Control of ciliary activity in paramecium—IV. Ca2+ modification of Mg2+ dependent dynein ATPase activity

Comp. Biochem.Physiol.. Vol. 64B. pp. 255 to 266

0305-0491/79/1001-0255$02.00/0

© Pergamon Press Lid 1979. Printed in Great Britain

C O N T R O L O F CILIARY ACTIVITY IN P A R A M E C I U M - - I V . Ca2+ M O D I F I C A T I O N O F Mg 2÷ D E P E N D E N T D Y N E I N ATPase ACTIVITY MIKE J. DOUGHTY* Department of Molecular Sciences, University of Warwick, Coventry CV4 7A1, U.K. (Received 3 January 1979)

Abstract--1. Dynein proteins were solubilized from demembranated cilia of Paramecium by extraction at high ionic strength. 2. Mg2+-dependent ATPase (EC 3.6.1.3) activity of crude dynein extracts was inhibited by micromolar concentrations of Ca 2+ ions. 3. Sepharose 4B chromatography of the crude extracts yields three dynein fractions. The major fraction contains a single protein and is insensitive to Caz + ions. Two other fractions, both heterogeneous in composition, show opposing Ca2+ ion sensitivity expressed as a Ca2+ dependent alteration in MgATPz- dependent ATPase activity. The Ca2+ ion sensitive forms show altered electrophoretic mobility on native polyacrylamide gels in the presence and absence of Ca 2+ ions. 4. The data provides evidence for a Ca2+ ion dependent concomitent alteration in both molecular form and hydrolytic activity of the dyneins. The results are discussed in terms of a possible molecular mechanism for Ca ion regulation of ciliary activity in terms of the sliding microtubule model.

INTRODUCTION

The free-swimming ciliated protozoan, Paramecium, responds to sudden increases in the concentration of inorganic monovalent cation salts in its environment by showing a temporary reversed swimming behaviour (Mast & Nadler, 1926; Doughty & Dodd, 1978). This behavioural response, which is the result of a 180° reorientation of the effective power stroke of the locomotory cilia (ciliary reversal), is effected as a result of a graded regenerative depolarization of the ciliary membrane (Machemer & Eckert, 1973, 1975; Dunlap, 1977). Inward current is carried by Ca z + ions (Naitoh et al., 1972) to in turn effect an elevation of the intraciliary free Ca 2+ ion concentrations (Eckert, 1972). Membraneless "models". of Paramecium, reactivated with MgATP 2-, swim forwards in the presence of free Ca 2+ ion concentrations below I0-6 M and backwards at higher Ca 2+ ion concentrations (Naitoh & Kaneko, 1972). Intraciliary free Ca 2+ ion concentrations are therefore believed to be maintained in forward swimming cells at concentrations less than that required to effect ciliary reversal (Naitoh & Kaneko, 1972; Saiki & Hiramoto, 1975) through the activity of a ciliary membrane located Ca 2+ ion pump (Eckert, 1972; Machemer, 1974; Browning & Nelson, 1976; Doughty, 1978b). A Ca 2+ ATPase enzyme has been demonstrated by biochemical techniques to be present in the ciliary membrane (Baugh et al., 1976; Doughty, 1978a; Andrivon et al., 1977). Dentler (1977) and Burnasheva & Jurzina (1968) report histochemical localization of phosphatase or ATPase (respectively) reaction product on the ciliary membrane. The rate of Ca 2 + ion influx, through voltage sensitive Ca 2+ ion chan* Present address: Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221, U.S.A. 255

nels in the ciliary membrane (Dunlap, 1977; Ogura & Takahashi, 1976), is believed to determine both the ciliary beat frequency and its relative orientation during the cycle of ciliary reversal activity effected by membrane depolarization (Machemer, 1975; Machemer & Eckert, 1975). The molecular mechanism(s) by which micromolar concentrations of Ca 2+ ions can alter either ciliary (Naitoh & Kaneko, 1972) or flagella (Holwill & McGreggor, 1976) activity is unknown. Ciliary (Gibbons, 1965, 1966)and flagellar (Gibbons, 1965; Mohri et al., 1969; Gibbons & Fronk, 1972; Gibbons & Gibbons, 1972, 1973, 1976) axonemes contain Mg 2÷dependent adenosine triphosphatase proteins termed dyneins (Gibbons & Rowe, 1965). Several studies, particularly on sea urchin sperm flagella (Kincaid et al., 1973; Gibbons & Gibbons, 1976; Ogawa et al., t977) and on cilia from Tetrahymena (Gibbons, 1965; Warner et al., 1977) have shown that one form of dynein (30S or dynein 1) constitutes, at least the major component of, the outer "arms" extending from each of the pair of peripheral nine doublet microtubules of the axoneme. Each pair of peripheral doublets is linked to a central pair of microtubules (enclosed in a central sheath) by radial spokes (Allen, 1968; Warner, 1970). Each spoke has a thickened region for which the term spoke heads is generally used (Warner, 1970, 1976). According to the sliding microtubule hypothesis (Satir, 1972; Brokaw, 1972) as currently interpreted (Warner & Satir, 1974; Satir, 1974; Warner, 1976; Witman et al., 1976, 1978), axonemal motion is effected as a result oi" a continuous MgATp2--utilizing mechanochemical cycle involving a relative sliding of the peripheral doublet microtubules with respect to each other. The dynein arms are thought to be primarily responsible for this process which is observed as a characteristic disinte-

256

MIKE J. DOUGHTY

gration of peripheral doublet organization in demembranated, trypsin treated ciliary (Sale & Satir, 1976, 1977) and flagellar (Summers & Gibbons, 1971, 1973; Witman et al., 1976; Brokaw & Simonick, 1977) axonemes. The bending of the cilium, such that the normal three dimensional profile is effected (Parducz, 1967; Machemer, 1972; Tamm, 1970; Kuznicki et al., 1970) is currently considered to be effected as a result o f a continuous cycle of interactions, based on the radial spoke linkages, between the peripheral doublets and the central sheath (Warner & Satir, 1974; Warner, 1976; Witman et al., 1976, 1978). Extracted dynein proteins from either cilia (Gibbons, 1966; Hoshino, 1974, 1975) or flagella (Mohri et aL, 1969; Ogawa & Mohri, 1972; Hayashi & Higashi-Fujime, 1972; Gibbons & Fronk, 1972; Hayashi, 1974; Watanabe & Flavin, 1976) show optimum activity in the presence of Mg 2 ÷ ions and ATP. However, most of these authors and others (Burnasheva et al., 1965; Daiya et al., 1972) report that dynein ATPase activity can be stimulated by Ca 2÷ ions as well. All available evidence indicates that ciliary (NaP toh & Kaneko, 1972; Verdugo et al., 1977) and flagella (Hyams & Borisy, 1975; Holwill & McGreggor, 1976; Doughty & Diehn, 1979) activity is altered once the intraaxonemal free Ca 2÷ ion concentration, at unknown sites, is elevated above mieromolar concentrations. However, intraaxonemal free Ca 2÷ ion concentrations in the millimolar range inhibit ciliary motion in Paramecium "models" (Naitoh & Kaneko, 1972) and arrest mussel gill cilia (Murakami & Takahashi, 1975; Satir, 1975). It is therefore questionable whether the Ca 2÷ ion sensitivity of the extracted dynein ATPase proteins (in the millimolar range) hitherto reported has any physiological relevance to the reorientation mechanism(s). Gibbons & Gibbons (1972) report an antagonistic action of Ca 2 ÷ ions on M g A T P 2- reactivated sperm flagella and concluded that C a A T P 2- may simply be competing for the same sites as M g A T P 2-. Since the presence of Mg z÷ ions (plus ATP) are an absolute requirement for ciliary motion (Naitoh & Kaneko, 1972; Saavedra & Renaud, 1975), in lieu of a specialized compartmentalization or isolation of the machinery responsible for ciliary reorientation and beat frequency alteration, it is possible that the Ca 2÷ ion sensitivity of the axonemal enzymes or proteins responsible for this change, may be expressed as a Ca 2÷ ion dependent alteration of M g A T P 2dependent activity in an in vitro system. Ca 2÷ ion alteration of Mg 2 + dependent dynein ATPase activity was considered as the most obvious candidate for the Paramecium ciliary "Ca ion motor" discussed in the recent literature (Eckert & Machemer, 1975) in view of the widely accepted role of dynein proteins in conferring motility to axonemes. MATERIALS AND METHODS Cultures Paramecium aurelia-syngen 4, stock 5 ls and Paramecium caudatum Ehb.-stock 1660/2 were obtained from the Cam-

bridge culture collection of algae & protozoa (Cambridge. * Abbreviations used: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane-sulphonic acid; Pipes, piperazine-N,N' bis-(2-ethanesulphonic acid); PCMB, para-chloromercuribenzoic acid.

England). Both organisms were cultured on mixed meadow hay-lettuce infusions (0.1 and 0.4~ w/v respectively) essentially as previously described (Doughty, 1978a). The P. aurella cultures were supplemented with l rag/1, fl-sitosterol and bacterized with controlled quantities of Klebsiella pneumoniae. The P. caudatum infusions were supplemented with 0.3~o w/v CaCOa and 0.05~o w/v hydrolysed caesin. These cultures were not inoculated with bacteria but simply left with loose fitting covers. Mid to late logarithmic phase of growth in 5 litre Ehlenmeyer flasks or l0 litre glass jars was achieved after either 6-8 days or 20-25 days for the two organisms respectively at laboratory temperature. Cells were harvested by the sinter filter technique (Doughty, 1978a). Chemicals

Chemicals were obtained from sources detailed previously (Doughty, 1978a). The oligomyicin, aurovertin and PCMB (all BDH Chemicals, Poole, England) were a gift from Dr Mick Partis of this department. The ATP grade (prepared by phosphorylation of adenosine: Sigma) was specifically chosen in view of a report at the start of this work (Nagata & Flavin, 1975) that some grades of ATP from Sigma contained a strong dynein inhibitor which has recently now been identified as vanadate (Kobayasi et al., 1978; Gibbons et al., 1978). The Hepes and Pipes* buffers were treated by passage through Chelex 100 (Biorad Laboratories, Richmond, CA) to remove heavy metals and Ca 2 + ions prior to addition of any other cations or chemicals. The pH was then adjusted down to the required values by addition of analytical grade HCI. All buffers were estimated contain a background Na (OH) concentration of 6-8 mM as a result of the ion exchange treatment. Deciliation

Washed (1 mM Tris, 0.1 mM CaCl 2, pH 7.1), concentrated suspensions of Paramecium 0-2.107 cells) were deciliated in batches using the salt-ethanol technique previously described (Doughty, 1978a). Cell bodies and trichocysts were removed by low speed centrifugation (~ 2000 g, 45 sec) and the cilia recovered from the supernatant by centrifugation at either 14,5000 (P. aurelia) or 23,5000 (P. caudatum) for l0 min at 4°C (Sorval RC2-B, SS34 head). The cilia from P. aurelia were further purified by differential centrifugation and the final pellet in both cases washed once in buffer (Doughty, 1978a). Membrane removal

Ciliary membranes were removed with 0.025~o v/v Triton X-100 either by vortexing (Doughty, 1978a) or repeated mixing with a pasteur pipette (for P. caudatum cilia) over 10rain at room temperature. Demembranated axonemes were then recovered by centrifugation at either 14,500 or 23,500 0 for 10min at 4°C. Dynein extraction

Dyneins were solubilized by dispersion of the demembranated axonemes in 1 ml of high ionic strength salt solutions (after Gibbons, 1965; Kincaid et al., 1973). The basic extraction buffer contained 0.5 M KC1, 5 mM Hepes, 5 mM Pipes, 5 mM MgCl 2, I mM EDTA with the pH adjusted to either 6.5 or 8.0. Some solutions contained an additional l mM ATP. Extraction was carried out at room temperature, with occasional vortexing of the mixture, over a period of 45 min and then the extracted axonemes recovered by centrifugation at either 14,500 or 23,5000 as above. The .supernatant was then routinely concentrated over an ice bath using Minicon Bl5 microconcentration units (Amicon Corporation: molecular weight cut off of - 5000 daltons).

Control of ciliary activity--IV

Sepharose 4B chromatography The concentrated extract (100#1) was applied to 40 x 0.5 cm columns of Sepharose 4B washed and previously equilibrated with deionized buffer (2.5 mM Hepes, 2.5 mM Pipes, pH 8.0). Elution, at 4°C, was achieved using the same buffer using a closed reservoir system with a flow rate of approx 2 ml/hr. Void volume was determined using blue dextran (M, 2,000,000 daltons). Elution was monitored,on line at 280 nm (L.K. Laboratories Uvicord optical unit) and fractions of 10 drops (~200#1) collected by means of an automated microfraction collector (Gilson TDC 80: Anachem Labs., Bedfordshire, England). Peak fractions from the elution profile were collected, pooled and concentrated on the Minicon B15 units to the required protein concentration using very finely drawn pasteur pipettes to effect quantitative transfer.

257

genase (BDH Chemicals: 150,000 and 37,000); catalase (Sigma: 232,000 and 57,000) (all data from Klotz & Darnail, 1969). Electrophoresis was carried out at either room temperature (SDS gels) of 4°C (native gels) using an initial stacking electrophoresis of 1 mA/gel for 60min followed by electrophoresis at 2.5 o r 3 mA/gel until the bromophenol blue had migrated to within 5 mm of the end of the gel. Gels were stained for protein or ATPase activity as previously described (Doughty, 1978a) except that the ATPase incubation buffer contained 10mM Tris-HCl, 1 mM ATP, 1 mM MgCI2, 1 mM PbCI 2, 20 mM KCI, pH 8.0). Gels were scanned at either 550 nm (Coomassie Blue) or 750 nm (ATPase reaction product stain) using a Gilford spectrophotometer fitted with a linear transporter. RESUL'IS

A TPase assays

Characterization of the salt-extracts

Enzyme activi.ty was determined by measurement of net inorganic phosphate release using the molybdate-malachJ.te green-Triton X-405 method (Doughty, 1978a). Samples (5-20 #1 containing 0.5-14,ug protein) were incubated at 22-23°C in 200#1 of a standard assay buffer containing 2.5 mM Hepes, 2.5 mM Pipes, l mM ATP, 20 mM KCI, pH 8.0 plus other cations as required. The free Ca 2+ ion concentrations in these solutions was not determined but can be expected to be below 10 -6 M in view of the deionization treatment. "Model" Paramecium swim forwards in similar solutions containing 4 mM ATP and 4 mM MgCI 2 in the absence of added EGTA. This bioassay additionally indicates that the levels of free Ca 2+ ions are less than 10 -6 M. The Ca 2+ ion concentrations reported below to alter dynein ATPase activity are those concentrations added to the assay mixtures. The use of CaCI2: EGTA mixtures was purposely not chosen since EGTA at millimolar concentrations may alter dynein activity. Sulfhydryl group protection reagents were left out for similar reasons. All assays on crude extracts were carried out 12-28 hr after preparation after storage on ice. Sepharose chromatography was routinely started within 3-6 hr after preparation and concentration of the extracts.

A careful analysis was initially carried out on the salt-extractable proteins from d e m e m b r a n a t e d cilia of both organisms to verify that the A T P a s e activity was due to the presence of dynein proteins. The results of this analysis are briefly summarized below. Figure 1 shows densitometer traces of isolated cilia, ciliary Triton extracts ( m e m b r a n e fraction) and

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Polyacrylamide yel electrophoresis Gel electrophoresis was carried out as previously described (Doughty, 1978a) except using low concentration acrylamide-N-methylene his acrylamide gels (T = 3.5%, c = 2.5%). Disc gels (7.5 x 0.9 cm) were either prepared containing 0.1% SDS (SDS gels) or without (native gels). Gel solutions and reservoir buffers {40 mM Tris, 20 mM sodium acetate, pH 8.0) were prepared in either double distilled water (SDS gels) or Chelex deionized double distilled water (native gels). The addition of 0.1 mM EGTA to the native gel solutions or the reservoir buffers had no discernible effect on the gel patterns reported. The native gels were routinely prepared containing 1 mM MgCIz and additional CaClz (4-65 #Molar final concentration) where required. Samples for SDS-gel electrophoresis were prepared by addition of 5 #1 of a sample preparation buffer (10% glycerol, 5~o v/v mercaptoethanol, 2% w/v SDS, 1 mM EDTA, 0.5% bromophenol blue, 20 mM Tris), 5 #1 mercaptoethanol (stock solution) plus 5 pl 10% SDS solution and then heated for 1-2 min on a boiling water bath. Despite this treatment however, some of the molecular weight markers used, failed to break down completely into their expected subunits. The slowest migrating band on the gels was taken to be the literature reported native molecular weight. Markers used were BSA monomer (68,000); cross-linked BSA (68,000-406,000), equine apoferritin (Serva Bio~:hemicals: 480,00if--only samples stored in gel buffer at 4°C for several days were reduced to a small subunit after the reduction); bovine thryroglobulin (Sigma: 665,000 and 335,000 plus several minor bands); yeast alcohol dehydro-

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Fig. 1. Representative gel traces to show protein composition of cilia from Paramecium aurelia and associated fractions. Arrow indicates dye front. 3.5% gels plus SDS. Vertical bar is 0.1 absorbance unit at 550nm. (a) Isolated cilia--62 #g; (b) Triton extract (membrane fraction)--36 #g; (c) Demembranated axonemes~44 #g. Arrows and numbers show positions of BSA oligomers.

258

MlgE J. DOUGHTY

demembranated axonemes. The Triton treatment qualitatively removes protein with an estimated molecular weight of 250,000-300,000 daltons (the membrane antigens presumably: Hansma & Kung, 1975). The relative quantity of this protein varied from 10-60~o of the Triton extract--a difference that seemed to correlate with the age of the cells. Late logarithmic or early stationary phase cells showed far s.maller quantities. Subsequent analysis of the initial ciliary supernatant following treatment with a 60--70~ ammonium sulphate treatment (to precipitate the proteins after centrifugation at 23,500g for 30 min) showed that this supernatant contained protein(s) with this molecular weight in addition to a band at ~450,000 daltons. Washing of isolated cilia in the standard buffer used (10mM Tris-maleate, 1 mM MgCI2, 0.5mM EGTA, 20mM KCI, pH 7,1) but not a similar buffer in which MgC12 was replaced by EDTA, also removes the 250,0130-300,000 dalton proteins. As shown in Fig. lc, the demembranated axonemes contain two major groups of proteins with molecular weights that appear to be in excess of 500,000 daltons and a second group around 110,000 daltons. The apparent annomalous migration of this second major group (which are presumably tubulins) cannot be explained. Although complete reduction to subunit size may be the reason for this, this gel pattern was maintained even after boiling of the axonemal samples for 2 min in the denaturing solutions. Native gels of the demembranated axonemes (samples solubilized in extraction buffer) stained for ATPase activity only in a diffuse band (usually partially resolved into 3 peaks) close to the top of the gel. If the samples were not solubilized in the extraction buffer, no material migrated into the gel (determined by protein stain). The salt extracts from demembranated axonemes differed slightly in composition in the presence or absence of ATP in the extraction solutions. No significant differences were observed following extraction at either pH 6.5 or 8.0 either in the presence or absence of ATP. The KC1-ATP extracts on SDS gels showed bands between thyroglobulin dimer and the undissociated apoferritin, a second band at ~ 450,000 (between apoferritin and BSA6: usually migrating close to BSA6 oligomer), and two prominent bands at ~110,000 and 55,000 daltons. The extracted axoneme in this case retained bands at the highest molecular weight and at ~ 110,000 daltons. The KCI extracts however showed 2-4 bands at the highest molecular weight and small quantities (routinely less than 20~ of the extract) at 110,000 and 55,000 daltons. A prominent difference between the two types of extract was the absence of a band for the KCI extracts in the region of 400,000 daltons--this protein remaining associated with the axonemes following centrifugation at 14,500g for 10min. Both the KCI-ATP and KC1 extracts, showed low ATPase activity in the absence of any added divalent cations in the assay buffer (generally in the order of 0.05 #M Pi. mg. minute). This activity was markedly stimulated by the addition of MgC12 with the highest activity being obtained at 1 mM MgCI2 in the presence of 1 mM ATP. Specific activities under these conditions were variable but in general the KCI-ATP extracts have activities of 1-1.4 and the KC1 extracts

0.3-0.5. The basal activity could be stimulated by addition of Ca 2 ÷ ions with maximal activity being obtained at 0.25-0.5 mM CaCI2 (tested over range of 4-4000/aM). This activity was always less than the maximal activity attained in the presence of added Mg 2÷ ions and usually in the order of 65 80~o. The basal activity of KCI-ATP extracts from P. caudatum cilia was found to be stimulated by Ni 2+ ions (0.8mM: 82~o of that observed with added Mg 2÷ ions), Zn 2+ ions (0.01 mM: 4l~o of that with Mg 2+ ions) but inhibited by Ba 2÷ ions. In common with both the basal activity and the Ca 2 ÷ stimulated activity of the Triton extract (membrane fraction), both the basal activity and the Mg 2÷ ion stimulated activity of either the KCI-ATP or KCI extracts from the cilia were insensitive to ouabain (10 -3 M), oligomycin (10 and 140/~g/mg protein for the salt extracts and Triton extracts respectively) and aurovertin (850#g/mg). The Mg 2+ ion stimulated ATPase activity of the KCI-ATP extracts from P. caudatum cilia was inhibited by PCMB (60~ inhibition by 8/~M). Hydrolysis by GTP of the KCI extracts was less than 5~o of that observed with MgATP 2- regardless of the Mg 2+ ion concentration (0.5~4 mM). It was therefore concluded that the salt extracts from Paramecium cilia contain Mg 2÷ dependent ATPase proteins of a type reported for other ciliary and flagellar axonemes and in accordance with the recommendations of Gibbons (Gibbons et al., 1976), these proteins were termed dyneins. The salt extracts are hereafter referred to as crude dynein extracts.

Kinetics of Mg 2÷ dependent A T P hydrolysis from crude dynein extracts in the presence and absence of Ca 2+ ions In initial experiments it was established that, regardless of the Mg 2÷ ion and Ca 2÷ ion concentrations used (all in the 10-4-4.10 -3 range), a linear release of inorganic phosphate with time was observed from both the KC1 and KCI-ATP extracts from both P. caudatum and P. aurelia cilia. These assays were carried out by taking samples at 5 or I0 min intervals over a 30rain time period. This kinetic activity was subsequently carefully checked for the KCI extracts (Fig. 2) which shows that Mg z~ dependent Pi release from ATP, either in the presence or absence of Ca 2÷ ions at micromolar concentrations, was linear with time.

Alteration of Mg 2÷ dependent ATPase activity of crude dynein extracts by Ca :+ In initial experiments on KCI-ATP extracts from cilia of P. caudatum it was noted that Ca 2+ ions (0.25-4mM) inhibited the Mg 2÷ dependent ATPase activity. The inhibition was dependent on the Mg 2÷ ion concentration. Subsequent assays of ATPase activity in the presence of stochiometric concentrations of ATP and Mg 2+ ions (1 mM each) of both KCI and KC1-ATP extracts showed that micromolar concentrations of Ca 2÷ ions inhibited this activity whilst higher concentrations stimulated it (Fig. 3). The free Ca 2÷ ion concentrations in these assays, as noted above, can be taken as being close to those concentrations added to the assays. The differences in sensitivity for the different types of extract (KC1 extracts are more sensitive than KCI-ATP extracts) cannot

Control of ciliary activity--IV

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Fig. 3. Effects of Ca 2÷ ions on the Mg2+-dependent ATPase activity of crude dynein extracts. Basic assay buffer contains 2.5raM Hepes, 2.5raM Pipes, I mM MgCI 2. 1 mM ATP, pH 8.0 plus additional KCI as given below. Specific activities in the basic assay buffer in the absence of Ca 2+ (100~o activity) are given in brackets and followed by the range of activities observed at tile most effective CaCI 2 concentration. (O) KCI-ATP, pH 6.5 extract from cilia of P. caudatum in the presence of 270 mM KCi, 1 mM EDTA and 1.25mM MgCI2, 21°C. (0.465:54-68~). (Q) KCI-ATP, ~H 8.0 extract from cilia of P. aurelia concentrated to l/8th of original volume of 1000/~1 and then diluted 1:20 in the assay buffer. KCI concentration ~3 raM. (0.205:44-52~). ([~) KCI extract from cilia of P. aurelia concentrated to 1/10th and diluted 1:20 in assay. KC1 concentration - 2.5 raM. (0.185:26--35~). (O)--lower curve--KCl extract from cilia of P. aurelia concentrated to l/Sth and then diluted 1:40 in assay medium which contained an additional 20 mM KCI. (0.395:3-12~o).

of tests to determine the reason for the differences and in an attempt to keep protein concentrations reasonably constant in the assays. In addition, as noted above, the KCI extracts do not contain the 400,000 dalton protein whereas the K C I - A T P extracts do. In the traces in Fig. 3, the lowest trace is probably the closest to physiological concentrations of all cations (1 m M Mg 2+, 1 m M ATP, 22raM KCI and 6-8 m M Na ÷) since the intracellular K + ion concentrations for Paramecium are reported to be in this range (Yamaguchi, 1963).

Effect of Ca 2 ÷ ions on the electrophoretic mobility of dyneins on native gels On native polyacrylamide gels, in the absence of Ca 2 + ions (see methods), the crude dynein extract resolved into 3 bands with additional material scarcely penetrating into the gel (top trace, Fig. 4). However, if the gel was prepared containing an estimated 4 #M CaCl 2 (and then run side by side with a gel prepared concurrently that did not contain CaCI2) samples from the same crude dynein extract showed different electrophoretic mobility. As seen in the bottom trace in Fig. 4, in the presence of Ca 2 + ions, some of the dy'neins show a reduced electrophoretic mobility. The differences are not very large but were consistently observed in over 20 tests although the resolution was not always as good as in the paired set shown in Fig. 4. Since on gels of this type, separation is probably equally due to both molecular seiving effects of the gel matrix in addition to the net charge on the proteins, this Ca 2 + ion induced difference is thought to represent a concomitant alteration in both net charge (due to bound Ca 2 + ions) and native molecular size, Sepharose 4B chromatography of the crude dynein ex tract The elution buffer (2.5 m M Hepes, 2.5 m M Pipes, pH 8.0--deionized) was chosen such that the true cationic dependence of the dyneins could be established. Fig. 5 shows representative elution profiles for the two types of extract used. In both cases, three peaks of ATPase activity were observed. This activity was Mg 2+ ion dependent with no activity being

MIKE J. DOUGHTY

260

detected in the absence of added Mg z ÷ ions. The elution profiles for the two types of extract differed both in resolution and in the relative quantities of protein in each peak. Tube by tube analysis of ATPase activity was carried out in initial studies (bottom trace, Fig. 5) on KC1-ATP extracts at either pH 6.5 or 8.0. In studies with the KC1 extracts, only the peak tubes were assayed since they all showed Mg z+ ion dependent ATPase activity. The horizontal bars over the elution profile show the tubes pooled for analysis. For both types of extract, three basic peaks were observed which are assigned an identity notation of dynein fraction I, dynein fraction II etc. in order of elution and thus descending native molecular size. This assignment is made for identity purposes only and does not imply complete purification of any of the species. Apart from the three major peaks, no further protein was eluted until tubes 80-90 and this did not show any ATPase activity. Dynein fraction I was always found in tubes 6-9; dynein fraction II in tubes 17-21 whereas dynein fraction III varied in elution position from tubes 26-27 to tubes 53-54. Tubes from the centre of each peak were pooled and tested for both Ca 2÷ ion sensitive Mg 2÷ dependent ATPase activity and then for Ca 2÷ ion sensitive electrophoretic mobility on native polyacrylamide gels. The results from both types of extract are combined and full details are given in the figure legends. Dynein fraction I (Fig. 6) shows Ca 2+ ion stimulated ATPase activity and resolved into three bands

Fig. 4. Electrophoretic mobility of proteins in crude dynein extracts (KCI extract) on 3.5~ native gels in the presence and absence of CaCI2. Gels stained with Coomassie blue and scanned at 550 nm. Pair of gels run concurrently and loaded with 16.7/~g protein. Top trace--no added CaCI2, bottom trace--gel prepared containing 4~um CaCI2 final concentration (estimated).

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Control of ciliary activity--IV

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Fig. 6. Effects of Ca 2+ ions on dynein fraction I: Left panel: Effect of Ca 2+ ions on MgZ+-dependent ATPase activity. Assay buffer as for Fig. 3 with additional 20 mM KCI. Specific activities in the absence of Ca 2+ ions (100%) given in brackets. • (1.55), O (3.35), and • (4.83) are from KC1 extracts; A (1.37) is a KCI-ATP extract, pH 8.0. Right panel: Electrophoretic mobility of proteins in fraction I on 3.5% native gels. Lower of each pair of concurrently run gels contains 4 pm CaCI2. Top pair--KCIATP extract, tube 11 off column, 3.2#g; Bottom trace is fraction from KCI extract, tubes 6-8 off column, 2.4 pg and no CaCI2. b a n d o n native gels in either the presence or absence of C a 2 ÷ ions. Dynein fraction III (Fig. 8) shows Ca 2 ÷ ion inhibited M g 2+ ion dependent ATPase activity and resolves into two b a n d s on polyacrylamide gels in the absence of Ca 2÷ ions. These two proteins

on native gels in the absence of Ca 2+ ions a n d migrated as a single b a n d (scarcely penetrating the gel) in the presence of Ca 2÷ ions. Dynein fraction II (Fig. 7) appears to have largely insensitive M g 2+ dependent ATPase activity a n d resolves as a single

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Fig. 7. Effects of Ca z÷ ions on dynein fraction II: Left panel: Effect of Ca 2÷ ions on Mg2+-dependent ATPase activity. Assay buffer and details as in Figure 6. • (0.416), O (0.605) are fractions from KCI extracts./x (1.0) and • (0.49) are fractions from KC1-ATP extracts, pH 8.0. Right panel: Electrophoretic mobility of proteins in fraction II on 3.5% native gels. Lower of each pair of concurrently run gels contains 4 # M CaCI 2 . Top pair: KCI-ATP extract, pH 8.0, tube 20, 4.2 #g; middle pair: KCl-extract, tubes 17-18, 1.4 #g; Bottom pair: KCI-extract, tubes 19-20 (different experiment from middle traces), 1.6 #g.

262

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Fig. 8. Effect of C a 2+ ions on dynein fraction III: Left panel: Effect of Ca 2+ ions on Mg2+-dependent ATPase activity. Assay buffer and details as in Figure 6. O (0,305) is from a KCl-extract; A (0.50) and • (1.43) are from KCI-ATP extracts, pH 6.5 and [] (1.12) is from a KC1-ATP extract, pH 6.5. Rioht panel:. Electrophoretic mobility of proteins in fraction III on 3.5% native gels. Lower of each pair of concurrently run gels contains 4 #M CaC12. Top pair: KCl-extract, tubes 26--27, 1.4 #g; Bottom pair: KCI-ATP extract, pH 8.0, tubes 53-54, 1.4#g. apparently combine in the presence of micromolar concentrations of Ca 2÷ ions to give a single band with a lower electrophoretic mobility. In general, SDS polyacrylamide gel electrophoresis of any of the fractions reported above was not very successful. Protein bands usually only migrated 2-3 mm into the gel thus making quantitative determination of either composition or molecular weight unreliable and for this reason the data is not reported. In the first five experiments with the KC1 extracts, the resolution on the Sepharose column was not as good as presented in Fig. 5 (upper trace). Dynein fraction III was usually broad and showed little or no resolution. By slowing the flow rate down to approximately half that used in the previous experiments and by carrying out tube by tube protein analysis (both by Folin assay and absorbance at 280 nm), it was found tha¢ peak III was actually resolved almost quantitatively into two components whilst the other two peaks remained the same. The two subfractions of peak III show opposing Ca 2÷ sensitive Mg 2+ ion dependent ATPase activity. Fraction Ilia (eluting first) had a higher specific activity than the second fraction (IIIb) in three experiments. As shown in Fig. 9, dynein fraction IIIa shows Ca 2÷ stimulated Mg 2+ dependent activity whilst dynein fraction IIIb is inhibited by Ca 2÷ ions.

ATPase activity of the ciliary axoneme after dynein ex traction Even after overnight extraction of the demembranated axonemes with 0.5 M KCI at pH 8.0, considerable ATPase activity remains with the axonemal pellet even after washing. The extracted pellet was resuspended in an alternative extraction buffer (0.5 M KCI, 0.5mM EGTA, 0.5mM EDTA, 10mM Tris-HC1, pH 8.0) after washing in EDTA or EDTA plus EGTA buffers used to wash the isolated cilia (see above). Samples of the extracted axonemes were then assayed for ATPase activity in the standard

buffer. The ATPase activity was stimulated by addition of Mg 2+ ions with optimal activity again being observed in the presence of 1 mM ATP and l mM MgCI2. The specific activity is however considerably higher than that of the crude extract and ranged from 1.8-2.6 in the presence of optimum concentrations of Mg 2+ ions and ATP. On native polyacrylamide gels, the proteins in the extract stained for ATPase activity in a broad band close to the top of the gels. As with the crude extract, the extracted pellet shows signifi-

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Fig. "9. Effect of Ca 2+ ions on dynein fractions IIla and IIIb: Effect of Ca 2+ ions on Mgz +-dependent ATPase activity. Assay buffer and details as in Fig. 6. Fraction IIIa: • (1.54) and • (3.69). Fraction IIIb: • (0.46) and • (0.65).

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Fig. 10. Kinetics of MgATP2--dependent inorganic phosphate release from KCl-extracted ciliary axonemes from P. aurelia in the presence (0) and absence (O) of CaCI 2 (7.8 ,uM). cant Ca 2 + ion stimulated ATPase activity. Peak activity was however observed to occur in the presence of 1 m M added CaCI 2 (although this difference may not be real in view of the presence of 2 . 5 . 1 0 - 5 M EGTA in the assays). This Ca 2+ stimulated activity was observed to be either close to or higher than the activity in the presence of 1 m M MgCI2 and even higher in the presence of 1 m M MgCI2. The ATPase activity in the axonemes was not observed to be very sensitive to Ca 2 + ions in the presence of Mg 2÷ ions. Of particular note however, is the fact that the kinetics of ATP hydrolysis were subsequently found not to be linear over the normal assay period. As shown in Fig. 10, a discontinuity in the kinetic plots is observed either in the presence or absence of Ca 2+ ions (see discussion). DISCUSSION

These studies show that cilia from Paramecium contain dynein ATPase proteins. The results also show that the M g A T P 2- dependent activity of both the crude extract and some of the semi-purified forms of dynein is markedly affected by Ca 2+ ions at low concentrations. Such changes in enzyme activity would appear to be related to an alteration in the molecular state of the dyneins. Blum & Hayes (1977) report a Ca 2+ ion dependent alteration in the density of demembranated ciliary axonemes from Tetrahymena (the pellet height response) although, in the presence of EGTA, they found no signific.ant alteration in the M g '+ ion dependent ATPase activity by Ca 2+ ions. It cannot be stated with any certainty that the Ca 2 ÷ ion sensitivity of solubilized dyneins reported in this paper represents the physiological mechanism by which Ca 2+ ions effect control of both ciliary beat frequency and orientation in vivo. There is however a close correlation between those Ca 2+ ion concentrations (estimated) that effect maximal alteration of dynein activity and those that effect maximum velocity rearward swimming in "model" Paramecium (Naitoh & Kaneko, 1972). In addition, such Ca 2 + ion sensitivity of the solubilized dyneins shows that these proteins have not become insensitive to low concen-

263

trations of Ca 2 ÷ ions (an argument which might be raised in support of previous studies on Ca 2÷ ion sensitivity of dyneins). F r o m a theoretical point of view, Ca 2 + ions may act either on the basic mechanochemical cycle that effects interdoublet sliding or upon a secondary mechanochemical cycle that effects ciliary bending. A Ca 2÷ ion stimulation of cross-bridge (inter-doublet) activity could be the reason why high concentrations of Ca 2÷ ions effect an enhancement of ciliary beat frequency. C a A T P 2- promoted disintegration of trypsin treated axonemes has been reported (Summers & Gibbons, 1968; Mitchell & Warner, 1977). Ciliary inactivation (observed at the end of the period of reversed beating and prior to return of forward beating: Machemer, 1974; Machemer & Eckert, 1975) could conceivably be the result of Ca 2 ÷ ion inhibition of dynein (and thus cross-bridge) activity except that complete supression of soluble dynein activity is not effected even at 1 0 - 2 M Ca 2÷ ions (Hoshino, 1974). In addition, if Ca 2 ÷ ion alteration of the cross-bridge cycle was the cause of ciliary beat augmentation and ciliary reversal, it is difficult to envisage how Ca 2+ ion could effect such a change unless the kinetics of Ca 2 + movement to these sites was specifically regulated and/or that each "arm" had a different composition and thus affinity for Ca 2÷ ions. Outer "arm" dynein appears to be relatively homogeneous in composition (Kincaid et al., 1973; Ogawa et al., 1977; Warner et al., 1977). In addition, in cilia that show an arrest response (instead of reversal), Ca 2 + ions do not apparently inhibit interdoublet sliding (Walter & Satir, 1977). A second site for the Ca 2+ dependent mechanism that effects both ciliary and flagella activity has been discussed in the recent literature (Holwill & McGreggor, 1976; Blum & Hayes, 1977). At the present time, there is little direct evidence that peripheral doublet-central sheath interactions do occur. However, as pointed out by Warner (Warner & Satir, 1974; Warner, 1976), the small changes in the relative angles of the spokes to the central sheath even in extremes of bending, suggest that intermittent radial spoke attachment to the central sheath may have occurred. There have been a few claims (see Summers, 1976; Warner, 1970, 1976) of.motile n + 0 axonemes but, as noted or implied by so"~e(al authors (Warner, 1970, 1976; Summers, 1976; Witliaan et al., 1978), the evidence needs substantiation. It is therefore pertinent to note the presence of Ca 2 + ion dependent electron dense deposits in Paramecium ciliary axonemes in the vicinity of the spoke heads (Tsuchiya & Takahashi, 1976; Fisher et al., 1976) and the report of ATPase reaction product deposition "'in areas between the central and double outer fibres" in Tetra, hymena (Burnasheva & Jurzina, 1968). Therefore, since KC1-ATP extraction appears to predominantly extract a homogeneous form of dynein and since the KC1 extract does not remove a 400,000 dalton protein from the axoneme, it is proposed that dynein fraction II is predominantly "'arm" dynein. The cross-bridge cycle is furthermore proposed, in view of the lack of Ca 2 + ion sensitivity in vitro, to be Ca 2+ ion insensitive at least at concentrations below 10-SM. The site of dynein fraction III, although definative localization will have to be carried out to substantiate this, is proposed to be at

MIKE J. DOUGHTY

264

the spoke heads. It is uncertain from these studies whether dynein fractions I and III are related or whether they represent two different sub classes of dyneins (the term isoenzymes is not chosen since purity has not been established). The presence of one or both of these classes of dyneins on the spoke heads would serve as an excellent basis for both a self regulating and Ca 2+ ion sensitive "motor". Changes in both hydrolytic activity and the conformationai state of these dyneins would serve to both facilitate peripheral doublet to central sheath interactions (and thus account for the enhanced ciliary beating and increased sweep angle at the onset of ciliary reversal) and perhaps also redirect these interactions to effect ciliary reversal. At higher Ca 2+ ion concentrations, alteration in conformational state and the association of some or all of these proteins with the structures to which they are normally presumably bound, could serve to "lock" the cilium (inactivation) as a result of physically preventing or destroying the effectiveness of the normal cycle that converts interdoublet sliding into bending and thus motion of the cilium. This inactivation would be gradual since current evidence, albeit circumstantial, suggests that the sites of Ca 2+ influx into the intraciliary space are located near the base of the cilium (Plattner, 1975; Dute & Kung, 1978; Byrne & Byme, 1978). Ca 2+ efflux has been proposed to occur across the same region (Baugh et al., 1976). Therefore, following Ca 2 + ion influx, ciliary reversal, either based upon a Ca 2 + ion sensitive dynein interaction or perhaps through a Ca 2+ ion dependent interaction at an apparent discontinuous linkage between one of the central doublets and the axosome (Ehret & McArdle, 1974; Dute & Kung, 1978), occurs concomitant with augmentation of ciliary frequency as a result of a facilitation of doublet to central sheath interactions through one or both sets of Ca 2+ ion sensitive dyneins. Inactivation is proposed to occur as a result of the gradual spread of Ca 2 + ions up the ciliary shaft to more distal spoke head sites. Renormalization is effected by the reverse process-cilia are gradually returned to forward beating orientation along with a gradual rise in beat frequency and sweep angle as the cilia become less stiff and fully active again as the Ca 2+ ions are pumped out of the intraciliary space. As a final note, the distinct linear kinetics of ATP hydrolysis by the crude dynein extracts used here and the discontinuity noted in samples containing significant quantities of structural protein (the extracted axonemes) indicates that the recently reported initial fast burst of soluble dynein activity (Nakamura & Masuyama, 1977) is not a property of the dyneins per se but due to their association with other structural units (probably tubulin) within the axoneme. However, such a change in activity may well provide additional reasons why the ciliary activity at the time of onset of ciliary reversal is initially dramatically enhanced and then gradually inactivated. SUMMARY

Dynein ATPase proteins were extracted from demembranated ciliary axonemes of Paramecium by treatment with high ionic strength salt solutions. Differences in the proteins extracted were noted in the

presence or absence of ATP. Salt extraction yields two basic types of dynein (separated by Sepharose 4B chromatography). One fraction appears to be homogeneous with respect to protein composition and shows Ca 2 + insensitive Mg 2 + dependent ATPase activity. The other dynein fractions are heterogeneous with respect to composition and show both Ca 2 + ion sensitive Mg 2+ ion dependent ATPase activity and a Ca 2+ ion dependent alteration in their molecular form. This differential sensitivity is thought to reflect the existence of different classes of dyneins within the ciliary axoneme and that Ca 2 + ion interaction at the sensitive sites provides a basis for a physiological mechanism by which Ca 2+ ions may effect control of both ciliary beat frequency and orientation. Acknowledgements--I should like to express my gratitude to both Dr Hans Machemer (University Bochum, West Germany) and Dr Ian Gibbons (University of Hawaii, USA) for both useful discussions and for critical reading of the first draft of this manuscript. I should also like to thank Dr Roy Burns (University of Edinburgh, Scotland) for initial advice on electrophoresis of the dynein proteins. This work was supported by a grant from the Medical Research Council, U.K. REFERENCES ALLEN R. D. (1968) A reinvestigation of cross-sections of cilia. J. Cell Biol. 37, 825-83 I. ANDRIVON C., WYROBA E. & PATTERSON D. J. (1977) Researches pr61iminaires sur les ATPases de Paramecium aurelia. J. Protozool. 24, 55A, Abstract. BAUGH L. C., SATIR P. & SATIR B. (1976) A ciliary membrane Ca 2+ ATPase: a correlation of structure and function. J. Cell Biol. 70, 66a, Abstract. BLUM J. J. & HAYES A. (1977) Effect of calcium on the pellet height response of Tetrahymena cilia. J. Supramol. Struct. 7, 205-211. BROKAW C. J. (1972) Flagellar movement: a sliding filament model. Science, N.Y. 178, 455-462. BROKAW C. J. & SIMONICKT. F. (1977) Motility of Tritondemembranated sea urchin sperm flagella during digestion by trypsin. J. Cell Biol. 75, 650-665. BROWNING J. L. & NELSON D. L. (1976) Biochemical studies of the excitable membrane of Paramecium aurelia. 1. 4SCa2 + fluxes across the resting and excited membrane. Biochim. biophys. Acta 448, 338-351. BURNASHEVAS. A., EEREMENKOM. V., CHUMAKOVAL. P. & ZUEVA L. V. 0965) Isolation of the contractile proteins from the cilia of Tetrahymena pyriformis and investigation of their properties. Biokhimiya. Engl. Trans. 30, 765-771. BURNASHEVA S. A. ~¢. JURZINA G. A. (1968) The ultrastructure and localization of ATPase activity of Tetrahymena cilia. 3rd Int. Conor. Histochem. Cytochem. New York. Abstract. BYRNE B. J. & BYRNEB. C. (1978) A ultrastructural correlate of the membrane mutant "Paranoic" in Paramecium. Science, N.Y. 199, 1091-1093. DAIYA O. Y., BURNASHEVA S. A. & LYUmMOVA M. N. (1972) Zsslyedovanie adyenozintrifosfatazikh svoistv byelov ryesnickyek Tetrahymena pyriformis. In Myekhanizmy Myshyechnooo Sokrashchyniya, pp. 167-174. Akad. Nauk SSSR, Moscow. DENTLERW. L. 0977) Fine structural localization of phosphatases in cilia and basal bodies of Tetrahymena pyriformis. Tissue Cell 9, 209-222. DOUGHTY M. J. (1978a) Ciliary Ca 2+ ATPase from the excitable membrane of Paramecium. Some properties and purification by affinity chromatography. Comp. Biochem. Physiol. 60B, 339-345.

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HOLW1LL M. E. J. & MCGREGGOR J. L. (1976) Effects of calcium on flagellar movement in the trypanosome Crithidia oncopelti. J. exp. Biol. 65, 229-242. HOSHINO M. (1974) Preparation and characterization of a dissociated 14S form from 30S dynein of Tetrahymena cilia. Biochim. biophys. Acta 351, 142-154. HOSHINO M. (1975) Dissociation of Tetrahymena 30S dynein into 14S subunit by sonication. Biochim. biophys. Acta 403, 544-553. HYAMS J. S. & Boglsv G. G. (1975) The dependence of the waveform and direction of beat of Chlamydomonas flagella on calcium ions. J. Cell Biol. 67, 186a, Abstract. KINCAXD H. L. JR, GmaONS B. H. & GmBONS I. R. (1973) The salt extractable fraction of dynein from sea urchin sperm flagella: an analysis by gel electrophoresis and by adenosine triphosphatase activity. J. Supramol. Struct. 1, 461-470. KLOTZ I. M. & DARNALL D. W. (1968) Protein subunits. A table. 2nd edn. Science, N.Y. 166, 126-128. KOBAYASHI T., MARTENSEN T., N^T8 J. & FLAVIN M. (1978) Inhibition of dynein ATPase by vanadate and its possible use as a probe for the role of dynein in cytoplasmic motility. Biochem. biophys. Res. Commun. 81, 1313-1318. KUZNICKI L., JAHN T. L. & FONSECA J. R. (1970) Helical nature of the ciliary beat of Paramecium multimicronucleatum. J. Protozool. 17, 16-24. MACHEMER H. (1972) Properties of polarized ciliary beat in Paramecium. Acta. Protozool. 11,297-300. MACHEMER H. (1974) Frequency and directional responses of cilia to membrane potential changes in Paramecium. J. Comp. Physiol. 92, 293-316. MACHEMER H. (1975) Modification of ciliary activity by the rate of membrane potential changes in Paramecium. J. Comp. Physiol. 101, 343-356. MACHEMER H. & ECKERT R. (1973) Electrophysiological control of reversed ciliary beating in Paramecium. J. Gen. Physiol. 61, 572-587. MACHEMER H. & ECI,:ERT R. (1975) Ciliary frequency and orientational responses to clamped voltage steps in Paramecium. J. Comp. Physiol. 104, 247-260. MAST S. O. & NADLER J. E. (1926) Reversal of ciliary action in Paramecium caudatum. J. Morph. 43, 105 117. MITCHELL D. R. & WARNER F. D. (1977) Dynein arm cross-bridges and the generation of sliding forces in Tetrahymena cilia. J. Cell Biol. 75, 295a, Abstract. MOHRI H., HASEGAWAS., YAMAMOTOM. & MURAKAMI S. (1969) Flagella adenosine triphosphatase (dynein) from sea urchin spermatozoan. Sci. Pap. Coll. Gen. edn. Univ. Tokyo, 19, 195-217. MURAKAMIA. & TAKAHASHI K. (1975) The role of calcium in the control of ciliary movement in Mytilus. II. The effects of calcium inonophores X537A and A23187 on the lateral gill cilia. J. Fac. Sci. Univ. Tokyo, Sect. IV. Zool. 13, 251-256. NAGATA Y. & FLAVIN M. (1975) Specific inhibitor or activator of dynein ATPase. J. Cell Biol. 300a, Abstract. NAITOH Y., ECKERT R. & FRIEDMAN K. (1972) A regenerative calcium response in Paramecium. J. exp. Biol. 56, 667-681. NAITOH Y. & KANEKO H. (1972) Reactivated Triton. extracted models of Paramecium: modification of ciliary movement by calcium ions. Science, N.Y. 176, 523-524. NAKAMURA K-I. & MASUYAMA E. 0977) Studies on the initial phase of dynein ATPase activity. Biochim. biophys. Acta 481, 660-666. OGAWA K. & MOnRI H. (1972) Studies on flagella ATPase from sea urchin spermatozoa. I. Purification and some properties of the enzyme. Biochim. biophys. Acta 256, 142-155. OGAWA K., MOHR1 T. & MOHm H. (1977) Identification of dynein as the outer arms of sea urchin sperm axonemes. Proc. Natn. Acad. Sci., U.S.A. 74, 5006-5010.

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