- Email: [email protected]
Response of the Cortical Cytoskeleton of Paramecium caudatum to Merotomy
Arch. Protistenkd. 140 (1991): 321-333 Gustav Fischer Verlag lena
Department of Biology, Medical Faculty, Masaryk University, Brno, Czechoslovakia
Response of the Cortical Cytoskeleton of Paramecium caudatum to Merotomy By ROMAN JANISCH With 10 Figures Key words: Cytoskeleton: Regeneration: Ultrastmcture: Paramecium
Summary Changes occurring within 2 min of merotomy in the cortical cytoskeleton of fragments cut from Paramecium caudatum were studied in thin sections. None of the microtubular components of the cortical cytoskeleton showed alterations suggestive of functional reorganization of microtubules and attributable to the mechanical damage made to the cell. Marked changes, however, were found in the amount and distribution of the infraciliary lattice. The bands of microfilaments were thicker and the meshes between them were reduced. This thickened layer penetrated deep into the wound under a newly-formed plasma membrane. The granulofibrillar meshwork, which fills the polygonal ridges in an intact cortex, was completely disorganized in fragments. Its remnants were always found in association with the plasma membrane or alveolar membranes. Cross-striated bands of microfilaments were retained only at the margin of the wound where they were attached to alveolar debris. Kinetodesmal fibrils maintained their integrity but their regular pattern of longitudinal bands was disturbed. The epiplasm (membrane skeleton) in the remnants of alveolar membranes was preserved and found also in some regions of the newly-formed plasma membrane. Little information is available on the chemical composition of these microfilamentous components and their function could only be the object of speculations. For instance the character of changes in the infraciliary lattice might suggest that its thickening was due to polymerization of new microfilaments which , together with wound contraction, were involved in the process of healing.
Introduction The cytoskeleton is a complex system based on proteins forming fibrillar supramolecular structures. Relationships between structural cytoskeletal components and their interaction with associated proteins allow the cell to perform the principal functions of the cytoskeletal system, which are structural support, movement and the flow of information. The structure and the function of each cytoskeletal component have been studied using a variety of models and experimental objects. Among these, protozoa assume a special position because of broad species diversity, high specialization of cellular structures and variability. Great diversity in the differentiation of protozoan cells resulted, in the course of evolution, in the development of numerous forms of cytoskeletal componens, which has no parallel in cells of multicellular organism (for review see GRAIN 1986). A considerable body of information has been obtained on the cytoskeletal structures of Paramecium, an infusorian, in which detailed studies on the ultrastructure of the cortical cytoskeleton have been carried out (ALLEN 1971, review by WICHTERMAN 1987). The cortical cytoskeleton is a heterogenous system of fibrillar components, such as microtubules, microfilaments, periodic fibres and membrane skeleton (epiplasm) which, in protozoa, comprises the surface layer called the cortex in infusoria. In addition to these structures this 21
Arch. Protistcnkd. Bd. 140.4
322
R. JANISCH
layer also involves membranous structures, i.e. , plasma membrane, alveoli and trichocyst trips. The pattern of subsurface alveoli has also been referred to as the hydroskeleton (GRAIN 1986). ALLEN reported as early as in 1971 that, apart from various microtubular structures, such as kinetosomes, ciliary axonemes, bands of postciliary and transversal microtubules, the cortex of Paramecium consisted of microfibrillar systems (granulofibrillar meshwork, crossstriated bands of microfilaments and infaciliary lattice), at that time beyond definition in terms of chemical structure, and periodic kinetodesmal fibrils and microfibrils associated with kinetosomes. The role of most of these structures is still not properly understood. Paramecium injured mechanically by cutting off its anterior or posterior parts in the range of Y3 to 1/4 of its body is capable of covering the exposed cytoplasm with a new plasma membrane within a few seconds. The process of plasma membrane formation and further healing of the defect have been studied by light and electron microscopy (JANISCH 1965 , 1967 , 1985 , 1987). One of the approaches to understanding the functions of the cytoskeletal structures in the cortex of Paramecium is the study of regenerating fragments. In this paper attention was focussed on changes in each component of the cortical cytoskeleton in Paramecium caudatwn. The investigation involved only the somatic region of the cortex without its specialized subsystems present at the sites of contractile vacuole pores, the cytoproct and the oral apparatus . For a better understanding , part 1 of Results gives a review on the present knowledge of the ultramicroscopic anatomy of the cortical cytoskeleton in intact Paramecium cells. A comparison with our findings following merotomy is presented in Part 2. The investigation into the nature and functions of the cytoske1eta1 components in Paramecium will later be completed with the results of our experiments involving specific antibodies to cytoskeletal proteins and inhibitors of protein polymerization which are under progress and will be reported elsewhere.
Material and Methods 1. Specimens A clone of Paramecium cauda tum isolated from a local source was grown in a medium with wheat grains 1940) and fed on Klebsiella aerogenes.
(VILLENEUVE-BRACHON
4
2. Merotomy Cell fragments were cut with a microscalpel made from a piece of razor blade fitted in a glass handle. Merotomy was performed by free hand under a dissecting microscope at a magnification of 40x. Fragments of about :y. of the original Paramecium cells were used for further studies.
Fig. I. Semitangetial section through the cortex of a normal cell of Paramecium caudatum showing various cytoskeletal structures. X 53,000 Fig. 2. A cross section of the Paramecium caudatum cortex from the area involving the cilium with a kinetosome . x73 ,000 Abbreviations in all figures: a - Alveolus; c - Ciliary axoneme; k - Kinetosome; pm - Plasma membrane: ia - Inner alveolar membrane ; oa - Outer alveolar membrane; e - Epiplasm; tp - Terminal plate; gf - Granulofibrillar meshwork; pc - Postciliary microtubules; tr - Transversal microtubules; sb - Bands of cross-striated microfilaments; if - Infraciliary meshwork: kd - Kinetodesma: fc - Connection linking kinetodesma to kinetosome; b - Connection between kinetosomes.
Response of the Cortical Cytoskeleton
21*
323
324
R.
JANISCH
Fig 3. A cross section through the wound margin with the cytoplasm overlapping the remnants of the marginal cortex. In some places the infraciliary lattice is seen as an almost continuous layer. The regular cortical pattern under the overlapping cytoplasm, i.e. , the system of alveoli, kinetosomes and other cytoskeletal structures, is completely disturbed. x 14,000
Response of the Cortical Cytoskeleton
325
3 . Electron Microscopy Within 2 min of merotomy Paramecium fragments were fixed with 2 % glutaraldehyde in a cacodylate buffer. pH 7.2, for 15 min and postfixed in I % OS04 in the same buffer. Each fragment was processed using the method of oriented embedding; thi s technique enabled us to make ultrathin sections parallel to the longitudinal axis of the fragment (JANISCH 1974). Ultrathin sections were made on a Reichert Ultracut E ultramicrotome , stained with lead and uranyl acetate (REYNOLDS 1963) and examined and photographed with a Tesla BS 500 electron microscope.
Results 1 . Ultrastructure of the Cortical Cytoskeleton The cortical cytoskeleton of Paramecium caudatum includes microtubular and microfilamentar structures, mostly chemically unidentified, periodic fibres and the membrane skeleton (epiplasm) of the plasma membrane and of alveoli.
1.1. Microtubules of the Cortical Cytoskeleton Microtubules in the Paramecium cortex are found mainly in kinetosomes and ciliary axonemes. Each single kinetosome or a pair of them have postciliary and transversal microtubules in their vicinity (Fig . 1). Most frequently 4 of them are assembled into flat bands . At division, during the ShOl1 period of cytodieresis (10 to 15 min), longitudinal bundles of 4 to 16 microtubules are formed in the cortical ridges; they are referred to as cytospindle or, according to their position over kinetodesmal fibrils, as suprakinetodesmal microtubules (JURAND & SELMAN 1969; EHRET & McARDLE 1974; SUNDARARAMAN & HANSON 1976). None of these structures studied in Paramecium fragments showed changes which could be related to surface defect healing or to cell regeneration.
1.2. Microfilamentous Structures The somatic cortex of Paramecium involves 4 categories of microfilamentous structures that have been gradually discovered and , under different names, described by the authors of many electron microscopic studies (PITELKA 1961 , 1965, 1969 ; EHRET & POWERS 1959; HUFNAGEL 1969; EHRET & McARDLE 1974) . Terms and classification used in this study have been introduced by ALLEN (1971). They are: granulofibrillar meshwork, cross-striated bands of microfilaments, infraciliary lattice, microfilaments linked to single kinetosomes or their pairs (Figs. 1, 2).
1.2 . 1. Granulofibrillar Meshwork It is made up of microfilaments, 30 to 40 nm long, linked to electron dense granules , 10 nm in diameter. The meshwork fills the upper parts of the polygonal ridges (Fig. 2). Along the sides, it is in contact with the membrane skeleton of the inner alveolar membrane and in the crests of the ridges it may communicate also with the membrane skeleton of the plasma membrane since alveoli there are slightly apart (about 150 nm). The chemical composition of
Fig. 4. A cross-section through the wound in the region where the wound edge is overflown with cytoplasm. The infraciliary lattice occasionally forms a continuous layer and is accumul ated in the centre of the wound. The pattern of kinetosomes. alveoli and kinetodesmal fibrils is disorganized. x 14 ,000
326
R.
JANISCH
Fig. 5. Ciliary axonemes embedded in the spilt cytoplasm. The centre of the picture is occupied with an axoneme still maintaining its typical arrangement of 9 + 2. In the left upper corner the remains of an axoneme are in the form several single and double microtubules. x 77 ,000 Fig. 6. Remains ofaxonemes embedded in the spilt cytoplasm. X40 ,OOO
Response of the Cortical Cytoskeleton
327
the granulofibrillar meshwork is not known. Indirect immunofluorescence methods, however, have shown the presence of proteins (130 and 50 kD) reacting with antimyosin antibody (COHEN et al. 1987) and some other components like actin (JANISCH 1988) and calmodulin (MOMAYEZI et al. 1986). The function of this structure is still discussed. Its position suggests involvement in maintaining the shape of polygonal ridges and perhaps the position of alveoli. COHEN et al. (1987) have shown that during division the granulofibrillar meshwork grows by longitudinal elongation without disruption of pre-existing meshes.
1.2.2. Cross-Striated Bands of Microfilaments The bands are 100 to 500 nm wide, oriented parallel to the organism's surface in the plane of the inner alveolar membranes where they are connected to the alveolar membrane skeleton (Figs. 1, 2). Each band, when cross-sectioned, shows a circular shape and is composed of microfilaments 6 nm in diameter. Electron-dense and electron-transparent parts alternate along the band due to the overlapping of microfilaments in darker parts (ALLEN 1971). Occasionally, fine bridges , about 10 nm long, are seen linking microfilaments lying close to each other. Nothing is known about their chemical composition but the presence of actin has been suggested (KERKSEN et al. 1986a, b).
1. 2.3. Infraciliary Lattice This structure was first described in silver-treated preparations by GELEI (1937) under the light microscope, later by many authors using electron microscopy (SEDAR & PORTER 1955; ROTH 1958; PITELKA 1965; HUFNAGEL 1969; JURAND & SELMAN 1969). The most detailed study was made by ALLEN (1971). This system is composed of bundles , 70 to 100 nm thick, which branch forming a lattice of irregular polygons. Each of these involves a single kinetosome or a pair of them or a single trichocyst (Fig . I) . At the site of bifurcation the bundles may become up to 400 nm wide . An electron-opaque rod of triangular cross-section, called a "post" by ALLEN (1971), is present at the centre of the branching site. The basic element of the bundle is a microfilament about 4 nm wide. These filaments are organized into tubules that appear in cross-sections as closely packed hexagons. The infraciliary lattice is situated parallel to the surface in the plane of proximal kinetosomal ends. GARREAU DE LOUBRESSE et al. (1988) have found two (23 to 24 kD) polypeptides which were shown to cross-react with a polyclonal antibody raised against 22 to 23 kD Ca2 + -binding proteins isolated from the microfibrillar ectoplasmic boundary of lsotricha. The authors suggest that the polypeptides belong to the class of contractile proteins.
Fig. 7. A cross-section through the cortex in the marginal part of the wound. The course of cross-striated kinetodesmas is unusual but their more or less parallel pattern is still preserved. X42.000 Fig. 8. A detailed view of the thickened infraciliary lattice shows a widening of bands up to 160 nm and increase in microfilament density within the band. x58.000
328
R.
JANISCH
Fig. 9. The granulofibrillar meshwork at the edge of the wound is disorganized. It consists of various irregular aggregates under the remnants of alveoli and plasma membrane. x 64,000 Fig . 10. Cross-striated bands of microfilaments are associated with alveoli at the wound edge. The granulofibrillar meshwork and kinetodesmas are disarranged. x 35,000
Response of the Cortical Cytoskeleton
329
1.2.4. Microfilaments Originating from Single or Paired Kinetosomes In the somatic part of the cortex, kinetosomes are found in pairs. A fine microfilamentous link extends from the right side of the anterior kinetosome and joins a kinetodesmal fibril radiating from the posterior kinetosome of the same pair (Fig. 1). The microfilaments are found in the proximal plane of kinetosomes. So far there are no data on either their function or chemical nature. Two more types of filamentous structures associated with kinetosomes that have a periodic character are reported under the following headings.
1.3. Periodic Fibres All three types of periodic fibres in the somatic part of the Paramecium cortical cytoskeleton are connected with kinetosomes. The most conspicuous, and also known for the longest time, are kinetodesmal fibrils or kinetodesmas (Fig. 1). They arise from the kinetosome of each kinety extending from its anterior right quadrant. If two kinetosomes are present, the kinetodesmal fibril starts from the rear one (DIPPEL 1964). The kinetodesma runs anteriorly and rightwards taking a longitudinal course at the kinety's right side over a distance of 8 nm, always passing 5 to 6 kineties located ahead. At the end attached to the kinetosome it is wider, gradually getting narrower. On a longitudinal section, each kinetodesma shows a cross-striated pattern with a periodicity of 23 to 27 nm. Each kinetodesmal fibril consists of many elementary microfilaments (HUFNAGEL 1969) of unknown function and chemical composition. The two other types of periodic fibres (Fig. 1) are seen as links, pursuing an oblique course, between the kinetosomes of the same pair (ALLEN 1971). These fibres are located in the plane of the proximal ends of kinetosomes. Their function and chemical composition are not known.
1.4. Epiplasm - the Membranous Skeleton It occurs as an electron dense layer, fibrogranular in appearance, situated on the cytoplasmic side of the inner alveolar membrane (Fig. 2). It continues into the terminal plate of the kinetosome. It is less distinct between the plasma membrane and the outer alveolar membrane mostly due to a reduced space between the membranes. In Paramecium its chemical structure is unknown. In some other infusoria like Tetrahymena, the epiplasm has been reported to contain proteins of 122 to 145 kD (VAUDAUX 1976: VAUDAUX et al. 1977). A small amount of actin is also likely to be present (WILLIAMS et al. 1979). The role for the membrane skeleton in infusoria is still open to speculation. It can be assumed that it is involved in maintaining the overall architecture of membranous components of the cortex and in mediating contact with other cytoskeletal components (kinetosomes, transversal and postciliary microtubules, bands of cross-striated microfilaments) and possibly with some other organelles (mitochondria, trichocysts) situated closely under the organism's surface.
2. Changes in the Cortical Cytoskeleton after Merotomy Removal of a part of the Paramecium cell with a microscalpel was a severe intervention which brought about partial damage to or complete destruction of many of the cell's structures. In spite of it, most of the fragments were capable of covering the exposed cytoplasm with a new plasma membrane within a few seconds. In some fragments (Figs. 3, 4) the cytoplasm with the new membrane spilled and overlaid the damaged cortex (JANISCH 1985).
330
R.
JANISCH
2.1. Microtubules The microtubular component of the cortical cytoskeleton did not show any changes indicative of functional reorganization. All alterations in the tubular structures found in fragments in the area of injury consisted in disorganization and dislocation due to mechanical pressure produced by the microsurgical instrument. The cortex round the injured area had only a few kinetosomes which were unevenly distributed and assumed various atypical positions. Their integrity, however, was retained. In the displaced kinetosomes it was not possible to identify postciliary and transverse bands of microtubules (Figs. 5, 6). Such kinetosomes also lacked cilia. Occasional ciliary axonemes embedded in the cytoplasm were seen in the affected area, with their typical pattern of 9 + 2 still preserved. 2.2. 1nfraciliary Lattice The most marked changes in fragments were seen in the structure of the infraciliary lattice. In contrast to the intact cortex with a thin meshwork including kinetosomes or trichocyst, fragments showed a much thicker layer of the infraciliary lattice at the margins of the injured region; it penetrated deep into the wound under the newly formed plasma membrane (Figs. 3, 4). Bundles of infraciliary lattice were wider and denser (Fig. 8). On sections cut perpendicularly to the plane of the infraciliary lattice, this structure appeared as a wide electron-dense layer interrupted with short gaps. Reduced meshes of the lattice in this region did not contain kinetosomes or trichocysts. From these results it was not possible to draw a conclusion as to the mechanism controlling the changes in the structure of the lattice. Immediately after merotomy it was observed that the area of defect, boundered by a partly damaged cortex, contracted to a certain degree. This was related to the thickening of the infraciliary lattice, but the high degree of thickening of all bundles associated with an increase in electron density cannot be explained by mere contraction. It is open to question to what extent the increased density of the infraciliary lattice can be due to polymerization of new components. 2.3. Other Cytoskeletal Components The granulofibrillar meshwork, which is present in the crests of polygonal ridges of an intact cortex, was completely disorganized in fragments (Figs. 9, 10). Only occasionally it was still attached to the remnants of plasma membrane or alveolar membranes at the wound edges. The extent of changes in the whole amount of meshwork resulting from injury could not be estimated in ultrathin sections. Cross-striated bands of microfilaments were found in injured areas only in association with alveolar remnants (Fig. 10). Since they were never seen in the absence of alveoli, it can be concluded that, once separated from alveoli by merotomy, they disintegrated. Kinetodesmal fibrils retained integrity in the injured region but their regular pattern of longitudinal bundles was disturbed completely in both regularity and orientation (Figs. 7, 10). The epiplasm of alveolar membranes in the injured area remained unaffected (Figs. 9, 10). Of interest was the finding of the epiplasm in some parts of the newly formed plasma membrane covering the cytoplasm exposed by injury. The response to merotomy of the other components of the cortical cytoskeleton (postciliary and transversal bands of microtubules, microfilaments associated with kinetosomes) could not be estimated by means of the technique used.
Response of the Cortical Cytoskeleton
331
Discussion Merotomy in Paramecium results in an extensive defect in the cell surface the healing of which is a prerequisite for further viability of the fragment. Factors most important for healing are: rapid formation of a new plasma membrane covering the exposed cytoplasm, contraction of the wound, and quick reaction of the cytoskeletal structures preventing the cell content from spilling in the first moments after merotomy. The first step, i.e. plasma membrane formation, has been the subject of our previous studies (JANISCH 1965, 1967, 1985, 1987). The contraction of the wound and the response of cytoskeletal structures to injury appear to be closely related processes. One of our earlier studies on fragments of Paramecium employing ultrathin sectioning (JANISCH 1964) showed the presence of a discontinued layer of osmiophilic material at the boundary between the necrotic and intact cytoplasm in the injured area. However, the application of methacrylate as an embedding medium and an insufficient resolution of the electron microscope employed at that time did not allow us to identify the structures more exactly. From examination of ultrathin sections in this study it is now apparent that the osmiophilic layer is a thickened and enlarged infraciliary lattice (Figs. 3, 4, 8). The response of this cytoskeletal component is the most marked of all. Its function however, is still very poorly understood. Actin detected by immuno-fluorescence in the cortex of Paramecium has often been explained as its mere accumulation in the infraciliary lattice (TIGGEMANN & PLATTNER 1981; TIGGEMANN et al. 1981; PLATTNER et al. 1982). COHEN et al. (1984), on the other hand, failed to prove actin labelled with heavy meromyosin in the infraciliary lattice. As seen in Fig. 11 of the work by KERKSEN et al. (J 986 a) a thick layer involving bands of microfilaments was produced after microinjection with CaCl 2 in Paramecium tetraurelia. These cortical microfilaments most probably correspond to the enlarged bands of the infraciliary lattice seen in the fragments under study. The thickening of the infraciliary lattice in Paramecium after microinjection may be related to the contraction of the cortex at the site of injection, as is evident from the picture in the paper by KERKSEN et al. It is yet to be decided what is the role of Ca2 + -ions in the contraction and what is the involvement of mechanical injury by a microinstrument, which must also be taken into consideration. Structural changes in the infraciliary lattice during reparation of fragments may be due to contraction of the wound circumference immediately after merotomy or to polymerization of new microfilaments. The appearance and intensity of enlargement of the infraciliary lattice may be the result of both. GARREAU DE LOUBRESSE et al. (1988) have shown that the infraciliary lattice displays contractile properties and that its assembly is Ca2+ -dependent. Perhaps the infraciliary lattice is a new example of the non-actin contractile system found in lower eukaryotes. The authors have demonstrated a disorganization-reorganization cycle of the infraciliary system during division and regarded the electron-opaque "posts" at branching sites as organizing centres for assembling the lattice. It is possible that a similar process is involved in the infraciliary lattice assembly during regeneration of fragments. Cross-striated bands of micro filaments in the cortex of Paramecium are attached to the epiplasm of the inner alveolar membrane linking the bottoms of adjacent depressions. Their striated pattern with a periodicity of 100- 200 nm, straight course and regular arrangement indicate that they have contractile or supportive functions. It is evident that their existence is associated with their attachment to the membrane skeleton of the inner alveolar membrane (Fig. 1) since in fragments of Paramecium the remnants of these bands were found only at the margin of the wound together with alveolar debris (Fig. 10) while they were never observed free in the partly damaged cytoplasm. Their chemical composition has not been unraveled yet. Studies looking for actin in the cortex of Paramecium tetraurelia with the use of indirect immunofluorescence, labelling with heavy meromyosin and DNAase I (TIGGEMANN & PLATTNER 1981; COHEN et al. 1984), and R-phalloidin (KERKSEN et al. 1986b) all
332
R. JANISCH
demonstrated the presence of actin in the cortical layer. Of all the known cortical cytoskeletal structures, only areas close to kinetosomes were clearly marked for actin. Because crossstriated bands of microfilaments are attached to the alveolar membrane in the bottom of the depression close to the kinetosome, with 4-6 bands gathering at each kinetosome, it cannot be excluded that actin is part of just these structures. For a definite decision a technique with a higher degree of resolution will have to be employed. The granulofibrillar meshwork of an intact cortex fills the polygonal ridges delimiting kineties. It was severely disorganized in the area of defect (Fig. 9) reflecting the overall impainnent of the regular structure of polygons in the cortex adjacent to the wound, while being absent under the new plasma membrane covering the wound. The chemical composition of this cytoskeletal component still remains unknown but it has been suggested by some authors that several different proteins may be involved. Recent studies based on immunofluorescence techniques employing anti myosin antibody showed the presence of two proteins (130 kD and 50 kD) in Paramecium tetrauretia (COHEN et al. 1987). Repeated experiments with different antimyosin antibodies, however, failed to confirm the relation of these proteins to myosin. MOMAYEZI et al. (1986) investigated the presence of calmodulin by immunocytochemistry in Paramecium tetraurelia and they demonstrated it, apart from other sites, in the ridges of kinetides. Our recent studies (JANISCH 1988) with a polyclonal antibody prepared against actin from eggs of Xenopus showed that in Paramecium caudatum actin was also present in the cortex at the sites corresponding to the ridges of kinetides. COHEN et al. (1987) have demonstrated growth of the granulo-fibrillar meshwork during division without disruption of its framework. This growth is original and probably related to submembrane localization and function of the granulofibrillar meshwork as part of the membrane skeleton. They suggest that the meshwork stabilizes the cortex during division and channels surface growth and basal body positioning. Its own organization would, in tum, be dependent upon basal body duplication and spacing. Immunofluorescence studies following redistribution of actin in regenerating fragments of Paramecium caudatum (JANISCH et al. 1988) proved the accumulation of actin in the injured area within 2 min of merotomy. The reason for the increased amount of actin has not been found yet. It may be that mechanical injury induced the depolymerization of some cytoskeletal components which may have released actin, if this was present, which was subsequently used in the polymerization of new material. The formation of actin gel in the injured area could be involved in maintaining the fragment's integrity.
References ALLEN, R. D. (1971): Fine structure of membraneous and microfibrillar system in the cortex of Paramecium caudatum. J. Cell BioI. 49: 1-20. COHEN, J., GARREAU DE LOUBRESSE, N., & BEISSON, J. (1984): Actin microfilaments of Paramecium: localization and role in intracellular movements. Cell Mati!. 4: 443-468. - - KLOTZ, C., RUIZ, F., BODES, N., SANDOZ, D., BORNENS, M., & BEISSON, J. (1987): Organization and dynamics of a cortical fibrous network of Paramecium: the outer lattice. Cell Mati!. Cytoskeleton 7: 315-324. DIPPEL, R. V. (1964): Perpetuation of cortical structure and pattern in P. aurelia. Proc. 11th Int. Congr. Cell BioI.: 16-17. EHRET, C. F., & McARDLE, E. W. (1974): The structure of Paramecium as viewed from its constituent level of organization. In: W. J. VAN WAGTENDONK (ed.), Paramecium, A Current Survey, pp. 263-338. Amsterdam. - & POWERS, E. L. (959): The cell surface of Paramecium. lnt. Rev. Cytol. 8: 97 -133. GARREAU DE LOUBRESSE, N., KERYER, G., VIGUES, B., & BEISSON, J. (1988): A contractile cytoskeletal network of Paramecium: the infraciliary lattice. J. Cell Sci. 90: 351- 364.
Response of the Cortical Cytoskeleton
333
GRAIN, J. (1986): The cytoskeleton in protists: nature, structure and functions. Int. Rev. Cyto!. 104: 153-249. HUFNAGEL, L. (]969): Cortical ultrastructure of Paramecium aurelia: Studies on isolated pellic1es. J. Cell Bio!. 40: 779-801. JANISCH, R. (1964): Sub-microscopic structure of injured surface of Paramecium caudatum. Nature 204: 200-201. (1965): Regeneration of surface structures in Paramecium caudatum. Acta Protozoo!' 3: 363 - 367. (1967): Electron microscopy study of regeneration of surface structures of Paramecium caudatlllll. Folia Bio!. 13: 386- 392. (1974): Oriented embedding of single-cell organisms. Stain Techno!. 49: 157-160. (1985): Formation of the plasma membrane during regeneration of Paramecium cal/datum. Protoplasma 125: 94-102. (1987): Biomembranes in the life and regeneration of Paramecium. Bmo: 1. E. Purkyne University, 159 pp. (1989): The presence of actin in the cortex ofregenerating Paramecium caudatwll. Vest. Spo!. Zoo!. 53: 73. JURAND, A" & SELMAN, G. G. (1969): The Anatomy of Paramecium aurelia. New York. KERKSEN, H., MOMAYEZI, M., BRAUN, C., & PLATTNER, H. (l986a): Filamentous actin in Paramecium cells: functional and structural changes correlated with phalloidin affinity labelling in vivo. J. Histochem. Cytochem. 34: 455-465. VILMART-SEUWEN, J. , MOMAYEZI, M., & PLATTNER, H. (l986b): Filamentous actin in Paramecium cells: mapping by phalloidin affinity labelling in vivo and in vitro. J. Histochem. Cytochem. 34: 443-454. MOMAYEZI, M., KERKSEN, H., GRAS, U., VII.MART-SEUWEN, 1., & PLATTNER, H. (1986): Calmodulin localization from the in vivo to the ultrastructural level. J. Histochem. Cytochem. 34: 1621-1638. PITELKA, D., R. (1961): Fine structure of the silverline and fibrillar systems of three tetrahymenid ciliates. 1. Protozoa!' 8: 75-89. (1965): New observations on cortical ultrastructure in Paramecium. J. Microsc. 4: 373-394. (1969): Fibrillar structures of the ciliate cortex: the organization of kinetosomal territories. Proc. 3rd Int. Congr. Protozool. (Leningrad): 44-45. PLATTNER, H., WESTPHAL, C., & TIGGEMANN, R. (1982): Cytoskeleton-secretory vesicle interaction during the docking of secretory vesicles at the cell membrane in Paramecium aurelia cells. J. Cell BioI. 92: 368-377. REYNOLDS, E. S. (\ 963): The use of the lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell BioI. 17: 208-216. ROTH, L. E. (1958): A filamentous component of Protozoan fibrillar systems. J. Ultrastruct. Res. 1: 223-234. SEDAR, A. W., & PORTER, K. R. (1955): The fine structure of cortical components of Paramecium l1luitimicrolluc/eatum. J. Biophys. Biochem. Cytol. 1: 583-604. SUNDARARAMAN, V., & HANSON, E. D. (1976): Longitudinal microtubules and their functions during asexual reproduction in Paramecium tetraureiia. Genet. Res. Camb. 27: 205-211. TIGGEMANN, R., & PLATTNER, H. (1981): Localization of actin in the cortex of Paramecium tetraurelia cells by immuno- and affinity-fluorescence microscopy. Eur. J. Cell BioI. 24: 184-190. - - RASCHED, I., BAUERLE , P., & WACHTER, E. (1981): Quantitative data on peroxidatic markers for electron microscopy. J. Histochem. Cytochem. 29: 1387-1396. VAUDAUX, P. (1976): Isolation and identification of specific cortical proteins in Tetrahvmella pvriformis GL. J. Protozool. 23: 458-464. - WILLIAMS, N. E., FRANKEL, J., & VAUDAUX, C. (1977): Interstrain variability of structural proteins in Tetrahymena. J. Protozool. 24: 453-458. VILLENEUVE-BRACHON, S. (1940): Recherches sur des Cilies heterotriches. Arch. Zool. Exp. Gen. 82: 1-100. WICHTERMAN, R. (1987): The biology of Paramecium. New York, London. WII.LIAMS, N. E., VAUDAUX, P. E" & SKIVER, L. (1979): Cytoskelctal proteins of the cell surface in Tetrahymena. I. Identification and localization of major proteins. Exp. Cell Res. 123: 311- 320. Author's address: Dr. ROMAN JANISCH, Department of Biology, Faculty of Medicine, Masaryk University, Jostova 10, 66244 Bmo, Czechoslovakia.
Department of Biology, Medical Faculty, Masaryk University, Brno, Czechoslovakia
Response of the Cortical Cytoskeleton of Paramecium caudatum to Merotomy By ROMAN JANISCH With 10 Figures Key words: Cytoskeleton: Regeneration: Ultrastmcture: Paramecium
Summary Changes occurring within 2 min of merotomy in the cortical cytoskeleton of fragments cut from Paramecium caudatum were studied in thin sections. None of the microtubular components of the cortical cytoskeleton showed alterations suggestive of functional reorganization of microtubules and attributable to the mechanical damage made to the cell. Marked changes, however, were found in the amount and distribution of the infraciliary lattice. The bands of microfilaments were thicker and the meshes between them were reduced. This thickened layer penetrated deep into the wound under a newly-formed plasma membrane. The granulofibrillar meshwork, which fills the polygonal ridges in an intact cortex, was completely disorganized in fragments. Its remnants were always found in association with the plasma membrane or alveolar membranes. Cross-striated bands of microfilaments were retained only at the margin of the wound where they were attached to alveolar debris. Kinetodesmal fibrils maintained their integrity but their regular pattern of longitudinal bands was disturbed. The epiplasm (membrane skeleton) in the remnants of alveolar membranes was preserved and found also in some regions of the newly-formed plasma membrane. Little information is available on the chemical composition of these microfilamentous components and their function could only be the object of speculations. For instance the character of changes in the infraciliary lattice might suggest that its thickening was due to polymerization of new microfilaments which , together with wound contraction, were involved in the process of healing.
Introduction The cytoskeleton is a complex system based on proteins forming fibrillar supramolecular structures. Relationships between structural cytoskeletal components and their interaction with associated proteins allow the cell to perform the principal functions of the cytoskeletal system, which are structural support, movement and the flow of information. The structure and the function of each cytoskeletal component have been studied using a variety of models and experimental objects. Among these, protozoa assume a special position because of broad species diversity, high specialization of cellular structures and variability. Great diversity in the differentiation of protozoan cells resulted, in the course of evolution, in the development of numerous forms of cytoskeletal componens, which has no parallel in cells of multicellular organism (for review see GRAIN 1986). A considerable body of information has been obtained on the cytoskeletal structures of Paramecium, an infusorian, in which detailed studies on the ultrastructure of the cortical cytoskeleton have been carried out (ALLEN 1971, review by WICHTERMAN 1987). The cortical cytoskeleton is a heterogenous system of fibrillar components, such as microtubules, microfilaments, periodic fibres and membrane skeleton (epiplasm) which, in protozoa, comprises the surface layer called the cortex in infusoria. In addition to these structures this 21
Arch. Protistcnkd. Bd. 140.4
322
R. JANISCH
layer also involves membranous structures, i.e. , plasma membrane, alveoli and trichocyst trips. The pattern of subsurface alveoli has also been referred to as the hydroskeleton (GRAIN 1986). ALLEN reported as early as in 1971 that, apart from various microtubular structures, such as kinetosomes, ciliary axonemes, bands of postciliary and transversal microtubules, the cortex of Paramecium consisted of microfibrillar systems (granulofibrillar meshwork, crossstriated bands of microfilaments and infaciliary lattice), at that time beyond definition in terms of chemical structure, and periodic kinetodesmal fibrils and microfibrils associated with kinetosomes. The role of most of these structures is still not properly understood. Paramecium injured mechanically by cutting off its anterior or posterior parts in the range of Y3 to 1/4 of its body is capable of covering the exposed cytoplasm with a new plasma membrane within a few seconds. The process of plasma membrane formation and further healing of the defect have been studied by light and electron microscopy (JANISCH 1965 , 1967 , 1985 , 1987). One of the approaches to understanding the functions of the cytoskeletal structures in the cortex of Paramecium is the study of regenerating fragments. In this paper attention was focussed on changes in each component of the cortical cytoskeleton in Paramecium caudatwn. The investigation involved only the somatic region of the cortex without its specialized subsystems present at the sites of contractile vacuole pores, the cytoproct and the oral apparatus . For a better understanding , part 1 of Results gives a review on the present knowledge of the ultramicroscopic anatomy of the cortical cytoskeleton in intact Paramecium cells. A comparison with our findings following merotomy is presented in Part 2. The investigation into the nature and functions of the cytoske1eta1 components in Paramecium will later be completed with the results of our experiments involving specific antibodies to cytoskeletal proteins and inhibitors of protein polymerization which are under progress and will be reported elsewhere.
Material and Methods 1. Specimens A clone of Paramecium cauda tum isolated from a local source was grown in a medium with wheat grains 1940) and fed on Klebsiella aerogenes.
(VILLENEUVE-BRACHON
4
2. Merotomy Cell fragments were cut with a microscalpel made from a piece of razor blade fitted in a glass handle. Merotomy was performed by free hand under a dissecting microscope at a magnification of 40x. Fragments of about :y. of the original Paramecium cells were used for further studies.
Fig. I. Semitangetial section through the cortex of a normal cell of Paramecium caudatum showing various cytoskeletal structures. X 53,000 Fig. 2. A cross section of the Paramecium caudatum cortex from the area involving the cilium with a kinetosome . x73 ,000 Abbreviations in all figures: a - Alveolus; c - Ciliary axoneme; k - Kinetosome; pm - Plasma membrane: ia - Inner alveolar membrane ; oa - Outer alveolar membrane; e - Epiplasm; tp - Terminal plate; gf - Granulofibrillar meshwork; pc - Postciliary microtubules; tr - Transversal microtubules; sb - Bands of cross-striated microfilaments; if - Infraciliary meshwork: kd - Kinetodesma: fc - Connection linking kinetodesma to kinetosome; b - Connection between kinetosomes.
Response of the Cortical Cytoskeleton
21*
323
324
R.
JANISCH
Fig 3. A cross section through the wound margin with the cytoplasm overlapping the remnants of the marginal cortex. In some places the infraciliary lattice is seen as an almost continuous layer. The regular cortical pattern under the overlapping cytoplasm, i.e. , the system of alveoli, kinetosomes and other cytoskeletal structures, is completely disturbed. x 14,000
Response of the Cortical Cytoskeleton
325
3 . Electron Microscopy Within 2 min of merotomy Paramecium fragments were fixed with 2 % glutaraldehyde in a cacodylate buffer. pH 7.2, for 15 min and postfixed in I % OS04 in the same buffer. Each fragment was processed using the method of oriented embedding; thi s technique enabled us to make ultrathin sections parallel to the longitudinal axis of the fragment (JANISCH 1974). Ultrathin sections were made on a Reichert Ultracut E ultramicrotome , stained with lead and uranyl acetate (REYNOLDS 1963) and examined and photographed with a Tesla BS 500 electron microscope.
Results 1 . Ultrastructure of the Cortical Cytoskeleton The cortical cytoskeleton of Paramecium caudatum includes microtubular and microfilamentar structures, mostly chemically unidentified, periodic fibres and the membrane skeleton (epiplasm) of the plasma membrane and of alveoli.
1.1. Microtubules of the Cortical Cytoskeleton Microtubules in the Paramecium cortex are found mainly in kinetosomes and ciliary axonemes. Each single kinetosome or a pair of them have postciliary and transversal microtubules in their vicinity (Fig . 1). Most frequently 4 of them are assembled into flat bands . At division, during the ShOl1 period of cytodieresis (10 to 15 min), longitudinal bundles of 4 to 16 microtubules are formed in the cortical ridges; they are referred to as cytospindle or, according to their position over kinetodesmal fibrils, as suprakinetodesmal microtubules (JURAND & SELMAN 1969; EHRET & McARDLE 1974; SUNDARARAMAN & HANSON 1976). None of these structures studied in Paramecium fragments showed changes which could be related to surface defect healing or to cell regeneration.
1.2. Microfilamentous Structures The somatic cortex of Paramecium involves 4 categories of microfilamentous structures that have been gradually discovered and , under different names, described by the authors of many electron microscopic studies (PITELKA 1961 , 1965, 1969 ; EHRET & POWERS 1959; HUFNAGEL 1969; EHRET & McARDLE 1974) . Terms and classification used in this study have been introduced by ALLEN (1971). They are: granulofibrillar meshwork, cross-striated bands of microfilaments, infraciliary lattice, microfilaments linked to single kinetosomes or their pairs (Figs. 1, 2).
1.2 . 1. Granulofibrillar Meshwork It is made up of microfilaments, 30 to 40 nm long, linked to electron dense granules , 10 nm in diameter. The meshwork fills the upper parts of the polygonal ridges (Fig. 2). Along the sides, it is in contact with the membrane skeleton of the inner alveolar membrane and in the crests of the ridges it may communicate also with the membrane skeleton of the plasma membrane since alveoli there are slightly apart (about 150 nm). The chemical composition of
Fig. 4. A cross-section through the wound in the region where the wound edge is overflown with cytoplasm. The infraciliary lattice occasionally forms a continuous layer and is accumul ated in the centre of the wound. The pattern of kinetosomes. alveoli and kinetodesmal fibrils is disorganized. x 14 ,000
326
R.
JANISCH
Fig. 5. Ciliary axonemes embedded in the spilt cytoplasm. The centre of the picture is occupied with an axoneme still maintaining its typical arrangement of 9 + 2. In the left upper corner the remains of an axoneme are in the form several single and double microtubules. x 77 ,000 Fig. 6. Remains ofaxonemes embedded in the spilt cytoplasm. X40 ,OOO
Response of the Cortical Cytoskeleton
327
the granulofibrillar meshwork is not known. Indirect immunofluorescence methods, however, have shown the presence of proteins (130 and 50 kD) reacting with antimyosin antibody (COHEN et al. 1987) and some other components like actin (JANISCH 1988) and calmodulin (MOMAYEZI et al. 1986). The function of this structure is still discussed. Its position suggests involvement in maintaining the shape of polygonal ridges and perhaps the position of alveoli. COHEN et al. (1987) have shown that during division the granulofibrillar meshwork grows by longitudinal elongation without disruption of pre-existing meshes.
1.2.2. Cross-Striated Bands of Microfilaments The bands are 100 to 500 nm wide, oriented parallel to the organism's surface in the plane of the inner alveolar membranes where they are connected to the alveolar membrane skeleton (Figs. 1, 2). Each band, when cross-sectioned, shows a circular shape and is composed of microfilaments 6 nm in diameter. Electron-dense and electron-transparent parts alternate along the band due to the overlapping of microfilaments in darker parts (ALLEN 1971). Occasionally, fine bridges , about 10 nm long, are seen linking microfilaments lying close to each other. Nothing is known about their chemical composition but the presence of actin has been suggested (KERKSEN et al. 1986a, b).
1. 2.3. Infraciliary Lattice This structure was first described in silver-treated preparations by GELEI (1937) under the light microscope, later by many authors using electron microscopy (SEDAR & PORTER 1955; ROTH 1958; PITELKA 1965; HUFNAGEL 1969; JURAND & SELMAN 1969). The most detailed study was made by ALLEN (1971). This system is composed of bundles , 70 to 100 nm thick, which branch forming a lattice of irregular polygons. Each of these involves a single kinetosome or a pair of them or a single trichocyst (Fig . I) . At the site of bifurcation the bundles may become up to 400 nm wide . An electron-opaque rod of triangular cross-section, called a "post" by ALLEN (1971), is present at the centre of the branching site. The basic element of the bundle is a microfilament about 4 nm wide. These filaments are organized into tubules that appear in cross-sections as closely packed hexagons. The infraciliary lattice is situated parallel to the surface in the plane of proximal kinetosomal ends. GARREAU DE LOUBRESSE et al. (1988) have found two (23 to 24 kD) polypeptides which were shown to cross-react with a polyclonal antibody raised against 22 to 23 kD Ca2 + -binding proteins isolated from the microfibrillar ectoplasmic boundary of lsotricha. The authors suggest that the polypeptides belong to the class of contractile proteins.
Fig. 7. A cross-section through the cortex in the marginal part of the wound. The course of cross-striated kinetodesmas is unusual but their more or less parallel pattern is still preserved. X42.000 Fig. 8. A detailed view of the thickened infraciliary lattice shows a widening of bands up to 160 nm and increase in microfilament density within the band. x58.000
328
R.
JANISCH
Fig. 9. The granulofibrillar meshwork at the edge of the wound is disorganized. It consists of various irregular aggregates under the remnants of alveoli and plasma membrane. x 64,000 Fig . 10. Cross-striated bands of microfilaments are associated with alveoli at the wound edge. The granulofibrillar meshwork and kinetodesmas are disarranged. x 35,000
Response of the Cortical Cytoskeleton
329
1.2.4. Microfilaments Originating from Single or Paired Kinetosomes In the somatic part of the cortex, kinetosomes are found in pairs. A fine microfilamentous link extends from the right side of the anterior kinetosome and joins a kinetodesmal fibril radiating from the posterior kinetosome of the same pair (Fig. 1). The microfilaments are found in the proximal plane of kinetosomes. So far there are no data on either their function or chemical nature. Two more types of filamentous structures associated with kinetosomes that have a periodic character are reported under the following headings.
1.3. Periodic Fibres All three types of periodic fibres in the somatic part of the Paramecium cortical cytoskeleton are connected with kinetosomes. The most conspicuous, and also known for the longest time, are kinetodesmal fibrils or kinetodesmas (Fig. 1). They arise from the kinetosome of each kinety extending from its anterior right quadrant. If two kinetosomes are present, the kinetodesmal fibril starts from the rear one (DIPPEL 1964). The kinetodesma runs anteriorly and rightwards taking a longitudinal course at the kinety's right side over a distance of 8 nm, always passing 5 to 6 kineties located ahead. At the end attached to the kinetosome it is wider, gradually getting narrower. On a longitudinal section, each kinetodesma shows a cross-striated pattern with a periodicity of 23 to 27 nm. Each kinetodesmal fibril consists of many elementary microfilaments (HUFNAGEL 1969) of unknown function and chemical composition. The two other types of periodic fibres (Fig. 1) are seen as links, pursuing an oblique course, between the kinetosomes of the same pair (ALLEN 1971). These fibres are located in the plane of the proximal ends of kinetosomes. Their function and chemical composition are not known.
1.4. Epiplasm - the Membranous Skeleton It occurs as an electron dense layer, fibrogranular in appearance, situated on the cytoplasmic side of the inner alveolar membrane (Fig. 2). It continues into the terminal plate of the kinetosome. It is less distinct between the plasma membrane and the outer alveolar membrane mostly due to a reduced space between the membranes. In Paramecium its chemical structure is unknown. In some other infusoria like Tetrahymena, the epiplasm has been reported to contain proteins of 122 to 145 kD (VAUDAUX 1976: VAUDAUX et al. 1977). A small amount of actin is also likely to be present (WILLIAMS et al. 1979). The role for the membrane skeleton in infusoria is still open to speculation. It can be assumed that it is involved in maintaining the overall architecture of membranous components of the cortex and in mediating contact with other cytoskeletal components (kinetosomes, transversal and postciliary microtubules, bands of cross-striated microfilaments) and possibly with some other organelles (mitochondria, trichocysts) situated closely under the organism's surface.
2. Changes in the Cortical Cytoskeleton after Merotomy Removal of a part of the Paramecium cell with a microscalpel was a severe intervention which brought about partial damage to or complete destruction of many of the cell's structures. In spite of it, most of the fragments were capable of covering the exposed cytoplasm with a new plasma membrane within a few seconds. In some fragments (Figs. 3, 4) the cytoplasm with the new membrane spilled and overlaid the damaged cortex (JANISCH 1985).
330
R.
JANISCH
2.1. Microtubules The microtubular component of the cortical cytoskeleton did not show any changes indicative of functional reorganization. All alterations in the tubular structures found in fragments in the area of injury consisted in disorganization and dislocation due to mechanical pressure produced by the microsurgical instrument. The cortex round the injured area had only a few kinetosomes which were unevenly distributed and assumed various atypical positions. Their integrity, however, was retained. In the displaced kinetosomes it was not possible to identify postciliary and transverse bands of microtubules (Figs. 5, 6). Such kinetosomes also lacked cilia. Occasional ciliary axonemes embedded in the cytoplasm were seen in the affected area, with their typical pattern of 9 + 2 still preserved. 2.2. 1nfraciliary Lattice The most marked changes in fragments were seen in the structure of the infraciliary lattice. In contrast to the intact cortex with a thin meshwork including kinetosomes or trichocyst, fragments showed a much thicker layer of the infraciliary lattice at the margins of the injured region; it penetrated deep into the wound under the newly formed plasma membrane (Figs. 3, 4). Bundles of infraciliary lattice were wider and denser (Fig. 8). On sections cut perpendicularly to the plane of the infraciliary lattice, this structure appeared as a wide electron-dense layer interrupted with short gaps. Reduced meshes of the lattice in this region did not contain kinetosomes or trichocysts. From these results it was not possible to draw a conclusion as to the mechanism controlling the changes in the structure of the lattice. Immediately after merotomy it was observed that the area of defect, boundered by a partly damaged cortex, contracted to a certain degree. This was related to the thickening of the infraciliary lattice, but the high degree of thickening of all bundles associated with an increase in electron density cannot be explained by mere contraction. It is open to question to what extent the increased density of the infraciliary lattice can be due to polymerization of new components. 2.3. Other Cytoskeletal Components The granulofibrillar meshwork, which is present in the crests of polygonal ridges of an intact cortex, was completely disorganized in fragments (Figs. 9, 10). Only occasionally it was still attached to the remnants of plasma membrane or alveolar membranes at the wound edges. The extent of changes in the whole amount of meshwork resulting from injury could not be estimated in ultrathin sections. Cross-striated bands of microfilaments were found in injured areas only in association with alveolar remnants (Fig. 10). Since they were never seen in the absence of alveoli, it can be concluded that, once separated from alveoli by merotomy, they disintegrated. Kinetodesmal fibrils retained integrity in the injured region but their regular pattern of longitudinal bundles was disturbed completely in both regularity and orientation (Figs. 7, 10). The epiplasm of alveolar membranes in the injured area remained unaffected (Figs. 9, 10). Of interest was the finding of the epiplasm in some parts of the newly formed plasma membrane covering the cytoplasm exposed by injury. The response to merotomy of the other components of the cortical cytoskeleton (postciliary and transversal bands of microtubules, microfilaments associated with kinetosomes) could not be estimated by means of the technique used.
Response of the Cortical Cytoskeleton
331
Discussion Merotomy in Paramecium results in an extensive defect in the cell surface the healing of which is a prerequisite for further viability of the fragment. Factors most important for healing are: rapid formation of a new plasma membrane covering the exposed cytoplasm, contraction of the wound, and quick reaction of the cytoskeletal structures preventing the cell content from spilling in the first moments after merotomy. The first step, i.e. plasma membrane formation, has been the subject of our previous studies (JANISCH 1965, 1967, 1985, 1987). The contraction of the wound and the response of cytoskeletal structures to injury appear to be closely related processes. One of our earlier studies on fragments of Paramecium employing ultrathin sectioning (JANISCH 1964) showed the presence of a discontinued layer of osmiophilic material at the boundary between the necrotic and intact cytoplasm in the injured area. However, the application of methacrylate as an embedding medium and an insufficient resolution of the electron microscope employed at that time did not allow us to identify the structures more exactly. From examination of ultrathin sections in this study it is now apparent that the osmiophilic layer is a thickened and enlarged infraciliary lattice (Figs. 3, 4, 8). The response of this cytoskeletal component is the most marked of all. Its function however, is still very poorly understood. Actin detected by immuno-fluorescence in the cortex of Paramecium has often been explained as its mere accumulation in the infraciliary lattice (TIGGEMANN & PLATTNER 1981; TIGGEMANN et al. 1981; PLATTNER et al. 1982). COHEN et al. (1984), on the other hand, failed to prove actin labelled with heavy meromyosin in the infraciliary lattice. As seen in Fig. 11 of the work by KERKSEN et al. (J 986 a) a thick layer involving bands of microfilaments was produced after microinjection with CaCl 2 in Paramecium tetraurelia. These cortical microfilaments most probably correspond to the enlarged bands of the infraciliary lattice seen in the fragments under study. The thickening of the infraciliary lattice in Paramecium after microinjection may be related to the contraction of the cortex at the site of injection, as is evident from the picture in the paper by KERKSEN et al. It is yet to be decided what is the role of Ca2 + -ions in the contraction and what is the involvement of mechanical injury by a microinstrument, which must also be taken into consideration. Structural changes in the infraciliary lattice during reparation of fragments may be due to contraction of the wound circumference immediately after merotomy or to polymerization of new microfilaments. The appearance and intensity of enlargement of the infraciliary lattice may be the result of both. GARREAU DE LOUBRESSE et al. (1988) have shown that the infraciliary lattice displays contractile properties and that its assembly is Ca2+ -dependent. Perhaps the infraciliary lattice is a new example of the non-actin contractile system found in lower eukaryotes. The authors have demonstrated a disorganization-reorganization cycle of the infraciliary system during division and regarded the electron-opaque "posts" at branching sites as organizing centres for assembling the lattice. It is possible that a similar process is involved in the infraciliary lattice assembly during regeneration of fragments. Cross-striated bands of micro filaments in the cortex of Paramecium are attached to the epiplasm of the inner alveolar membrane linking the bottoms of adjacent depressions. Their striated pattern with a periodicity of 100- 200 nm, straight course and regular arrangement indicate that they have contractile or supportive functions. It is evident that their existence is associated with their attachment to the membrane skeleton of the inner alveolar membrane (Fig. 1) since in fragments of Paramecium the remnants of these bands were found only at the margin of the wound together with alveolar debris (Fig. 10) while they were never observed free in the partly damaged cytoplasm. Their chemical composition has not been unraveled yet. Studies looking for actin in the cortex of Paramecium tetraurelia with the use of indirect immunofluorescence, labelling with heavy meromyosin and DNAase I (TIGGEMANN & PLATTNER 1981; COHEN et al. 1984), and R-phalloidin (KERKSEN et al. 1986b) all
332
R. JANISCH
demonstrated the presence of actin in the cortical layer. Of all the known cortical cytoskeletal structures, only areas close to kinetosomes were clearly marked for actin. Because crossstriated bands of microfilaments are attached to the alveolar membrane in the bottom of the depression close to the kinetosome, with 4-6 bands gathering at each kinetosome, it cannot be excluded that actin is part of just these structures. For a definite decision a technique with a higher degree of resolution will have to be employed. The granulofibrillar meshwork of an intact cortex fills the polygonal ridges delimiting kineties. It was severely disorganized in the area of defect (Fig. 9) reflecting the overall impainnent of the regular structure of polygons in the cortex adjacent to the wound, while being absent under the new plasma membrane covering the wound. The chemical composition of this cytoskeletal component still remains unknown but it has been suggested by some authors that several different proteins may be involved. Recent studies based on immunofluorescence techniques employing anti myosin antibody showed the presence of two proteins (130 kD and 50 kD) in Paramecium tetrauretia (COHEN et al. 1987). Repeated experiments with different antimyosin antibodies, however, failed to confirm the relation of these proteins to myosin. MOMAYEZI et al. (1986) investigated the presence of calmodulin by immunocytochemistry in Paramecium tetraurelia and they demonstrated it, apart from other sites, in the ridges of kinetides. Our recent studies (JANISCH 1988) with a polyclonal antibody prepared against actin from eggs of Xenopus showed that in Paramecium caudatum actin was also present in the cortex at the sites corresponding to the ridges of kinetides. COHEN et al. (1987) have demonstrated growth of the granulo-fibrillar meshwork during division without disruption of its framework. This growth is original and probably related to submembrane localization and function of the granulofibrillar meshwork as part of the membrane skeleton. They suggest that the meshwork stabilizes the cortex during division and channels surface growth and basal body positioning. Its own organization would, in tum, be dependent upon basal body duplication and spacing. Immunofluorescence studies following redistribution of actin in regenerating fragments of Paramecium caudatum (JANISCH et al. 1988) proved the accumulation of actin in the injured area within 2 min of merotomy. The reason for the increased amount of actin has not been found yet. It may be that mechanical injury induced the depolymerization of some cytoskeletal components which may have released actin, if this was present, which was subsequently used in the polymerization of new material. The formation of actin gel in the injured area could be involved in maintaining the fragment's integrity.
References ALLEN, R. D. (1971): Fine structure of membraneous and microfibrillar system in the cortex of Paramecium caudatum. J. Cell BioI. 49: 1-20. COHEN, J., GARREAU DE LOUBRESSE, N., & BEISSON, J. (1984): Actin microfilaments of Paramecium: localization and role in intracellular movements. Cell Mati!. 4: 443-468. - - KLOTZ, C., RUIZ, F., BODES, N., SANDOZ, D., BORNENS, M., & BEISSON, J. (1987): Organization and dynamics of a cortical fibrous network of Paramecium: the outer lattice. Cell Mati!. Cytoskeleton 7: 315-324. DIPPEL, R. V. (1964): Perpetuation of cortical structure and pattern in P. aurelia. Proc. 11th Int. Congr. Cell BioI.: 16-17. EHRET, C. F., & McARDLE, E. W. (1974): The structure of Paramecium as viewed from its constituent level of organization. In: W. J. VAN WAGTENDONK (ed.), Paramecium, A Current Survey, pp. 263-338. Amsterdam. - & POWERS, E. L. (959): The cell surface of Paramecium. lnt. Rev. Cytol. 8: 97 -133. GARREAU DE LOUBRESSE, N., KERYER, G., VIGUES, B., & BEISSON, J. (1988): A contractile cytoskeletal network of Paramecium: the infraciliary lattice. J. Cell Sci. 90: 351- 364.
Response of the Cortical Cytoskeleton
333
GRAIN, J. (1986): The cytoskeleton in protists: nature, structure and functions. Int. Rev. Cyto!. 104: 153-249. HUFNAGEL, L. (]969): Cortical ultrastructure of Paramecium aurelia: Studies on isolated pellic1es. J. Cell Bio!. 40: 779-801. JANISCH, R. (1964): Sub-microscopic structure of injured surface of Paramecium caudatum. Nature 204: 200-201. (1965): Regeneration of surface structures in Paramecium caudatum. Acta Protozoo!' 3: 363 - 367. (1967): Electron microscopy study of regeneration of surface structures of Paramecium caudatlllll. Folia Bio!. 13: 386- 392. (1974): Oriented embedding of single-cell organisms. Stain Techno!. 49: 157-160. (1985): Formation of the plasma membrane during regeneration of Paramecium cal/datum. Protoplasma 125: 94-102. (1987): Biomembranes in the life and regeneration of Paramecium. Bmo: 1. E. Purkyne University, 159 pp. (1989): The presence of actin in the cortex ofregenerating Paramecium caudatwll. Vest. Spo!. Zoo!. 53: 73. JURAND, A" & SELMAN, G. G. (1969): The Anatomy of Paramecium aurelia. New York. KERKSEN, H., MOMAYEZI, M., BRAUN, C., & PLATTNER, H. (l986a): Filamentous actin in Paramecium cells: functional and structural changes correlated with phalloidin affinity labelling in vivo. J. Histochem. Cytochem. 34: 455-465. VILMART-SEUWEN, J. , MOMAYEZI, M., & PLATTNER, H. (l986b): Filamentous actin in Paramecium cells: mapping by phalloidin affinity labelling in vivo and in vitro. J. Histochem. Cytochem. 34: 443-454. MOMAYEZI, M., KERKSEN, H., GRAS, U., VII.MART-SEUWEN, 1., & PLATTNER, H. (1986): Calmodulin localization from the in vivo to the ultrastructural level. J. Histochem. Cytochem. 34: 1621-1638. PITELKA, D., R. (1961): Fine structure of the silverline and fibrillar systems of three tetrahymenid ciliates. 1. Protozoa!' 8: 75-89. (1965): New observations on cortical ultrastructure in Paramecium. J. Microsc. 4: 373-394. (1969): Fibrillar structures of the ciliate cortex: the organization of kinetosomal territories. Proc. 3rd Int. Congr. Protozool. (Leningrad): 44-45. PLATTNER, H., WESTPHAL, C., & TIGGEMANN, R. (1982): Cytoskeleton-secretory vesicle interaction during the docking of secretory vesicles at the cell membrane in Paramecium aurelia cells. J. Cell BioI. 92: 368-377. REYNOLDS, E. S. (\ 963): The use of the lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell BioI. 17: 208-216. ROTH, L. E. (1958): A filamentous component of Protozoan fibrillar systems. J. Ultrastruct. Res. 1: 223-234. SEDAR, A. W., & PORTER, K. R. (1955): The fine structure of cortical components of Paramecium l1luitimicrolluc/eatum. J. Biophys. Biochem. Cytol. 1: 583-604. SUNDARARAMAN, V., & HANSON, E. D. (1976): Longitudinal microtubules and their functions during asexual reproduction in Paramecium tetraureiia. Genet. Res. Camb. 27: 205-211. TIGGEMANN, R., & PLATTNER, H. (1981): Localization of actin in the cortex of Paramecium tetraurelia cells by immuno- and affinity-fluorescence microscopy. Eur. J. Cell BioI. 24: 184-190. - - RASCHED, I., BAUERLE , P., & WACHTER, E. (1981): Quantitative data on peroxidatic markers for electron microscopy. J. Histochem. Cytochem. 29: 1387-1396. VAUDAUX, P. (1976): Isolation and identification of specific cortical proteins in Tetrahvmella pvriformis GL. J. Protozool. 23: 458-464. - WILLIAMS, N. E., FRANKEL, J., & VAUDAUX, C. (1977): Interstrain variability of structural proteins in Tetrahymena. J. Protozool. 24: 453-458. VILLENEUVE-BRACHON, S. (1940): Recherches sur des Cilies heterotriches. Arch. Zool. Exp. Gen. 82: 1-100. WICHTERMAN, R. (1987): The biology of Paramecium. New York, London. WII.LIAMS, N. E., VAUDAUX, P. E" & SKIVER, L. (1979): Cytoskelctal proteins of the cell surface in Tetrahymena. I. Identification and localization of major proteins. Exp. Cell Res. 123: 311- 320. Author's address: Dr. ROMAN JANISCH, Department of Biology, Faculty of Medicine, Masaryk University, Jostova 10, 66244 Bmo, Czechoslovakia.