarization under some circumstances. The regenerative depolarization, in veiw of the alterations in activity of the demembranated cilium by calcium ions (see Section 4.5), would therefore appear to be a requirement per se for any ciliary reorientation leading to a reversal of the effective power stroke of the cilium and thus reversed swimming behavior of the cell, i.e. for counterclockwise reorientation of the cilium (see below). The regenerative spike amplitude is graded with respect to the applied stimulus, i.e. the injected current intensity (Naitoh et al., 1972; Machemer and Eckert, 1973). The same approximate relationship holds for the concentration of extracellularly applied cations and the duration of the sensory (ciliary reversal) response (see Sections 4.1 and 4.2). Machemer (1974a) found that all changes in the beat frequency of the cilia in reverse mode were clearly related to the angular orientation of the cilia, i.e. the beat direction for the effective power stroke. An increase in beat frequency following depolarization of the membrane was found to be accompanied by a counterclockwise shift in the orientation of

74

M.J. DOUGHTYand S. DRYL

the power stroke (moving towards and eventually readopting the reversed beating mode). At the end of ciliary reversal, the ciliary beat frequency, after an inactive stage, gradually increased back to prestimulus levels with accompanying clockwise shift in orientation. Machemer (1975) found that the time course (latency) of transition of the cilia from forward to reversed beating was related to the rate of membrane depolarization. Application of short duration, 20 mV voltage, ramps (160-650 mV sec- 1 change in membrane voltage) to cells under clamp, effects an "immediate" (latency of less than 4 msec) ciliary reversal response with the cilia beating at enhanced frequency. The ciliary reversal ends immediately after removal of the voltage ramp under these conditions. With application of longer duration voltage ramps (40-2.5 mV sec-1), the latency for ciliary reversal onset increased from a few milliseconds to several seconds, although the ciliary reversal activity still ended within less than 4msec after removal of the ramp. With depolarizations exceeding a rate of 40 mV sec- 1 the cilia thus switch directly from normal beating to a high frequency reversed beating mode whilst with lower rates of voltage change, a depression of forward beating frequency occurs prior to a far more gradual onset of the higher frequency reversed beating activity. Relationships between ciliary activity and the applied voltage were studied through use of long duration command pulses (64 sec), i.e. the voltage ramp was held throughout the period of change of ciliary activity and as such is analogous to an in vivo situation in which ciliary activity goes through a cycle of change under a constantly applied stimulus such as an elevation in extracellular cation concentration. With voltage changes of greater than 6 mV, the majority of the cilia of the cell assumed reversed beating mode within one second after application of the command pulse. The frequency of reversed beating declined over the next 16-32 sec although the cilia are still in reversed mode. After this time, the majority of the cilia assumed forward beating except for very large depolarizations (Machemer and Eckert, t975). Inward current was not measured in these experiments but it was found that changes in outward current reached a maximum in a similar time course as the reversed beating frequency and declined over a similar time course, i.e. the character of ciliary beating appears to show some relationship to the activation and inactivation of late outward (K ÷) current although activation of inward (Ca 2 +) conductance and thus inward calcium current are required for the induction of the ciliary reversal activity. Brehm and Eckert (1978a) made further studies of changes in ciliary beat frequency following application of 3 sec, 10-I°A current pulses. In agreement with earlier studies (Machemer and Eckert, 1975), Brehm and Eckert (1978a) found marked enhancement (2-4 fold) of ciliary beat frequency (in reversed mode) with depolarizations greater than 5 mV from the resting potential. It should be noted that a similar enhancement of ciliary beat frequency is observed with step hyperpoiarizations of similar magnitude (Machemer, 1974a, 1975; Machemer and Eckert, 1975; Brehm and Eckert, 1978a) although the cilia remain in forward beating mode and shift, in a clockwise direction, in orientation. A quantitative study of the ciliary responses to hyperpolarizing stimuli was made by Machemer (1976). Brehm and Eckert (1978a) reanalyzed this data (Machemer, 1976) and found that there existed a closer relationship between inward (electrotonic) current amplitude and ciliary beat frequency changes, following hyperpolarizing voltage changes, than for membrane resting potential. The inward current for the hyperpolarizationinduced frequency changes is essentially independent of the cation composition of the medium. Relationships between inward (regenerative) current and changes in ciliary (reversed) beating frequency remain to be established. The factor controlling ciliary reorientation and frequency changes following depolarization may be the rate of regenerative calcium influx, i.e. the rate of calcium conductance activation (Machemer and Eckert, 1975), although the lifetime of calcium conductance activation (and inactivation) does change as a function of the resting potential (Brehm and Eckert, 1978b) (see following section). For hyperpolarizing step potential changes, the rate of electrotonic inward current does not appear to be significant in determining the ciliary response--the frequency being suggested to be dependent on electrophoretically induced cation permeability changes (Brehm and Eckert, 1978a).

CONTROL OF CILIARY ACTIVITYIN PARAMECIUM

75

Data on demembranated Paramecium "models" indicates that free calcium at less than micromolar concentrations effects an elevation in ciliary beat frequency in forward mode (Naitoh and Kaneko, 1972). Calcium may thus determine the degree of augmentation of ciliary frequency following hyperpolarization of the membrane although the possibility that, for example, other species such as Mg 2+ or H + are responsible cannot be ruled out at this time. The ciliary reversal response in Paramecium follows a regenerative depolarization of the ciliary membrane. This depolarization, which is calcium ion dependent, is a requirement for the ciliary reversal response. The magnitude of the regenerative depolarization, and thus the inward calcium current, is graded with respect to the applied stimulus. The resultant response (change in ciliary activity to reversed mode of beating and the duration of the response) is also graded with respect to the applied stimulus. 4.4. C a 2 + GATING: ACTIVATION, INACTIVATION AND CONTROL

Ciliary reversal is preceded by an elevation in the calcium ion conductance of the ciliary membrane (see Section 4.3). The subsequent influx of calcium ions effects an alteration of mechanism(s) controlling ciliary frequency and power stroke orientation with resultant enhancement of beating frequency and reversal of the effective power stroke. The cell swims backwards for a finite period of time. The extracellular calcium ion concentrations used for most sensory experiments on Paramecium (both behavioral and electrophysiological) are routinely between 10 -4 and 10 -3 M. Data on demembranated "model" cells (Naitoh and Kaneko, 1972) and from iontophoretic injection experiments (Saiki and Hiramoto, 1975) suggest that, in unstimulated resting state, intraciliary calcium ion concentrations in Paramecium are maintained below 10-6M since, at higher calcium ion concentrations, reversal of ciliary activity occurs. This low level of intraciliary calcium ion activity is believed to be maintained through the activity of a membrane site "Ca-ion pump" (Eckert, 1972; Machemer, 1974a; Doughty, 1978a, b). Due to the extracellular calcium ion concentrations, a large electrochemical gradient for calcium ions between the medium and the intraciliary space can be expected. Thus, on activation or elevation of the calcium ion conductance of the ciliary membrane by suitable depolarization, calcium ions can be expected to move rapidly down the electrochemical gradient into the intraciliary space. The influx of calcium ions results in continued depolarization of the membrane. The depolarization is thus regenerative. Measurements of the character and kinetics of the regenerative depolarization (especially dV/dt measurements) show that the inwardly going current is short circuited shortly afterwards (a few msec) according to the duration and magnitude of the injected current used to elicit the regenerative depolarization in microelectrode impaled cells (Naitoh and Eckert, 1968a; Naitoh et al., 1972; Friedman and Eckert, 1973; Machemer, 1974a, 1975; Eckert and Machemer, 1975; Brehm and Eckert, 1978a, b). The Ca -'+ regenerative depolarization is normally graded with respect to the applied stimulus. The ciliary membrane can, however, respond to injected current or suitable cation stimulation by showing either graded or all-or-nothing electrogenesis. It will be convenient to first of all establish the nature of the depolarization under conditions where the response is not graded since, by current understanding, the graded characteristic normally exhibited by the Paramecium membrane is presumably the result of additional controls that are either bypassed or inactivated under conditions where all-or-nothing behavior is exhibited, rather than the latter activity proceeding by an independent component(s) of the ciliary membrane. Kinosita et al. (1964a, c, 1965) first reported that, upon bathing Paramecium in Ca 2+/Ba 2+ mixtures, the membrane responded with a series of regular spike depolarization episodes. These spikes show a distinct firing threshold and are constant in amplitude after activation. The membrane exhibits all-or-nothing behavior. If Paramecium are bathed in solutions of Ca 2 + (10-3 M) and low concentrations of Ba 2 +, the resting potential is usually stable (Naitoh and Eckert, 1968b). However, in these solutions or in similar

76

M . J . DOUGHTY and S. DRYL

solutions containing slightly higher concentrations of Ba 2 +, the resting potential can shift in a depolarizing direction to reach a critical level (firing level) at which spontaneous firing (all-or-nothing) activity of the membrane occurs. However, if the resting potential is below the firing level, injected current of suitable magnitude is sufficient to induce all-or-nothing behavior. Such induced all-or-nothing spikes show a defined latency and threshold for activation and are identical to those of a spontaneous type. The spikes are not graded with respect to injected current magnitude as are those for the membrane in the absence of Ba 2+ (Naitoh and Eckert, 1968a, b). In the presence of Cao2÷, the amplitude of the all-or-nothing spikes increases as a function of [Ba 2 +]0 suggesting that Ba 2 + are more effective charge carriers than Ca 2+ (Naitoh and Eckert, 1968b). At constant Ba 2+, elevation in I-Ca2+]o, does not alter the amplitude of the all-or-nothing spikes (although the threshold level for firing is altered) (Naitoh and Eckert, 1968b). In the presence of Ca 2+, increase in [Ba2+]0 (i.e. elevation of the Ba 2+ :Ca z+ ratio) results in progressively longer sustained depolarization indicating a delay in the activation of repolarization (rectification) mechanisms. This latter character has been taken as evidence that Ba 2 + cannot substitute for Ca 2 +, at least to the same degree of effectiveness, in activating repolarization mechanisms (Eckert et al., 1976). Increasing [Ca2+]0 at constant [Ba 2 +]o, reduces the duration of the all-or-nothing depolarization episodes but does not convert the membrane to graded control (Naitoh and Eckert, 1968b). Extracellular K + ions (1-4 mM) in the presence of constant calcium (1 raM) and barium (1 or 8 raM) ions, also effects a marked reduction in the duration of the all-or-nothing events as well as reducing the threshold (resting) level towards zero potentials. Application of Na +. Mg 2+ or Mn 2+ has insignificant effect on the injected current induced all-or-nothing events. Procaine HCI (0.1-1 raM) has negligible effects on the resting potential in the presence of barium and calcium ions or the firing level for the all-or-nothing depolarizations. However, procaine, in the presence of high extracellular barium (8 mM), prolongs the duration of the all-or-nothing depolarizations although no effect is seen following treatment with either tetraethylammonium ions or tetrotoxin (Naitoh and Eckert, 1968b). These results collectively indicate that the magnitude of positive outwardly directed current (inactivation current) is reduced in the presence of barium ions and that the graded response is converted into an all-or-nothing type through alteration in this rectification property. The nature of the alteration however remains obscure as is the nature of the charge carrying species since it is unknown if barium ions will induce activation of the ciliary reversal mechanism at the axonemal level. Under normal conditions, inward current appears to be carried by calcium ions. In theoretical terms it can be expected that the membrane conductance for a predominant charge carrying species will be proportional to the difference between the resting potential and the equilibrium potential of the charge carrying species in the sense that the persistence of conductance activation will depend on the magnitude of the relevant electrochemical gradients. Due to the steep electrochemical gradient, for calcium ions, maintained across the plasma membrane (10-a to less than 10-6 M), the calcium equilibrium potential can be expected to be greater than + 9 0 m V and thus the difference between the resting potential and the equilibrium potential (Eca) will exceed 100 inV. The inward calcium current (/ca) would thus be expected to be a steep function of Ec~. In Ca 2 + or Ca 2 + plus K ÷ solutions, such a steep function is found (Naitoh and Eckert, 1968a; Naitoh et al., 1972; Brehm and Eckert, 1978b; Eckert and Brehm, 1979). At room temperature (20-21°C), short duration current injection under voltage clamp, results in activation of lca. Ica is fully activated and then inactivates (i.e. subsides) within a total period of 5-10msec (Brehm and Eckert, 1978b; Satow and Kung, 1979; Oertel et al., 1977). A plot of this early current vs V,, shows a region of negati~ce resistance since progressive depolarization activates progressively larger calcium conductance until maximum calcium current is achieved at 30-35 mV positive to the resting potential (holding potential in these experiments)(Brehm and Eckert, 1978b; Eckert and Brehm, 1979). The early current plot then bends up towards the voltage axis on I-Vplots crossing the axis at an estimated potential far less than the predicted Eca. At all positive potentials (to V,,)

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

77

of greater than 25-30mV, there is a steep rise in outward current (Brehm and Eckert, 1978b; Eckert and Brehm, 1979), i.e. maximal inward current is observed with potential shifts smaller than those required to significantly activate outward current. As noted in previous sections, the amplitude of the regenerative depolarization increases as a function of extracellular calcium ion concentration in a manner close to that predicted by the Nernst equation for a membrane showing pure calcium ion conductance (Naitoh and Eckert, 1968a; Naitoh et al., 1972; Eckert and Machemer, 1973) (Fig. 53b). The Ca 2+ regenerative response is only slightly affected by [K+]o (Fig. 53b)--a sensitivity that supports opinion that the "action potential" in Paramecium is a Ca 2 + rather than K + ion dependent phenomena and favors a concept that the inward Ca z + current (Ic~) was short circuited (inactivated) by outward leakage of K ÷ (Naitoh et al., 1972). This leakage current could be reduced with elevation in [K+]o so thus conveniently explaining the [K +]o-dependent increase in the ciliary response duration as being due to depolarization by K + to induce elevation of membrane calcium conductance (9c,) followed by a [K+]0-dependent inactivation of membrane potassium conductance (gK) (Naitoh et al., 1972; Machemer and De Peyer, 1977). In support of such a hypothesis, a recognized K + conductance antagonist, tetraethylammonium ion, TEA, (Hille, 1967: Armstrong, 1971; Thompson, 1977; Kenyon and Gibbons, 1979), alters active electrogenesis in Paramecium. Extracellularly applied TEA slows the repolarizing phase of the "action potential" (Naitoh et al., 1972) and enhances the amplitude of the regenerative response (Friedman and Eckert, 1973; Naitoh et al., 1972). Iontophoric injection of TEA intracellularly enhances membrane resistance and blocks outward (K +) currents (Friedman and Eckert, 1973). The activation and inactivation of inward current is dependent on the magnitude of the injected current, with the fastest kinetics being observed at potential shifts at which significant activation of outward current has occurred (Schein et al., 1976)--evidence taken to suggest that activation of 9K is a predominant factor contributing to the initial decline of active inward current (Ic,) (Schein et al., 1976). However, the characteristics of inward current activation and inactivation in Paramecium have recently been examined in more detail. Under voltage clamp, Ic~, is found to be fully activated within a few msec (2-15 depending on injected current intensity) and declines over a similar time course, before significant activation of late, outward current (Brehm and Eckert, 1978b; Eckert and Brehm, 1979; Oertel et al., 1977~ Satow and Kung, 1979). Activation and inactivation of lc,, is very dependent on the injected current intensity, being slower at lower current intensities (Schein et al., 1976; Schein, 1976: Brehm and Eckert, 1978b). Peak inward current is achieved at depolarizations of 20-40mV positive from resting potential (Schein et al., 1976; Brehm and Eckert, 1978b; Eckert and Brehm, 1979; Oertel et al., 1977)--i.e. is voltage sensitive. In addition, the kinetics and amplitude of early inward current is very dependent on [Ca 2+ ]o (Eckert and Brehm, 1979; Satow and Kung, 1979). Iontophoric injection of TEA and Cs ÷ (in combination) has little effect on the kinetics or amplitude of lc, (Brehm and Eckert, 1978b) whilst strongly inhibiting late outward currents. Significant sustained inward current is observed in EGTA-injected cells (Eckert and Brehm, 1979); in cells bathed in Sr 2+ or Ba 2+ solutions in the presence of low [Ca2+]o (Brehm and Eckert, 1978b); in cells bathed in La 3 + solutions (as determined from dV/dTtraces; see Friedman and Eckert, 1973) and extracellular TEA does not affect the amplitude or kinetics of early inward current (Satow and Kung, 1979). The voltage sensitivity of early inward current, i.e. the potential displacement from the resting potential at which peak inward current is observed, is unaffected by [Ca2+]o (Satow and Kung, 1979) although in the unclamped or current clamped membrane, since the resting potential, V,,, shifts with increasing [CaZ+]o, the absolute potential at which peak inward current is observed should get progressively more positive. Eckert and Brehm (1979) report that lc, increases with [ Ca2 +]o elevation over two orders of magnitude concentration (10-4-10 - 2 M), although Satow and Kung (1979) provide data to show that the system "saturates" at approximately 10 -3 M [Ca2+]o . It should be noted that in the former case, K-Ca solutions were used and in the latter, Ca-citrate or Ca-K-citrate solutions were used. "Ca-inactivation".

78

M . J . DOUGH'rv and S. DRYL

(Tillotson and Horn, 1978) (decrease in lc~ effected by a depolarizing conditioning prepulse) appears to be present in Paramecium, bathed in K+-free solutions containing either Ca 2 ÷ or Sr 2+. Ba 2÷ however fails to support inactivation indicating that depolarization per se is not responsible for the inactivation. This indicates that lc~ is selfinactivating and perhaps controlled by inward Cai attained as a result of passage of Ca 2 ÷ across the membrane during an early phase of lc~ (Oertel et al., 1977; Brehm and Eckert, 1978b; Brehm et al., 1978; Eckert and Brehm, 1979; Satow and Kung, 1979). Therefore, inactivation of Ica is currently understood to be principally dependent on the entry of Ca 2 ÷ and not on the depolarization per se nor on the change of driving force for the ion flux that would be predicted with change in [Ca2+]o, although V,, will determine at which potential displacement maximum /ca will occur and thus indirectly influence Cai and its action on inactivation of lc~. The action of K~ on the inactivation of lc~ (Satow and Kung, 1979) may in part be explained by an effect of K ÷ on V,,. The role of outwardly directed late currents on calcium channel activity (responsible for /ca) thus remains uncertain. The late currents appear to be predominantly due to activation of potassium conductance, gK, since these currents can be almost completely suppressed by intracellular injection of a combination of TEA + and Cs + (Brehm and Eckert, 1978b; Eckert and Brehm, 1979) and results in linearization of steady state current-voltage relationships (Brehm et al., 1978). Since lc~ is unaffected, such linearization of I - V plots reflects reduction in other currents with K + current being the only certain candidate at this time (Brehm et al., 1978; Schein et al., 1976; Schein, 1976). Iontophoric injection of EGTA has a similar action to TEA/Cs injection but additionally introduces a marked discontinuity in I - V plots at extreme potential displacements (Satow, 1978; Brehm et al., 1978), The action of injected EGTA might indicate that gK is controlled by Ca~ as has been implicated in a variety of other excitable membranes (Meech and Strumwasser, 1970; Krjevic and Lisiewicz, 1972; Meech, 1972; Baker et al., 1973; Meech and Standen, 1975; Standen, 1975; Mounier and Vassort, 1975; Isenberg, 1975; Krnjevic et al., 1975; Clusin et al., 1975; Heyer and Lux, 1976; Clusin and Bennett, 1977). It has been suggested that ~ in Paramecium is controlled by Ca~ (Eckert et al., 1976; Nelson and Kung, 1978; Eckert and Brehm, 1979). However, since in other systems, the action of intracellularly injected TEA has been interpreted in terms of voltagedependent potassium conductance inactivation (Thompson, 1977; Herman and Gorman, 1979; Heyer and Lux, 1976), the mode of activation of gK remains uncertain. We can conclude, at this time, that unless there are normally large calcium leakage currents across the ciliary membrane, that persistent activation of g~: cannot be maintained or regulated by Ca~ since mutants with defective lc~ (absent) show rectification (identical I - V plots at positive potentials in steady state) very similar to normal cells (Kung and Eckert, 1972; Schein et al., 1976; Oertel et al., 1977) and other mutants defective (reduced) in lc~ show only slightly altered rectification (Schein et al., 1976). It should be noted that, if gc~ is inactivated as a consequence of passage of calcium ions through the channel then the effects of EGTA, for example, may be in part due to interference with lc~ inactivation. Therefore, Ic~ in Paramecium would appear to be controlled by Cai or the passage of calcium ions and not significantly dependent on ~ . The potassium conductance thought to be primarily responsible for late currents (since available evidence, albeit scanty, suggests that the Paramecium membrane is relatively anion insensitive) appears to be in part controlled by Ca~ but not exclusively so. The calcium sensitive component of potassium conductance activation may be in part responsible for conferring graded character to the normal membrane active electrogenesis. This latter conclusion is supported by the following experimental observations: iontophoric injection of either sodium citrate (Kamada, 1938), potassium citrate (Saiki and Hiramoto, 1975) induces ciliary reversal in Paramecium bathed in calcium ion containing solutions. Iontophoric injection of another calcium ion chelating agent, EGTA, causes a series of sustained plateau depolarizations near zero potential level (Brehm et al., 1978) with the cilia remaining in reverse for the entire duration of the plateau. A reduction in Ca~ thus appears to convert the membrane from graded to all-or-nothing behavior. Following EGTA injection, the occurrence of

CONTROL OF CILIARY ACTIVITY1N PARAMECIUM

79

spontaneous all-or-nothing behavior declines to zero after a short time. Subsequent current injection elicits all-or-nothing behavior and with large depolarizations, the all-ornothing spike depolarizations are converted into depolarizations with a developed plateau lasting up to 20 sec (Brehm et al., 1978). Elevation in extracellular KCI prolongs the rate of activation and inactivation of all-or-nothing spikes in EGTA injected cells (Brehm et al., 1978). The amplitude of the spikes is unaffected by [K÷]0 or [Na+]0 (Satow, 1978; Brehm et al., 1978) but increases as a function of [Ca2+]o (Satow, 1978; Brehm et al., 1978) in common with Ba 2÷ treated cells (Naitoh and Eckert, 1968b). Ca-inactivation does not appear to be supported by Ba 2÷ (Brehm and Eckert, 1978b). However, the opposite actions of [K÷]o on all-or-nothing behavior in Ba 2 ÷ treated cells and EGTA injected cells indicates that, although Cai plays a significant role in control of the response character of the membrane, a simple mechanism is unlikely. In view of the different voltage sensitivities of Ba 2÷ treated and EGTA-treated cells, rectification properties that determine normal graded electrogenesis are probably regulated by both V,, and Cal as well as the net electrochemical gradient for K ÷ ions. Discrete compartmentalization of calcium ions would appear to play a critical role in the control of ciliary activity, whether this be at the surface membrane (screening effects), in the intraciliary space (Ca control of ciliary orientation) and in adjacent regions (Ca control of regenerative response character). It is significant, however, that all processes appear to be controlled by two cations alone (Ca 2÷ and K÷). Before a suitable understanding or explanation can be found to explain both the nature and control of Ica and the interrelationships between graded and all-or-nothing electrogenesis in the P a r a m e c i u m membrane, further studies will need to be carried out. Behavioral studies and electrophysiological analysis indicates that the ciliary reversal types induced by stimulation of cells with either K ÷, Na ÷ or Ba 2÷ are effected by different mechanisms, although the final consequence of the stimulation is presumably to elevate the ciliary membrane conductance to calcium ions. Indications that the mechanisms are subtly different come from behavioral studies on the cell. Controlled treatment of P a r a m e c i u m with low concentrations of a cross-linking reagent (glutaraldehyde, Doughty and Dodd, 1978; bifunctional imido esters, Doughty, unpublished) results in differential alteration of Na + and K ÷ induced behavior of the cell. Treatment of Paramecium with K ÷ and glutaraldehyde results in complete suppression of ciliary reversal behavior for many hours, whilst treatment with Na ÷ and glutaraldehyde effects overreaction to stimuli (Doughty and Dodd, 1978). K+-induced ciliary reversal behavior (continuous ciliary reversal sequence) is unaffected by cholinergic antagonists (gallamine and tubocurrarine), but is suppressed by agonists (nicotine) (Doughty, 1978c) whilst Na÷-induced ciliary reversal (PCR behavior) is suppressed by antagonists and enhanced by agonists (Doughty, unpublished). The nature, role or significance of either the cholinergic sensitivity or the differential action of the cholinoceptive reagents remains to be elucidated. In conclusion, current evidence indicates that three distinct electrophysiological events occur in the P a r a m e c i u m membrane: (1) Depolarization induced activation of an inward calcium current, Ic~. Ic~ is, at least in part, selfinactivating and is responsible for elevation of the free calcium ion concentration (activity) in the intraciliary space to effect a change in ciliary beat frequency and orientation. (2) Voltage-dependent and/or calcium-dependent activation of outwardly directed potassium current, IK. (3) Intraciliary calcium, Cai, activation/inactivation of mechanisms determining graded or all-or-nothing electrogenesis in the membrane--the former mechanism being the physiologically relevant one. The interrelationships of (2) and (3) with (1) remain to be established. The kinetics and gK activation and inactivation with respect to Ca~ would appear however to be a dominant factor in controlling (2) and (3). 4.5. C a 2 +-MODIFICATION OF THE CILIARY MECHANOCHEMICAL CYCLE

At the present time, the mechanisms by which Ca 2 ÷ ions alter the ciliary mechanochemical cycle to effect changes in ciliary beat frequency and the relative orientation of

80

M.J. DOUGHTY and S. DRYt.

the ciliary power stroke are unknown. We have however several reports on the gross effects of calcium ions on the ciliary axoneme activity and on the fine structure of the axoneme and some clues as to the localization of labile sites within the axoneme where C a 2+ may act. The ciliary axoneme (i.e. a demembranated cilium either still attached to the cell or isolated from the cell) can be activated in the absence of the ciliary membrane so allowing studies of the axonemal mechanochemistry in the absence of membrane control. Seravin (1961) reported that several ciliated protozoans could be treated with low concentrations of a detergent (saponin) with resultant loss of ciliary activity and thus cellular motility although the cilia remain attached to the cells. On transfer of these extracted cells into solutions containing high concentrations of adenosine triphosphate (ATP) and divalent cations effects reactivation, i.e. the cells start swimming again. Seravin (1961) thus proposed that each individual cilium contained its own energy-utilizing contractile system and the activity of this system was responsible for cellular locomotion. This study prompted a search for contractile proteins in cilia of Tetrahymena (Burnasheva et al., 1963, 1965). Prolonged exposure of Paramecium for several days at 4°C to high concentrations of glycerol also produces non-motile cells that can be subsequently partially reactivated with ATP. Glycerol treated cells, after transfer to ATP solutions, although failing to become motile again, still possess the mechanisms for ciliary reorientation since the cilia can be placed in either forward or reverse mode orientation by the absence or presence of calcium ions (Naitoh, 1969). Treatment of Paramecium with very low concentrations of a non ionic detergent, Triton X-100, results in destruction of the functional integrity of the plasma/ciliary membrane as evidenced by loss of membrane potential and loss of ATP into the medium (Naitoh and Kanelo, 1973). Such "model" Paramecium, obtained by Triton treatment, can be reactivated by transfer to solutions of ATP and Mg 2÷ ions. At a constant concentration of ATP and in the absence of free calcium ions, the cilia of the reactivated cell beat in forward mode and the cell swims forward. The ciliary beat frequency, and thus the swimming velocity of the cell, increases as a function of Mg 2÷ concentration (Naitoh and Kaneko, 1973) (Fig. 54). Triton-extracted sea-urchin sperm (flagella) can be also reactivated with MgATP 2- (Gibbons and Gibbons, 1972). For both Paramecium "models" and detergent extracted sea-urchin sperm, ATP is the only nucleoside triphosphate capable of supporting motility and all other nucleoside diphosphates are ineffective apart from ADP (see later). Similar results for reactivation have been found for isolated, glycerol-extracted Tetrahymena cilia (Wincur, 1967; Saavedra and

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FIG. 54. Ciliary mechanochemical activity in Paramecium: Sensitivity of adenosine triphosphatedependent activity on magnesium ion concentration. Effect of MgC12 concentration on the ciliary beat frequency (0) and cell swimming velocity (O) of demembranated (Triton-extract¢~l) "models" of Paramecium caudatum reactivated in a solution containing 4ram ATP, 3 mM EGTA, 10mr,I Tris-maleate, pH 7.0 19-21°C. Redrawn from Naitoh & Kaneko, 1973 with permission of Dr. Naitoh and J. experimental Biology/Cambridge University Press.

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

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[co](., FIG. 55. Ciliary mechanochemical activity in Paramecium: Sensitivity of MgATP 2+-dependent activity to Ca 2 + concentration. Effect of free calcium ion concentration on the cell swimming velocity and swimming direction of demembranated "models" of Paramecium caudatum reactivated in a solution of 4 m u ATP, 4mM MgCI2, 10mM Tris-maleate, 50mM KCI, pH 7.0 plus various concentrations of Ca 2÷ obtained either by use of CaCI2: EGTA mixtures or by addition of CaCI 2 19-21°C. From Naitoh and Kaneko, 1972. Science 176, 523-524. Copyright-Association for the advancement of Science.

Renaud, 1975). The isolated cilium can function in isolation from the cell. Ciliary reorientation can be effected in glycerol-treated Paramecium, in the presence of ATP (but not other nucleosides), by raising the concentration of calcium ions (10 mM) or by addition of low concentrations of zinc ions (10-5 M) (Naitoh, 1968). Calcium ions, again at millimolar concentrations, interfere with reactivation of Triton-extracted sea-urchin sperm flagella by MgATP 2- (Gibbons and Gibbons, 1972). For Paramecium "models" (prepared by extraction of the cells with Triton X-100), in the presence of optimum concentrations of MgATP 2- required for reactivation, micromolar concentrations of free calcium ions effect reorientation of the cilia and the reactivated models swim backwards instead of forwards (Naitoh and Kaneko, 1972) (Fig. 55). Maximum velocity rearward swimming was found to occur at approximately 5 x 10-6 u with the threshold for ciliary reversal occurring at I0-6 M. A similar threshold concentration of calcium ions for induction of ciliary reversal in Paramecium was found by Saiki and Hiramoto (1975) from inotophoric injection methods. The axonemal free calcium ion concentrations are thus controlled by the ciliary membrane since Paramecium fail to show ciliary reversal when presented with a calcium ion stimulus (Mast and Nadler, 1926; Oliphant, 1938). As a result of the activity of the ciliary membrane, therefore, intraciliary free calcium ion concentrations are apparently maintained below 1 0 - 6 M in the absence of stimulation since the cells swim forwards under these conditions and the threshold for induction of ciliary reversal, at the axonemal level, is 10 - 6 M. The regenerative depolarization therefore delivers at least sufficient calcium ions into the intraciliary space to raise the concentration above that required to induce reversal. The role of the membrane in determining the intraciliary space calcium ion concentrations is illustrated by studies on a mutant cell. The pawn phenotype swims forwards in the absence of stimulation but, unlike the wild-type cell, fails to show ciliary reversal behavior (backwards swimming) in response to K + stimulation (Kung, 1971a, b). Electro-

82

M.J. DOUGHTY and S. DRYL

physiological analysis shows that, although steady state current-voltage relationships appear normal, the membrane fails to respond to injected current and only electrotonic shift in membrane potential is observed rather than the regenerative depolarization (Kung and Eckert, 1972). However, "models" of pawn cells can be prepared. These models swim forwards in MgATP 2- solutions and backwards in similar solutions containing greater than 10-6M free calcium ions (Kung and Naitoh, 1973). The lack of regenerative depolarization thus indicates lack of calcium influx into the intraciliary space. The influx of calcium ions alters both the beat frequency and relative orientation of the ciliary power stroke affecting the swimming velocity and direction of swimming of the "model" Paramecium. Analogous studies on the calcium ion sensitivity of the ciliary mechanochemical cycle have been carried out on isolated cilia of Tetrahymena. Glycerol or Triton treated cilia can be pelleted by moderate centrifugation, in graduated tubes, to give a characteristic "pellet height". In the presence of ATP, the pellet height increases (Raft and Blum, 1966). In the presence of constant concentrations of ATP (1 mu) and Mg 2+ (2.5 mM), addition of calcium ions effects a further marked increase in the pellet height (Blum and Hayes, 1977) (Fig. 56). Before trying to ascertain the site and/or mechanism of Ca 2+-dependent control of ciliary (and flagellar) activity, it is necessary to reexamine what is known about the structural components of the ciliary axoneme and the overall activity of the axoneme. As an overview statement, it is generally recognized that the dynein proteins from both cilia and flagella are responsible for conferring motility to these organelles. Several studies, particularly on sea-urchin sperm flagella (Gibbons and Fronk, 1972; Gibbons and Gibbons, 1973, 1976; Ogawa et al., 1977b; Kincaid et al., 1973) and also on cilia from Tetrahymena (Gibbons, 1963, 1965, 1966; Warner et al., 1977) have shown that one form of dynein, dynein 1 (the major protein of the 30S dynein fraction obtained by sucrose gradient fractionation of dynein proteins) constitutes at least the major part of the outer row of arm-like projections extending from the a-subfibers of each of the nine peripheral doublet microtubules of the axoneme (see Fig. 13). The inner arms are also composed of dynein proteins, but of different subunit molecular weight to dynein 1, which are found in the 30S synein fraction (Gibbons, 1963, 1965; Takahashi and Tonomura, 1978). It is not known if the principal dynein, dynein 1, is identical in ciliary and flagellar axonemes especially since the 30S oligomeric form of dynein(s) is not obtained from flagellar axonemes by conventional fractionation procedures. Mitchell and Warner (1978) showed that KCl-extracted arm dyneins could be almost completely restored to the a-subfiber of KCl-extracted ciliary axonemes in the presence of Mg 2+ ions and other divalent cations. In the presence of ATP, the reconstituted ciliary axonemes (from Tetra-

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CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

83

hymena) disintegrated. Tris-EDTA extracted dyneins from Tetrahymena have also been found to recombine with both a- and b-subfibers of the stripped axonemes (Takahashi and Tonomura, 1978). On addition of ATP, detachment of the arms from the b-subfiber occurs (Takahashi and Tonomura, 1978) and this activity presumably forms the basis of the sliding disintegration characteristic of ATP-treated axonemes. At least in Tetrahymena ciliary axonemes, the inner and outer arms do not appear to occur at the same level: although the inner and outer arms are spaced some 23-24 nm apart, they appear to be staggered in longitudinal disposition (Chasey, 1972). A similar staggered arrangement of the inner and outer dynein arms is apparent in flagellar axonemes (Amos et al., 1976) and metazoan gill cilia (Warner, 1976). In flagella, both inner and outer arms can serve as sources of mechanochemical activity to drive inter-doublet sliding, in sea-urchin sperm flagella, the beat frequency drops as the outer dynein arms are extracted with KC1. When all of the outer arms are extracted, the beat frequency approximates 50~o of that seen in non-extracted axonemes although the waveform remains unchanged (Gibbons and Gibbons, 1976). Addition of the KCl extract back to the axonemes restores the arms and the beat frequency back to nearly normal (Gibbons and Gibbons, 1976). Removal of the outer arms by NaC1 extraction of sperm flagella does not apparently alter the ability of the axoneme to undergo sliding disintegration following trypsinization and subsequent exposure to MgATP 2- (Hata et al., 1980) indicating that the inner arms have a similar, if not identical, function as the outer arms and that the total number of active arms determines the rate of sliding in the intact axoneme (Zanetti et al., 1979). It should be noted, however, that it is unknown if both sets of arms are active simultaneously. Labelled antibodies, raised against a trysin fragment of dynein 1 of sea-urchin sperm flagella, associate with the distal ends of the outer arms and are not found associated with the axoneme if dynein outer arms are extracted prior to treatment of the axoneme with labelled antibody (Ogawa et al., 1977). The antibody inhibits dynein enzyme (ATPase) activity in vitro (Ogawa and Mohri, 1975). However, the antibody, in a dosedependent manner, could effect 100~o inhibition of the MgATp2--induced sliding disintegration suggesting that dynein 1 alone and not dynein 2 was responsible for the fixed polarity (base-tip) sliding of the peripheral doublet microtubules (Masuda et al., 1978). The antibodies raised against dynein 1 are specific for this dynein and do not inhibit dynein 2 (another distinct dynein found in flagellar axonemes: Ogawa and Gibbons, 1976) (Ogawa and Gibbons, 1976; Masuda et al., 1978) and since sliding disintegration can occur in the absence of dynein 1 (Hata et al., 1980) it would appear both that dynein 2 is sited elsewhere than the outer arms and that inactivation of dynein 1 associated with activity may influence (inhibit) dynein 2 associated activity. Axonemal bending, at least in flagellar axonemes, is possible with only inner arms (Gibbons and Gibbons, 1973, 1976; Bacetti et al., 1979) or only outer arms (Bacetti and Dallai, 1978). The 9 + 2 anoneme is both motile and the predominent naturally occurring form (Phillips, 1974). Structural variants on the 9 + 2 theme are widespread however (Phillips, 1974) and although 9 + 0 flagellar axonemes (of Chlamydomonas which lack both the radial spoke system and the central tubules) are immotile (Witman et al., 1976, 1978), the presence of a highly organized radial spoke/central pair system typified by the normal 9 + 2 axoneme does not appear essential for motility (Phillips, 1974). Even in nonmotile flagella, the peripheral doublet sliding disintegration, induced by MgATP 2- following trypsinization of the axoneme, still occurs (Witman et al., 1978) indicating that those axonemes that contain either inner or outer arms can retain functional motile elements. Thus, according to the sliding microtubule hypothesis of ciliary and flagellar motion (Satir, 1972; Brokaw, 1972) as currently interpreted (Warner and Satir, 1974; Satir, 1974b; Witman et al., 1976, 1978), axonemeal motion is effected as a result of a continuous MgATP 2--utilizing mechanochemical cycle involving a relative sliding of the peripheral doublet microtubules with respect to each other. The bending of the ciliary axoneme, such that the normal three-dimensional profile is effected, is currently considered to be effected as a result of a continuous (or discontinuous) cycle of interactions, based on the radial spoke linkages, between the peripheral doublets and the central

84

M.J. DOUGHTYand S. DRYL

sheath enclosing the central pair of microtubules. This current conclusion may require substantial revision since bending motion is apparently possible in the absence of a central sheath. Nakamura and Kamiya (1978) report bending motion in groups of 4 peripheral doublet microtubules of Chlamydomonas axonemes where the groups may or may not have contained a central pair. Therefore, either other structures must be implicated in the control of the sliding to bending process (e.g. the outer singtet microtubules surrounding the peripheral doublet microtubules in 9 + 9 + n axonemes: Phillips, 1974) or the radial spoke and sheath arrangement is required to perform a regulatory event related to calcium ion control of the axoneme motion characteristics. The microtubule sliding-disintegration process is that observed in either ciliary or flagellar axonemes that have first been demembranated with nonionic detergents and then usually treated with a proteolytic enzyme (trypsin) to digest all structures between the peripheral doublets (apart from the "arms") and across the matrix space between the peripheral doublets and the central sheath (Summers and Gibbons, 1973). Subsequent addition of ATP induces a relative sliding of the peripheral doublets with respect to each other such that the axonemes either slide apart (Summers and Gibbons, 1971, 1973; Sale and Satir, 1977; Satir and Sale, 1977) or fray apart in a characteristic manner (Sale and Satir, 1976). These processes can occur either in completely digested axonemes or, to a certain extent, in partially digested axonemes (Brokaw and Simonick, 1977). Warner and Mitchell (1978), in fact, report that trypsin treatment is not required for sliding-disintegration to occur in ATP-treated Tetrahymena ciliary axonemes. It is unknown why the dynein arms are relatively resistant to proteolysis in situ since they are degraded in vitro after extraction (Hoshino, 1977a; Ogawa, 1973). ProteolyticaUy digested axonemes do not bend to any appreciable extent, i.e. as a result of the digestion, it would appear that the structures or mechanisms that usually effect generation or control of active bending are destroyed. Since ciliary anf flagellar "orientation" appears to be a Ca 2 +-dependent process, it is necessary to note that no studies have been carried out to see if digested axonemes will bend or show different types of sliding-disintegration under physiological conditions, i.e. MgATP 2- + micromolar concentrations of calcium ions ( + u n k n o w n factors?). It is interest to note that isolated intranuclear spindles from protozoa and from sea-urchin eggs (contain tubulin and dynein-like proteins) apparently can undergo change of polymerization state following addition of micromolar concentrations of Ca 2 ÷ or Sr 2÷ in the presence of ATP (Salmon and Jenkins, 1977). Recent studies on ciliary axonemes however have indicated that, at least in Tetrahymena and metazoan gill cilia, calcium ions do not affect the dynein arm cross-bridge cycle per se at concentrations that may be reasonably considered to be physiologically significant, i.e. micromolar. Warner (1978) reported that the number (~o) of dynein cross-bridges (i.e. links between a- and b-subfibers made by dynein armst was very dependent on the concentrations of Mg 2÷ and ATP to which the axoneme was exposed. In these gill cilia, in the presence of 2 mM Mg 2÷. all doublets are found to be bridged with an 80-95~o efficiency being found for the doublets adjacent to the 5-6 structural link between the number 5 and 6 doublets or those opposite to this bridge. In the presence of an additional 10-4M ATP, the frequency and distribution of cross-bridging changes dramatically: apart from the permanent 5-6 bridge, bridging efficiency between all other doublets is reduced to 20-50~o with minimum bridging being observed at doublets 3-2 and 8-9, i.e. 2 removed from the 5-6 bridge (see Fig. 13). Subsequent studies on Tetrahymena ciliary axonemes confirms that bridging efficiency is dependent on Mg 2÷ ion concentration (Zanetti et al., 1979). In Tetrahymena axonemes, the 5-6 permanent bridge is absent. Numbering of the doublets is therefore based on their position relative to the plane of the central pair of microtubules. By numbering the doublets in this manner, an asymetric distribution of crossbridges round the axoneme is found, as in gill cilia. Both Mg 2÷ and Ca 2 ÷ are essentially equally efficient in promoting cross-bridge activity with 90--95~o activity being observed at > 2 mM divalent cation. Millimolar concentrations of either Ca 2 + (alone) or Mg 2÷ (alone) show differential efficiency in promoting ATP-induced sliding-disintegration: Ca 2 * being only half as efficient as Mg 2 + In the presence of Mg 2 ÷ (2 mM), very high

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

85

concentrations of C a 2 + ( > 3 mM) effect significant impairment of the ATP-induced disintegration although, as acknowledged by the authors (Zanetti et al., 1979), such effects may not reflect the physiological response, and in view of the high concentrations of Ca 2 + compared to ATP (10-4 M). limitation of available ATP may be in part responsible for the observed inhibition. In mussel gill cilia (Walter and Satir, 1978, 1979) elevation in intraaxonemal calcium levels effects inhibition of ciliary activity (arrest response). Similar results have been found earlier (Satir, 1975; Tsuchiya, 1976b; Murakami and Takahashi, 1975). Even high concentrations (10mM) of Ca 2÷ do not inhibit ATP-induced slidingdisintegration of cilia isolated from the gill tissue (Walter and Satir, 1978, 1979). It should perhaps be considered that there is no a priori reason why Ca 2÷ should be inhibitory with respect to cross-bridge activity in either Paramecium or Tetrahymena cilia since, unlike the gill cilia where Ca 2 ÷ effect arrest of the activity, stimulus induced reorientation of protozoan ciliary activity is associated with a marked increase in beat frequency. The differences in symmetry of beat cycle and the stimulus-induced responses of protozoan and metazoan cilia may be in part due to the presence of a persistent 5-6 bridge in the peripheral doublet microtubule arrangement in the metazoan cilia. It is not known whether this bridge contains dynein proteins. The bridge in metazoan cilia is stable (i.e. 100~o cross-bridge activity) under conditions where 10~o of the other crossbridges are present whereas in protozoan cilia, reduction in cross-bridge activity at other than the 5-6 site results in reduction of activity at the 5-6 site although the distribution is asymmetrical. Therefore, although Ca 2 + ions may regulate ciliary activity in Paramecium at the level of microtubule sliding, this regulation does not apparently result from an inhibition of dynein cross-bridge activity. Several authors (Allen, 1968; Warner, 1970; Shimizu and Kimura, 1974; Blum and Hayes, 1977; Doughty, 1979b; Blum and Hines, 1979) have considered that the Ca 2 + ion sensitive sites, in either ciliary or flagellar axonemes, may be located elsewhere in the axoneme. Cytochemical localization of ATPase reaction product has been reported in both cilia and flagella at the spoke/spoke head vicinity (see Section 2.4). While it has generally been reported that the dyneins show Ca 2 + sensitive activity, the stimulation of the enzyme activity by Ca 2÷ is generally less than that achieved with an equivalent concentration of Mg 2+ ions (Burnasheva et al., 1965; Gibbons, 1966; Daiya et al., 1972; Hoshino, 1974, 1975; Zanetti et al., 1979; Doughty, 1979b; Mohri et al., 1969; Ogawa and Mohri, 1972; Hayashi and Higashi-Fujime, 1972; Gibbons and Fronk, 1972; Hayashi, 1974; Watanabe and Flavin, 1976). However, such Ca 2 ÷ stimulation as has been reported is generally in the millimolar concentration range. All available current evidence indicates that in protozoan cilia (Naitoh and Kaneko, 1972; Saiki and Hiramoto, 1975); metazoan cilia (Satir, 1975; Tsuchiya, 1976b, 1977; Murakami and Takahashi, 1975; Walter and Satir, 1979); mammalian cilia (Verdugo et al., 1977; Verdugo, 1980) and flagella (Hollwill and McGreggor, 1976; Schmidt and Eckert, 1976; Besson et a l., 1978; Hyams and Borrisy, 1978; Doughty and Diehn, 1979) change in activity is associated with changes in the intraaxonemal calcium activity within the micromolar range. In the presence of MgATP 2-, millimolar concentrations of calcium ions, immobilize Paramecium "models" (Naitoh and Kaneko, 1972) although Naitoh (1969) reported that very high concentrations of calcium ions (10 mM) could effect reversal of immotile, glycerol-extracted, cilia of Paramecium. CaATP 2- is unable to support motility in Paramecium "models" (Naitoh and Kaneko, 1972, 1973). MgATP 2- is an absolute requirement for ciliary axoneme motion (Naitoh and Kaneko, 1972, 1973; Kung and Naitoh, 1973; Saavedra and Renaud, 1975). Micromolar concentrations of calcium ions alter the density (pellet height response) of Tetrahymena ciliary axonemes in the presence of Mg 2+ and EDTA (Fig. 56) although no Ca2+-dependent alteration of ATPase activity (dynein activity) of the ciliary axonemes is found apparently under these conditions (Blum and Hayes, 1977). In contrast, total Mg2+-dependent ATPase activity of crude salt extracts (dynein extracts) of Paramecium ciliary axonemes shows 'marked Ca: + sensitivity in which low concentrations (micromolar) of Ca 2 ÷ effect inhibition of the dynein activities (Fig. 57) (Doughty, 1979b). Similar inhibition of total dynein ATPase

86

M.J. DOUGHTYand S. DRYL

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FIG. 57. Ciliary mechanoehemical activity in Paramecium: Sensitivity of the activity of the mechanoehemieal proteins (the dynein ATPases) to Ca 2÷ and Mg 2+. Effect of Ca ~÷ (by addition of CaCI2) on the M g A T P ' - dependent ATPase activity of crude extracts of dynein proteins from isolated cilia of Paramecium assayed in 5 mM Hepes-Pipes, pH 8.0 23°C. The different curves show extracts obtained under different conditions. Inset effect of Mg 2÷ on ATPase activity in the nominal absence of free calcium ions, pH 8.0. (Doughty, unpublished). From Doughty, 1979b.

activity is found for salt-extracted dyneins from Euglena flagella axonemes (Doughty and Diehn, 1979, and unpublished). Fractionation of salt-extracted dyneins from Paramecium ciliary axonemes by agarose chromatography in the absence of Ca 2+ yields several fractions, the major one of which shows relative insensitivity to Ca 2 + ions whilst other fractions are either stimulated or inhibited by Ca 2+ ions (Doughty, 1979b). These preliminary results indicate that the dyneins show differential Ca 2÷ sensitivity. The relationship between the agarose separated dyneins and the 30S and 14S fractions obtained by sucrose gradient fractionation of dynein proteins is unknown. The location of these p o t e n t i a l l y C a 2 + sensitive dyneins is also unknown. Relative interactions between spoke/spoke head sites and the central sheath have been demonstrated in bent regions of fixed metazoan ciliary axonemes (Warner and Satir, 1974). Alteration in electrophoretic mobility of some dynein proteins of polyacrylamide gels in the presence and absence of Ca 2÷ ions indicates that some dyneins may undergo changes in conformational state or molecular size in the presence of micromolar concentrations of Ca 2÷ ions (Doughty, 1979b) and could perhaps serve as a basis for a Ca 2 +dependent regulatory event. Since the arm sited cross-bridge cycle may not be the site of Ca 2+ sensitive control of ciliary activity, the Ca 2 ÷ sensitive site may be at the sites where sliding-bending conversion is believed to occur, i.e. at the spoke head--central sheath domains (Blum and Hayes, 1977; Doughty, 1979b). Control of sliding--bending conversion could potentially form the basis of the Ca 2 ÷-dependent reorientation of the ciliary axoneme. A calcium ion sensitivity of cross-bridge activity, of interdoublet sliding activity or dynein ATP phosphohydrolase (ATPase) activity are probably not the only sources or sites of Ca 2÷-dependent control of ciliary activity at the axonemal level. Ciliary axonemes contain low levels of guanosine triphosphate phosphohydrolase (GTPase) activity (as so far assayed: Blum, 1973; Stevens and Levine, 1970; Gibbons, 1966; Doughty, 1979b). However, axonemcs have been reported to contain guanosine nueleotides (Yanagisawa et al., 1968) and a potential regulatory role of GTP has been reported for Tetra-

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87

hymena cilia (Saavedra and Renaud, 1975). At this time, it is unknown if any fine structural alterations occur in the tubulin skeleton during changes in ciliary activity. Both Ca 2+ and GTP have been shown, in a variety of systems, to have a marked effect on tubulin conformation/assembly (Weisenberg, 1972; Rosenfield and Weisenberg, 1974; Solomon, 1976; Arai and Kaziro, 1977; David-Pfeuty et al., 1977, 1978; Maekawa and Sakai, 1978; Farrell and Wilson, 1978). Scanty data is currently available on the effects of tubulin on dynein ATPase activity apart from the several reports that the characteristics of"bound" dynein (to tubulin) are different from those of soluble dynein. Several authors (Hayashi and Higashi-Fijume, 1972; Shimizu, 1975) report alterations in dynein-tubulin re-association such that "binding" was altered by both divalent cations and various nucleotides. Tubulin has been reported to alter the specific activity of 30S dynein from Tetrahymena cilia (Otokawa, 1972; Hoshino, 1977b). Limited evidence suggests that tubulin (or another nondynein component of the axoneme) is capable of altering the kinetics of ATP hydrolysis by dynein preparations (Nakamura and Masuyama, 1977, 1979; Doughty, 1979b; Shimizu et al., 1979). Since ciliary and flagellar activity is dependent of MgATP 2-, an alteration in the availability of either of the two components of the substrate can be expected to alter axonemal activity. Sperm flagella bend at regions of local application of ATP (Brokaw and Gibbons, 1973; Shingyoji et al., 1977) and abrupt removal of ATP induces a rigor state in flagellar axonemes (Gibbons and Gibbons, 1974). Such results indicate that bending activity may be an active, ATP requiring process rather than simply being a consequence of limitation of interdoublet sliding due to the nexin linkages between the peripheral doublets. Recently, Mg 2 +-dependent alteration in ciliary activity in Paramecium "models" in the presence of 10-SM Ca 2+ has been reported (Nakaoka and Toyotama, 1979). Since this alteration is also dependent on the relative ATP concentration, this sensitivity was interpreted as further evidence that the concentration of free ATP 4- may be a determinant for ciliary orientation. At this time, in lieu of any evidence to the contrary, it is assumed that the ATP in the axonemes is of cellular origin and is "transported" to the axoneme across the basal plate in protozoan ciliates. If this is the case, then perturbation of ATP levels in the axoneme by alteration of this process can be expected to be responsible only for long term alteration in axonemal motion. However, the apparent presence of at least three enzymes in either ciliary or flagellar axonemes that are theoretically capable of regulating intraaxonemal ATP concentrations (Yanagisawa et al., 1968; Watts and Bannister, 1970; Otokawa, 1974; Watanabe and Flavin, 1976) indicates that some form of regulation could occur at this level, although the presence of these enzymes as true axonemal rather than cellular components remains to be established (Watanabe and Flavin, 1976). Cyclic nucleotide dependent and stimulated protein kinases have been reported in ciliary and flagellar axonemes (Murofushi, 1973, 1974; Garbers et al., 1973; Hoskins et al., 1972). Their role remains to be determined, especially in view of reports of the effects of cyclic nucleotide phosphodiesterase inhibitors on sperm flagellar activity (Tamblyn and First, 1977; Lindemann, 1978). In conclusion, ciliary and flagellar axonemal activity is MgATP/--dependent and regulated by Ca 2 ÷ ions. This calcium ion sensitivity would not appear to be the result of Ca 2 + interactions at the dynein arm cross-bridge cycle (unless the inner and outer arms show differential Ca 2+ sensitivity due to heterogeneous composition: see Fay and Witamn, 1977). If Ca 2 ÷ alteration of the basic peripheral doublet cross-bridge cycle was the cause of Ca2+-dependent augmentation of ciliary activity and reversal of ciliary activity, it is difficult to envisage, at this time, how Ca z ÷ could effect such a gradual and sequential change unless the kinetics of Ca z+ movement to these sites was specifically regulated (rather than Ca 2+ simply flooding the intraciliary space) and/or that each "arm" had a different composition and thus affinity for Ca 2+ (Doughty, 1979b). At this time there is neither evidence for such control of Ca 2 ÷ movement or for significant heterogeneity in "arm" composition especially since the "arm" in ciliary axonemes appears to be composed of several dynein I subunits (Warner and Mitchell, 1978). The Ca 2 + ion sensitive sites would therefore appear to be sited elsewhere within the axoneme. J.P.N.

16~1

F

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M.J. DOUGHTY and S. DRYL

The identity of these sites and the mechanism(s) of Ca z + regulation of ciliary activity remains obscure. 4.6. RENORMALIZATION OF CILIARY ACTIVITY: EVIDENCE FOR A C a 2 + PUMP AND ITS CONTROL

The ciliary reversal response in either free-swimming cells or microelectrode impaled cells has a finite duration even if the stimulus that induced the response in maintained, i.e. elevation in the extracellular concentration of KCI or continued application of a voltage ramp in electrophysiological experiments. The cell thus has the obvious capability to adapt to stimulation and from a physiological point of view, to adapt to a new ionic environment and thus resumes forward mode ciliary activity and thus forward swimming (FLS) as a result of this adaptation. At the present time, we have insufficient data on the swimming behavior or the swimming velocity of Paramecium in different ionic environments to make any positive statements concerning the swimming velocity of Paramecium before, and after, recovery from stimulation. Several reports indicate that the swimming velocity does change and is maintained in different ionic environments (Andrejewa, 1931; Kinosita et al., 1964a; Nakatani, 1970; Fujii and Asai, 1976; Dryl, 1959, 1961a, b~, (see Section 3.1). Since ciliary reversal in Paramecium is generally accepted to be the result of an elevation in the intraciliary fr.ee calcium ion concentrations, a renormalization of ciliary activity is presumably the result of the opposite activity, i.e. removal of Ca 2 ÷ from the intraciliary space (Eckert, 1972). Three mechanisms could account for this adaptation. Firstly, the ciliary mechanochemical cycle itself is capable of adapting to higher levels of Ca2 +, i.e. by this sort of mechanism, a secondary effect of Ca -,+ would be to alter the regulation of ciliary axonemal biochemistry and as a consequence, a differing threshold for induction of ciliary reversal at the axonemal level would be anticipated. No studies have been carried out to explore this possibility, probably because of a lack of precedent from other contractile systems. Secondly, the Ca 2 + ions that enter the intraciliary space via the ciliary membrane are either sequestered (stored in bound state) or simply passively move or leak out of the axoneme. Thirdly, as proposed by Eckert (1972), the ciliary membrane exerts secondary control of ciliary activity by possessing mechanisms to effect active removal of Ca-' ÷ ions from the intraciliary space. In the latter two cases, movement of Ca 2 + through to the intracellular space could also occur. Ciliary activity recovers rapidly after inorganic cation stimulation of the cell (Mast and Nadler. 1926: Oliphant, 1938, 1942; Kamada and Kinosita, t940: Naitoh, 1968; Doughty, 1978b1. Similar recovery of ciliary activity is observed under maintained depolarizing stimuli in the form of an applied voltage ramp for microelectrode impaled cells (Machemer, 1975: Machemer and Eckert, 1975). The recovery of ciliary activity, according to the strength and nature of the applied stimulus, takes from a few to 100 seconds. However. recovery of ciliary activity (return to a forward beating mode from reversed orientation) can occur on a far more rapid time scale. Ciliary activity can be placed in reversed mode by application of a suitable voltage ramp. However, as soon as the same ramp is removed, the cilia return to forward beating in a period of milliseconds indicating both that the renormalization process is voltage dependent and that passive diffusion of Ca" + from the intraciliary space is unlikely to play any significant role in the renormalization process, unless an alternative mechanism is operative under these conditions. Browning and Nelson (1976a) provide evidence that, at ffC, elevation of extracellular concentrations of K ÷ raises the intracellular Ca 2 + concentrations in Paramecium (45Ca2+ flux measurements). While it is uncertain what percentage of this intracellular Ca 2÷ concentration increase reflects elevation in the intraciliary space Ca 2+ concentration (see Section 3.4), we can conclude that, since such elevation in Ca z * concentration in the cell could not be detected at room temperature, persistence of elevated intracellular Ca 2 + does not occur and that normally Ca: + are removed to permit restoration of forward ciliary activity. The mechanism of removal would appear to be tempera-

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g9

ture sensitive. There is no evidence, to date, that Ca 2 + are actually sequestered within the cilium under normal conditions although complete ciliary matrix occlusion with electron-dense material is observed in metazoan cilia subjected to osmotic shock--a treatment which results in immobilization of ciliary activity (Kilburn et al., 1977). Therefore, following initial influx of Ca 2 + into the intraciliary space due to activation of the ciliary membrane conductance to Ca 2 + (Dunlap, 1977; Ogura and Takahashi, 1976; Machemer and Ogura, 1979), Ca 2+ are subsequently removed such that normal ciliary activity is restored. The intraciliary space free Ca 2 + concentration/activity is therefore believed to be maintained at sub-micromolar concentrations. Since there is currently no evidence for passage of Ca 2 + through to the 4ntracellular space from the intraciliary space across the basal plate (e.g. the presence of Ca 2 +-dependent electron-dense deposits between these two compartments), we conclude that Ca 2 + moves out across the ciliary membrane and that the ciliary membrane posseses mechanisms to effect this transfer. If we consider that Ca 2 + are actively removed from the intraciliary space, there are two basic types of mechanism that could be operative. Firstly, Ca 2 + could be removed by an exchange mechanism dependent on the opposite flux of another ionic species-presumably Na + or K + since, although no exhaustive studies have been carried out, the membrane activity of Paramecium appears relatively insensitive to anions (see Satow and Kung, 1974). Increased Na~- produces a fall in membrane conductance perhaps due to reduction in gK (Naitoh and Eckert, 1968a). Thus, change in g~,. (if occurrent) could lead to a lower Cai and part of total calcium flux activity could be coupled to an electrochemical gradient for sodium ions. Small Z2Na fluxes have been reported in Paramecium that were dependent on [Ca2+]0 (Hansma, 1979). However, Paramecium will survive in solutions nominally free of sodium ions but not in calcium free solutions containing sodium ions (Doughty and Dodd, 1976 and unpublished). Ca Na exchange mechanisms involved in regulation of either internal Ca 2+ or Na + (or both) have been reported in squid axons (Baker et al., 1969; Blaustein, 1974); in barnacle smooth muscle (Dipolo, 1973); heart sarcolemnal membranes (Reuter and Seitz, 1968; Horackova and Vassort, 1979; Reeves and Sulko, 1979) and mammalian synaptosomes (Blaustein and Oborn, 1975; Blaustein and Ector, 1976). External sodium inhibits both [Na+]0-dependent and [Ca 2 *]0-dependent Ca 2+ efflux from ATP-depleted squid axons (Blaustein and Russell, 1975). Na Ca exchange in axons does not appear to be dependent on ATP (at least at high internal ionized Ca) although can be stimulated by ATP (Blaustein, 1976; Dipolo, 1976). Na Ca exchange appears to be membrane-potential sensitive (Baker and McNaughton, 1976a, b; Horackova and Vassort, 1979; Blaustein, 1974). It is obvious that considerable further work will have to be on Paramecium before an Na-Ca exchange mechanism can be ruled out but at this time, we consider that an Na-Ca exchange mechanism at the ciliary membrane level, as a means to renormalize intraciliary free calcium ion concentrations, is unlikely. The principal reason is that the ciliary responses of Paramecium are dominated by K + and Ca 2 + and the relative effects of these two cations are observed essentially independent of the sodium ion concentration of the solution in which the cells are studied (tap water, distilled water, double distilled water, deionized water or buffered solutions in which the pH has been adjusted by use of sodium hydroxide: the sodium ion concentrations in these solutions can be expected to be significantly different). Since K + flux activity in Paramecium appears dominated by outwardly directed current, K - C a exchange can be considered unlikely. Of relevance to any exchange mechanism pssibility is the possibility that the active removal of Ca 2+ could be electrogenic, i.e. involves active charge transfer and contributes to the membrane (somatic or ciliary) resting potential. Oliphant (1938) reported that the duration of ciliary reversal responses, of free-swimming Paramecium to a variety of inorganic cation salts, was inversely proportional to temperature. Both the initial response (CCR) and subsequent ciliary responses (PaCR), given by Paramecium to KC1 stimulation, are temperature sensitive (Doughty, 1978b). As detailed earlier (see Section 4.1), the overall duration of the sensory response (tR) shows an inverse, linear, relationship to temperature (Fig. 43a); the initial CCR response shows

90

M.J.

and S. DRYL

DOUGHTY

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FIG. 58. Depolarization-induced ciliary reversal in Paramecium: Sensitivity to temperature. Frequency of reversed beating cilia in Paramecium caudatum following stimulation with injected current as a function of temperature. Data from Macbemer, 1974a (with permission Dr. Machemer). Cells in I mM Tris, 1 mM KC1, 1 mM CaCI2, pH 7.2. Under these conditions, the change in amplitude of the active depolarization (regenerative response), with stimulus intensities of between 1-1.4.10- 9 A, is less than 5% (Naitoh et al., 1972) thus allowing comparison of all data on temperature sensitivity. Data presented with permission J. comparative Physiology/ Springer-Verlag.

an inverse, but discontinuous, relationship to temperature (Fig. 43b) and the recovery (PaCR) responses show a similar discontinuous, inverse, relationship to temperature as characteristic of the CCR response (Fig. 43c). The inverse relationship of CCR to temperature has been reported by several authors (Oliphant, 1938; Doughty, 1978a; Hildebrand, 1978). The reason for the sensitivity is uncertain but may, in part be attributed to a temperature dependent change in ciliary activity in the lower temperature range. The peak ciliary frequency attained by cilia beating in reversed mode following current injection into microelectrode impaled cells, increases with increasing temperature (Fig. 58) (Machemer, 1974a). An increased beating frequency and thus speed of translational motion of the cell in a rearward direction might be expected to result in a shortened response duration if the magnitude of the calcium influx was constant with respect to temperature (although this latter point is unknown). However, in addition to this change is a marked, temperature-related, alteration in the kinetics of ciliary activity change. This is readily seen in Fig. 40: the rate of decline in ciliary beat frequency, from the enhanced frequency in rearward mode and the rate of return of forward beating are considerably slowed at low temperature (Machemer, 1974a). As the temperature is reduced, the apparent rate of activation of membrane Ca 2 ÷ conductance is reduced (Machemer, 1974a). The temperature effects on the kinetics of ciliary activity change were interpreted as indicative of a temperature-sensitive Ca 2+ extrusion mechanism. Further indications that the renormalization of ciliary activity involves the operation of an active transport system for Ca 2 + come from the sensitivity of Paramecium to a Ca-ionophore antagonist. Ruthenium red blocks Ca z + translocation associated with membrane Ca 2+ ATPase activity, i,e. a Ca-pump (Shamoo et al., 1975; Watson et al., 1971; Ash and Bygrave, 1977; Caroni et al., t977). Ruthenium red, at micromolar concentrations, prolongs the duration of the secondary phases of the ciliary reversal response to K ÷ (PaCR; the recovery response:

CONTROL OF CILIARY ACTIVITY IN PARAMECIUM

91

Doughty, 1978b) (Fig. 47) and also inhibits the activity of a detergent solubilized CaATPase from Paramecium cilia (Doughty, 1978a). Ciliary renormalization thus appears to involve an ATP-dependent Ca-pump (Eckert, 1972; Machemer, 1974a; Andrivon, 1974a; Doughty, 1978b). As discussed above, there are two possible directions in which Ca 2+. may be removed, either across the ciliary membrane, or through to the intracellular space. Several authors have either suggested or implied that ciliary activity in Paramecium may be regulated and linked to the intracellular calcium ion concentrations (Eckert, 1972; Kamada, 1938; Wense, 1939a, b; Saiki and Hiramoto, 1975; Brehm et al., 1978; Patterson, 1978). Data from iontophoric injection experiments certainly indicate that intracellular calcium ion concentrations can affect ciliary activity in Paramecium (Kamada, 1938; Saiki and Hiramoto, 1975; Brehm et al., 1978; Satow, 1978). Similar alteration of flagellar activity by intracellular injection of either Ca 2 + or Mg 2+ has also been reported (Nichols and Rikmenspoel, 1978). However, although the actions of intraceilularly injected EGTA or EGTA-Ca 2+ mixtures have been largely interpreted in terms of a direct alteration in either the intraciliary calcium ion concentrations or the activation of late outward potassium currents, since the microelectrodes are intracellularly located, we cannot be certain that the injected solutions are actually reaching the intraciliary space or are acting directly on the inner face of the ciliary membrane. Brehm and co-workers (Brehm et al., 1978) have suggested that intracellular injection of EGTA may in fact alter the ciliary membrane calcium ion conductance. The question of compartmentalization of the intraciliary space from the intracellular space may require revision of the explanations for the effects of injection of Ca 2 +. The calcium efflux mechanism could conceivably occur across the ciliary membrane or across the basal plate region if the cilia is compartmentalized from the rest of the cell. At this time, the molecular identity of the "Ca-pump" is unknown. In other membrane systems, active transport of both Ca 2 + and Na÷/K + can be intimately associated with the activity of their respective ion stimulated ATPase enzymes (i.e. Ca 2÷ + Mg 2+-ATPase and Mg2+-dependent, Na + + K + stimulated ATPase respectively: Robinson, 1976; Korenbrot, 1977; Shamoo and Goldstein, 1977). For this reason, various studies have been carried out to determine if Ca 2 +-ATPase activity is associated with the excitable (ciliary) membrane of Paramecium. The presence of such an enzyme activity would support a theory that Ca 2 ÷ transport across the ciliary membrane involved the activity of such an energy requiring Ca-pump. Several reports indicate the presence of divalent cation stimulated (Ca 2+ stimulated predominantly) ATPase/phosphatase activities in protozoan cilia or ciliary membrane preparations (Baugh et al., 1976; Doughty, 1978a; Andrivon et al., 1977) but not monovalent cation stimulated ATPase activity or p-nitrophenyl phosphatase (K-phosphatase) activity (Doughty, 1978a and unpublished; Dentler, 1977). Histochemical techniques have revealed divalent cation stimulated phosphohydrolase activity in the vicinity of ciliary membranes of Tetrahymena (Dentler, 19771. In the same cell, unspecified ATPase activity was reported to be present "along the length of the outer fibres running into the basal body and within the kinetesome" (Burnasheva and Jurzina, 1968). Similar kinetesomal localization of monovalent cation stimulated phosphohydrolase activity in Tetrahymena has been reported (Dentler, 1977). Ca2+-ATPase activity is also present in the pellicle of Paramecium (Noguchi et al., 1979). It is unknown, at this time, if any of these ATPase activities are associated with transport activity although the sensitivity of the ciliary Triton-solubilized enzyme to ruthenium red has been taken as an indicator that this enzyme is a "'Ca-pump" (Doughty, 1978a). The regulation of this hypothetical ciliary membrane Ca pump and its activity in relation to the inward calcium conductance activation is unknown. However, it has been speculated that inward Ca fluxes (through a voltage sensitive, gated channel) and outward Ca fluxes (through the pump) are closely coupled through an, as yet unidentified, allosteric mechanism (Andrivon, 1974a, b; Doughty and Dodd, 1978; Doughty, 1978a, b). If the pump is electrogenic, then alteration (inhibition) of pump activity by membrane depolarization would be anticipated and could thus in part serve to explain the longevity of the ciliary reversal response. The close correlation found between the kinetics of change of ciliary.

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beating activity following depolarization and the kinetics of outward IK ÷) current in Paramecium supports an idea that the pump activity is indeed voltage sensitive. The renormalization of ciliary activity appears to involve the active removal of C 2 ~ from the intraciliary space. Current evidence suggests that this is achieved through the activity of a ciliary membrane-sited Ca 2÷ ion pump. A candidate for this Ca-pump is the Ca-ATPase enzyme found in the ciliary membrane. 4.7. CILIARY METACHRONY~ HYDRODYNAMIC VERSUS NEURAL CONTROL? In view of the sensitivity of the ciliary activity of Paramecium to certain recognized "neurochemicals" and agonists/antagonists of their action (see Sections 4.2-4.5), it seems necessary and useful to attempt to reconcile earlier ideas on the control of ciliary metachrony with our current knowledge and views on the control of ciliary activity. The cilium functions as an independent unit (since the isolated cilium can both move and is sensitive to perturbations that alter ciliary activity in vivo). However, each cilium operates in a defined relationship to its immediate neighbors and over the entire field of cilia covering the body surface, there is an organized pattern of activity. The control of the activity of adjacent cilia, and over the entire field of cilia, thus might be controlled by signals relayed from one cilia to another or from some central unit. The concept of an elementary type of external (to the cilia) (neural) control mechanism for control, of ciliary activity developed in the early years of this century. The origin of the "neuroid" hypothesis is uncertain and its development is, perhaps, to the contemporary reviewer, somewhat surprising. Schuberg (1905)documented the presence of fibrillar structures linking the kinetesomes (basal bodies) of the cilia. Since these structures linked cilia in longitudinal rows, it was suggested that they may have a transmission function. Independently, Gelei (1925, 1937a) and Klein (1926) published extensive reports on the presence of these and other fibrillar networks in Paramecium. The network of fibrillar structures revealed by Klein, using a silver nitrate fixation technique, are often referred to as the silverline system. Many workers have studied, and reported the presence of a variety of fibrillar networks in ciliated protozoa over the following ten years (Parker, 1929; Taylor, 1941). The actual "neuroid" hypothesis appears to have been born in 1914 (Sharp, 1914) on little more than a personal notion, and perhaps supported only by the proliferation of papers on the same structures and subject, without any real attempt being made to demonstrate a function towards the fibrillar network. The intricate fibrillar systems, revealed primarily by rather harsh fixation techniques, in both Paramecium (Rees, 1921) and Euplotes (Yocum, 1918; Taylor, 1920) appeared to be directed towards a center that was in close proximity to the nucleus of the cell. It is uncertain whether these early workers actually observed such a delicate fibrillar network in its entirety or drew its organization from partial visualization (Rees, 1921). The neuromotor organization first reported by Sharpe (1914) was localized in the gullet region of the cell (Diplodinium) and not the nucleus. The silverline system appears centered in the gullet region. Taylor (1920) reported that ciliary coordination was interrupted following incision into the membrane and pellicle between kinetesomal organizations in Euplotes but his data was refuted in subsequent similar work carried out on Paramecium (Worley, 1934) and Euplotes (Okajima and Kinosita, 1966). The "neuroid" or "neuromotorium" concept appears to have been developed in apparent ignorance (or disregard?) of most other Paramecium work carried out during the period 1900--1940. The concept appears to have gained common acceptance by many authors (in view of the many papers on this subject in the period 1914-1934), probably because of the many works published on the subject. However, other early experimenters had independently already provided unchallenged evidence against the neuroid hypothesis and it is their evidence that is in part responsible for contemporary rejection of the hypothesis. Jennings and Jamieson 0902) reported that fragments of Paramecium (cells cut into pieces with a needle) showed largely normal ciliary activity. Similar results were reported by other workers for Paramecium fragments (Alverdes, 1922; Worley, 1934; Horton, 1935) and Dileptus (Doroszewski, 1962). Ideas on

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a neuromotor system and a sensitivity of Paramecium to biogenic amines (Wense, 1934, 1935) prompted a search for the neurochemicals and their associated enzymes (Bayer and Wense, 1936a, b; Bullock and Nachmansohn, 1942; Seaman and Houlihan, 1951; Seaman, 1951; Tibbs, 1960; Aaronson, 1963) and exploration of the localization of the enzyme activity (Seaman, 1951; Schuster and Hershenov, 1969). Interest in the neuroid concept persisted until the mid-1960s (Sleigh, 1966; Okajima and Kinosita, 1966). At the present time, we can conclude that, although the role of the kinetesomal fiber network is still unknown, it is most unlikely that they serve to conduct neural type signals between adjacent cilia or ciliary domains. There is no evidence which indicates that the fibrils even have any conductile properties, although their presence is beyond doubt (Lund, 1933; Worley, 1933; Metz et al., 1953: Allen, 1967; Jurrand and Seaman, 1969; Ehret and McArdle, 1974). A full treatment of the evidence against the neuroid hypothesis, its development and later replacement by a mechanical interaction-hydrodynamic theory is outside the scope of this review. The reader is referred to the more extensive treatment of this matter given by Jahn and Bovee (1968, pp. 127-132) and to recent reviews (Sleigh, 1966; Parducz, 1967; Machemer, 1974b). Metachrony is not required for the motility of ciliated protozoa (Alverdes, 1922; Gelei, 1926, 1937b: Grebecki et al., 1967b). However, under conditions where ciliary metachrony is evident, cell swimming behavior is both more regular and generally' faster than in the absence of metachrony (Parducz, 1967; Machemer, 1969, 1974b; Grebecki et al., 1967b). Metachrony can be considered to effect an averaging and optimization of the power output of a field of cilia such that, in motile holotrich protozoan unicells at least, fine control of the motion of the cell through time and space can be expertly effected. In addition, since the normal forward left spiralling swimming mode (FLS) of Paramecium is considered the least favorable on hydrodynamic grounds (Ludwig, 1929; Grebecki et al., 1967b), the metachrony may also play an important role in both development and maintenance of this swimming mode. The true mechanisms underlying ciliary metachrony are still uncertain. However, in view of the marked alterations in both the preservation and type of ciliary metachrony in solutions of higher viscosity than normal (Machemer, 1969, 1972b, 1974b), it appears likely that metachrony is both the result of and maintained by a continuous mechanical and hydrodynamic interaction between adjacent cilia and ciliary rows. The fact that "model" Paramecium show both excellent ciliary synchrony and ciliary metachrony (Naitoh and Kaneko, 1972, 1973) both shows that the activity of the individual cilium is the result of its own mechanochemical cycle and argues against a neuroid transmission occurring at the membrane level. Metachrony is imposed upon a field of cilia by factors which are independent of those ciliary membrane sited mechanisms which control both ciliary beat frequency and orientation.

5. Summary and Perspectives The previous sections will hopefully have served to show that the holotrich ciliated protozoan unicell, Paramecium, is a free-living, motile sensory system capable of temporal perception of a variety of environmental signals. Such chemoreception, as has been so far demonstrated, is believed to be of physiological importance. At least for monovalent cation salts, such perception is reflected in an alteration in the membrane potential of the cells plasma membrane. The relative magnitude of such potential shifts would appear to change the probability of "firing" of the membrane although the ensuing active electrogenesis is graded (with respect to stimulus intensity) rather than all-or-nothing. The excitable response is graded, calcium ion dependent, regenerative depolarization of the membrane. This specific, voltage-dependent activation of the membrane conductance to calcium ions appears to be localized in that portion of the surface membrane covering the cilia. This calcium regenerative response is a prerequisite for the ciliary reversal response of the cell. The activation of the ciliary membrane calcium conductance results in a net influx of calcium ions into the intraciliary space. This calcium ion influx effects ciliary reversal. Counterclockwise reorientation of the many cilia, which effect trans-.

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lational motion of the cell, effects partial or extreme changes in the direction of the swimming of the cell--in the extreme, the cells swim backwards (the ciliary reversal response). Biochemical studies indicate that the influx of calcium ions effects a specific alteration of the mechanochemical processes that control both ciliary beat frequency and ciliary orientation, and in an analogous, but by no means identical, manner the calcium activation of smooth muscle contraction. The excitatory response, at the membrane level, lasts only a few msec and the calcium channels appear then to be inactivated as a consequence of passage of calcium ions through the channels. After inactivation of calcium influx and return of the membrane to a resting potential state, the cell possesses mechanism to effect renormalization of ciliary activity involving restoration of the intraciliary free calcium ion levels to prestimulus concentrations thus permitting return of forward swimming. This renormalization appears to involve a membrane sited calcium ion pump. The return to forward swimming thus represents adaptation of both the excitable membrane and the sensory system, in the presence of a maintained stimulus. Available data indicates that the processes of excitation and renormalization are tightly coupled. Biochemical studies, in addition to further electrophysiological studies, appear to be the next step in the dissection of excitation-contraction coupling in Paramecium. The ciliary membrane appears to contain the calcium channels of principal interest in excitation and renormalization and thus the isolated ciliary membrane (which can be fairly readily isolated in relatively pure form) provides a starting material hopefully suitable and amenable to such biochemical studies. Recent biochemical analyses reveal a complexity (protein composition) of the ciliary membrane and analysis of the mutant ciliary membranes indicates that the molecular activities of the membrane are also complex (since no dramatic compositional differences are present despite the dramatically altered active electrogenesis). The active electrogenesis and renormalization are finely controlled and both inter-related to voltage changes across the membrane. Preliminary evidence poses the somewhat provocative question, whether either of these processes in this unicellular eukaryotic system are mediated through the use of a neurotransmitter molecule usually attributed to the presence of spatial transmission and transduction in differentiated nervous tissues. Paramecium obviously lends itself to considerable further studies on the character and nature of sensory behavior and transduction. There has been recent renewed interest in the phenomenon of galvanotaxis and its control by extracellular calcium ion concentrations (Dryl and Kurdybacha, 1978b; Saito and Asai, 1979) in addition to the calcium dependence of chemosensory behavior (Dryl and Kurdybacha, 1978a). The role of membrane surface phenomena in determination of excitability is being explored. Since most Paramecium species are found in fresh water habitats, it can be anticipated that the excitable membrane will be sensitive to bulk solution changes in ionic strength. Such increases, especially in (cat)ionic strength may specifically or non-specifically alter surface charge domains (Eckert et al., 1976; Brehm and Eckert, 1979). The excitability of Paramecium is influenced, reversibly, by low concentrations of a cationic detergent, cetyl trimethylammonium bromide (CTAB) (Butzel et al., 1960; Dryl and Bujwid-Cwik, 1972a). The reversible suppression of K +-induced ciliary reversal by CTAB (Dryl and Bujwid-Cwik, 1972a, b) could be related to alteration in surface charge in the vicinity of membrane ion channels rather than any specific alteration in ionic conductance analogous to the action of other quaternary ammonium ion compounds. The importance of surface charge and zeta potentials in the chemoreception of Tetrahymena is illustrated by recent studies on lipid replacement in the surface membranes of this cell (Tanabe et al., 1979). The in vivo correlation between cationic stimulation of Paramecium and changes in membrane potential (both shifts in the resting potential and active electrogenesis) remains to be carefully investigated before the links between membrane potential changes and ciliary activity can be quantitatively defined. Studies by Van Houten (1977, 1978, 1979) do however indicate that the relative magnitude of the membrane potential changes determines the relative chemosensory response. A further investigation of the relationships between external cation (and anion) concentrations and the cells' swimming behavior and

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speed is also obviously required. Judicious use of a combination of behavioral and pharmacological methods should provide further insight into the nature of the "chemoreceptors" on Paramecium surface membranes as well as the identity and interaction between such receptors and ion channels controlling ciliary activity (Van Houten, 1978, 1979; Doughty, 1978a, b, 1979). The problem of the kinetics and magnitude of calcium fluxes into the intraciliary space still remains to be addressed. A recent report indicates that less than 50~o of 45Ca2 + uptake, induced by depolarization, into Tetrahymena cells occurs across the ciliary membrane (Kusamran and Thompson, 1980). The earlier differences in ion-uptake reported for wild-type Paramecium and several membrane excitation mutants may not necessarily be due to changes in ciliary membrane permeability. The relatively recent development of defined voltage-clamp microelectrode electrophysiological techniques for Paramecium will allow further dissection of the kinetics of activation and inactivation of early inward current (Ic~) in particular and its control by Ca 2 + and K +. Relationships between the kinetics of change of ciliary activity and the kinetics of inward current activation and its magnitude require further investigation. Further application of membrane potential sensitive dyes may prove useful in this respect and has recently been applied to Tetrahymena (Aiuchi et al., 1980) in an analogous manner to the application of the technique to Paramecium (Doughty and Dodd, 1976). The control of ciliary (and flagellar) activity at the axonemal level, in the first instance, by calcium ions is currently being investigated. The structural complexity of the 9 + 2 axoneme suggests that the molecular interactions effecting fine control of motion will prove to be complex. In addition to the dynein arm based cross-bridge activity control, a secondary cyclic regulatory site may exist (Omoto and Kung, 1979). The combination of cross-bridge activity and other interactions may not occur in a continuous manner. Since the sliding displacement of the peripheral doublets at any position along the length of the axoneme is related to the angular vector in that region (Machemer, 1974b; Satir, 1972; Warner and Satir, 1974), the relative base-tip sliding displacement at the peripheral doublets at any one position can be expected to be different (since the peripheral doublets themselves do not change in length). In metazoan cilia, the sliding displacement appears to occur in a series of discrete steps rather than in a continuous manner (Baba, 1979). An alternative approach to dissection of the control elements determining ciliary (and flagellar) orientation has received considerable attention in recent months. Following early reports that different commercial preparations of ATP give different dynein specific activity values (Gibbons, 1966; Nagata and Flavin, 1975), the presence of an inhibitory substance in some commercial ATP preparations was reported (Nagata and Flavin, 1978). This inhibitor was identified as vanadate (vanadium in + 5 oxidation state) (Canttey et al., 1977; Quist and Hokin, 1978). Vanadate has been found to be a potent inhibitor of relatively purified dynein from flagellar (Gibbons et al., 1978) or ciliary axonemes (Kobayashi et al., 1978); of crude ciliary or flagellar dynein preparations obtained either by salt extraction or dialysis (Gibbons et al., 1978; Kobayashi et al., 1978) or of axonemal ATPase (bound dynein) activity (Kobayashi et al., 1978). Vanadate inhibits the motion of MgATP Ereactivated, demembranated sperm flagella (Kobayashi et al., 1978; Gibbons et al., 1978). All of the above effects are observed around micromolar concentrations of vanadate. High concentrations of vanadate (10-3 M) have no effect on the motility of intact cilia in situ (Tetrahymena: Kobayashi et al., 1978) or flagella in situ (Chlamydomonas: Kobayashi et al., 1978; Euglena: Doughty, unpublished) or sperm flagella in intact sperm (Kobayashi et al., 1978; Gibbons et al., 1978). The vanadate therefore appears to act at dynein sites within the axoneme but is normally unable to cross the plasma membrane of both protozoan cilia and flagellar, or spermatozoa. Vanadate (5 × 10 -6 M) inhibits the sliding disintegration normally observed in trypsin-digested, ATP-treated flagellar axonemes (Sale and Gibbons, 1979). Vanadate appears to act at sites relatively sensitive to proteolytic digestion since, following more prolonged digestion of flagellar axonemes, vanadate, even at high concentrations, does not alter the sliding disintegration (Sale and Gibbons, 1979). These authors suggested that vanadate is altering a control element rather than the ATP-induced detachment of the dynein arm from the b-subfibers. How-

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ever, following more prolonged digestion of axonemes with trypsin, the percentage of arms remaining is more significantly changed than the percentage of intact spokes or nexin links (Summers and Gibbons, 1973). The difference in vanadate sensitivity in preparations digested to differing extents, may in part reflect a lack of critical numbers of functional, sensitive, dynein arms. It can, however, be anticipated that further investigations will yield further insight into the mechanisms controlling ciliary activity. For example, in metazoan gill cilia, vanadate has been used to probe the mechanisms determining ciliary orientation. Very high concentrations (10 mu) of vanadate effect an alteration in the arrest response of the cilia (independently known to be a Ca 2 ÷-dependent response: Section 4.5). Treatment of gill cilia with very high concentrations of Ca 2 ÷ (12.5 mM) in the presence of the calcium ionophore A23187 effects arrest of the cilia in the "hands up" position. Conversely, treatment of the cilia with vanadate induces arrest in a "hands down" position i.e. greater than 90 ° shift in orientation (Wais-Steider and Satir, 1979). It should be noted however that vanadate is by no means a specific inhibitor of dynein ATPase activity although it is an extremely effective one and appears to act at dynein sites in the axoneme to effect changes in axonemal activity. In addition to its now widely recognized inhibition of (Na, K)-ATPase activity in a variety of biological membranes, vanadate has also been reported to be an inhibitor, at similar concentrations, of a variety of other nucleotide utilizing enzymes (adenylate cyclase, phosphatases, some divalent cation dependent ATPases found in a variety of membranes: see O'Neal et al., 1979 for example). In addition, calcium has been reported to protect membrane Ca z ÷ ATPase from inhibition by vanadate (O'Neal et al., 1979). The actions of calcium and vanadate on axonemal activities and on dynein activities would seem of importance at this time before further conclusions can be drawn on the effect of vanadate on axonemal activity. While the vanadate studies on ciliary and flagellar axonemes do not appear to have been specifically directed at the calcium'control of axonemal activities, considerable effort is currently being expended to search for calcium regulatory proteins in ciliated and flagellated cells and more specifically in the ciliary and flagellar axonemes themselves. Calmodulin, one of a class of low molecular weight proteins serving an intermediate regulatory role in a large number of calcium regulatory processes (Wang and Waisman, 1979; Marx, 1979) has been found in several ciliated and flagellated and other unicellular protozoans. Uncertainty, however, exists at this time as to its localization. By biochemical analysis (typically identification by molecular weight and the sensitivity of the electrophoretic migration on polyacrylamide-urea gels), calmodulin has been identified in cell of Tetrahymena (Suzuki et al., 1979; Nagao et al., 1979: Jamieson et al., 1979; Kumagai et al., 1980; Aiuchi et al., 1980). Calmodulin has also been identified in ciliary preparations of Tetrahymena (Jamieson et al., 1979; Aiuchi et al., 1980). Using indirect immunofluorescent techniques (binding of fluorescent ligand-labelled anti-calmodulin antibody), calmodulin has been localized on the ciliary membranes of Paramecium in addition to other cellular sites (Maihle et al., 1979). A specific role for calmodulin in Tetrahymena ciliary axonemes has been proposed by Blum et al. (1980) who provide evidence that calmodulin can enhance the calcium sensitivity of sucrose fractionated dynein preparations and propose that calmodulin confers calcium sensitivity to the dyneins. The widespread occurrence of calmodulin in protozoa may indicate common functions: the presence of the protein (or other calcium regulatory proteins) in both the cilia and cell bodies will make assignment of function difficult. With the identification of calmodulin in cilia, earlier studies on the behavior of ciliated protozoa appear relevant. Since an activity attributed to calmodulins is their ability to bind a class of tranquilizer drugs--the phenothiazines (Levin and Weiss, 1977), the sensitivity of protozoa to these drugs is perhaps relevant to the recent biochemical studies. Stimulation of dynein ATPase activity, by calmodulin in the presence of calcium, is blocked by one of these drugs, chlorpromazine (Blum et al., 1980). Chlorpromazine inhibits the motility of Tetrahymena (Nathan and Friedman, 1962; Guttman and Friedman, 1963). An apparent limited protection of Paramecium, by chloropromazine, against osmotic shock has been reported (Boggs and Pottle, 1971). Dryl and Masnyk (1971) reported that sublethal

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concentrations of chlorpromazine induced a delayed PCR type behavior in Paramecium. The PCR behavior gives way to a very prolonged (20-30 min) CCR behavior after which slow FLS is regained. A short lasting (few sec) ciliary reversal behavior induced by high concentrations of chlorpromazine has also been reported (Schein, 1976b). As with related studies on potential calcium antagonists (ruthenium red, verapamik Ni 2+), considerable further studies are required to establish the specificity of action of chlorpromazine under the conditions tested. With chlorpromazine, we cannot, at this time, dissect ciliary membrane action from axonemal action of the drug but, as with other pharmacological studies, it shows promise as a tool to dissect control of ciliary activity. Parallel with physiological and biochemical dissection of membrane and axonemal function in Paramecium has been the development of heterozygous mutants of Paramecium aurelia (Kung, 1971a, b). Many lines of mutants, principally selected on the basis of altered chemosensory behavior, have been isolated in the last 10 years. Controlled mutagenesis with the point mutagen N-methyl-N'-nitro-N-nitrosoguanidine has produced several types of mutants (for reviews, see Kung et al., 1975: Byrne and Byrne, 1978: Nelson and Kung, 1978). Of principal interest at this time are mutants appearing to be selectively defective in activities related to either calcium or potassium ion channel function (pawn mutants: Kung and Eckert, 1972; Schein, 1976a, b; Schein et al., 1976: Chang et al., 1974, temperature sensitive pawn mutants; Chang and Kung, 1973a, b: Satow et al., 1974; Satow and Kung, 1976a, fast mutants; Satow and Kung, 1976b, TEA-insensitive mutants: Satow and Kung, 1976c; Chang and Kung, 1976: and paranoiac mutants, Satow et al., 1976; Hansma and Kung, 1976). At this time it is unknown if the mutants have altered ion channels, altered characteristics of channel activation or whether the changes in cell behavior and electrophysiology reflects differences in the number or relative numbers of channels operating at any one time. The pawn mutants have however provided powerful independent evidence that change of ciliary activity (reversal) is effected by a voltage dependent activation of ciliary membrane calcium conductance as a first step in the sensory transduction. The pawn phenotype is generally defective in this mechanism and thus fails to show backward swimming behavior in response to cationic stimulation and shows no calcium regenerative response to injected current (Kung and Eckert, 1972; Kung, 1971a, b). The extreme pawn phenotypes lack detectable inward current (Ic,) (Oertel et al., 1977). The axonemal mechanism that determines ciliary orientation however remains functional and apparently normal. MgATP 2 reactivated models of an extreme pawn mutant show ciliary reversal in the presence of calcium above 10 -6 M and exhibit forward swimming behavior in the presence of lower calcium concentrations (Kung and Naitoh, 1973). The conditional pawn (temperature sensitive) phenotypes can be induced by short term (5-8 hr) incubation at non-permissive temperatures (Satow et al., 1974). Membrane excitation mutants have also been produced in Paramecium caudatum (Takahashi and Naitoh, 1978: Takahashi, 1979). Axonemal mutants are also becoming available (Kung et al., 1975: Byrne and Byrne, 19781. The first biochemical studies on a slow swimming mutant of Paramecium caudatum indicate that considerable progress can be expected in this area. Hayashi and Takahashi (1979) found alteration in dynein activity in the slow swimming mutant. With respect to the membrane excitation mutants of Paramecium aurelia, no significant progress in biochemical dissection has so far been made. To date some minor quantitative differences in the protein composition of the ciliary membrane preparations of some of these mutants have been detected (Kung, personal communication; Merkel et al., 1980). Extensive analysis of the lipids of the cells and ciliary preparations of the principal mutant phenotypes compared to the parental stock has also revealed minor quantitative differences (Andrews and Nelson, 1979; Rhoads and Kaneshiro, 1979). It is unknown at this time if any of the differences can be assigned to the principal ciliary membrane electrophysiological defect. However, the work so far carried out provides us with a sound basis for further comparative analysis and also indicates that the expressed molecular defects in the mutants appear to be of a rather subtle nature. Further enzyme-based studies should provide insights into the molecular defects.

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In conclusion, Paramecium shows promise as a research system suitable for molecular dissection of a sensory transduction pathway at an eukaryotic level. Through use of mutants defective in the operation of the voltage dependent calcium channel, molecular dissection of the properties of a gated calcium ionophore should be possible. At this time considerable effort is being expended in both screening mutants and continuing characterization of the purity and stability of the isolated cilia and ciliary membrane preparations. Such studies are a prerequisite for any qualitative or quantitative molecular analysis of this sensory system. We have to acknowledge that although the ciliary membrane appears to contain the calcium channels mediating ciliary reversal, other related ion channels may be present on the somatic membrane--e.g, the rectification (K +) channels (see Machemer and Ogura, 1979). The use of a unicellular organism, of controlled quality, in analysis of a sensory transduction pathway offers several obvious advantages over use of tissues from multicellular orgamsms. Despite considerable progress on the electrophysiology and biochemistry of isolated cell lines from excitable tissues of higher organisms, especially in the recent past, it is anticipated that Paramecium will have a place in such molecular dissection studies of membrane excitation and sensory transduction.

Acknowledgements Our thanks go to our many colleagues who assisted with this review either by providing us with material for inclusion, by giving us permission to use their data or for permitting us to present their data in an alternative form to that originally published. We should also like to thank them for their valuable time given up in useful discussions and exchange of ideas. Our particular thanks go to the following: Drs. CI Andrivon; J. J. Blum; D. Cronkite; I. R. Gibbons; C. Kung; L. Kuznicki; M. Levandowsky; H. Machemer; S. Merkel; H. Mohri; Y. Naitoh; D. L. Nelson; A. Ogura; F. Oosawa; H. Plattner; J. Van Houten; F. D. Warner and T. Watanabe.

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