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Immunofluorescence detection of microtubular structures in Paramecium caudatum by means of monoclonal antibody TU-01
Arch. Protistenkd. 137 (1989): 309-316 VEB Gustav Fischer Verlag lena
Department of Biology, Medical Faculty, Purkyne University, Brno, Czechoslovakia
Immunofluorescence Detection of Microtubular Structures in Paramecium caudatum by Means of Monoclonal Antibody TU-Ol By ROMAN JANISCH With 8 Figures Key words: Immunofluorescence; Microtubules; Paramecium caudiltum
Summary Microtubular structures of the cortical cytoskeleton in Paramecium caudatum were identified by means of indirect immunofluorescence employing the monoclonal antibody TU-OJ. Specimens for observation were fixed with glutaraldehyde in a layer attached to a slide or made into a suspension and without fixation, permeabilized with Triton X-lOO. The antibody TU-Ol reacted with kinetosomes, bundles of postoral microtubules and microtubules of somatic cilia along their whole length. The cells on a slide were well preserved though often deformed in shape and poorly extracted. The cells in suspension, on the other hand, were better extracted but about 10 % of them became disrupted. The resultant fragments were used with advantage for studying organization of microtubular structures. Immunofluorescence labelling with TV-Ol is a useful tool in the study of the cortical cytoskeleton in, for instance, morphological mutants or abnormal cells of paramecia, and in investigations into the behaviour of the cytoskeleton during repair processes occurring in mechanically injured cells.
Introduction Visualization of various cellular structures by means of immunofluorescence techniques is a valuable and exact approach to research in cytology. These methods have also found broad application in investigation of the cell cytoskeleton, its structure and function. Among many model objects used in studying the cytoskeleton, infusoria of the genus Paramecium play an important role. Their advantages include a relatively large size and specific arrangement of the cortical cytoskeleton. On the other hand, the numerous microtubular structures present in the cytoplasm of a large cell interfere with immunofluorescent staining of the cortical structures. The cortex of Paramecium has been studied extensively and its ultrastructure has been described in great detail in many papers (PITELKA 1969; ALLEN 1971; EHRET & MAC ARDLE 1974). The model makes it possible to study changes in structure of the cortical cytoskeleton during cell reproduction (COHEN et a1. 1982), following treatment with various inhibitors of metabolism (COHEN et a!. 1984; KERKSEN et al. 1986a, b) or toxic substances, or after mechanical injury (JANISCH 1987). The cortical cytoskeleton of Paramecium consists of many microfilamentous and microtubular components. The latter include ciliary axonemes, kinetosomal microtubules, bundles of short postciliary and transverse microtubules (ALLEN 1971) and suprakinetodesmal microtubules, "a cytospindle" present at cytodieresis (EHRET & MAC ARDLE ]974; SUNDARARAMANN & HANSON 1976) and disappearing afterwards. The oral cytoskeleton is characterized mainly by bundles of microtubules originating from the right posterior wall of the buccal cavity and reaching the posterior cell pole (ALLEN 1974), referred to as postoral microtubules or earlier, postesophageal fibrils (LUND 1933). Electron microscopic studies have demonstrated two more units of 21
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microtubular components belonging to the oral apparatus, namely, bundles of 2 -4 microtubules of the right cytosome lip found below the right ribbed wall of the buccal cavity, and flat bands composed of 10-12 microtubules running along the left cytostome lip. The part of the buccal cavity shows 3 quadruple rows of cilia; one is calculated the quadrulus, the other two peniculi. During karyokinesis, microtubules have also been seen in the nucleus. Micronuclear division, which occurs without disruption of the nuclear envelope, involves successively two types of spindles: an orthodox mitotic spindle, and then a long separation spindl~ which pushes the two daughter micronuclei apart to the opposite poles of the cell (TUCKER 1979; GRANDCHAMP & BEISSON 1981). Segregation of genomes in the macronucleus is accompanied by the appearance of microtubule bundles inside the nucleus (TUCKER et al. 1980). These are apparently involved in the process of macronuclear elongation. All the structures described here have been identified by electron microscopy. Apart from them, the cytoplasm of Paramecium contains many more transient microtubular structures. The first attempt at detecting microtubular structures in Paramecium by means of immunofluorescence with the use of polyclonal antibody was published by COHEN et al. (1982). The authors were able to visualize some of the cortical microtubules and, for the first time demonstrate with the immunofluorescence technique dynamic changes in the microtubular system during the cell cycle in Paramecium. Preliminary results on the reaction of TU-OI monoclonal antibody with tubulin bands in Paramecium caudatum were published as an abstract in the proceedings of a conference (JANISCH & HASEK 1985). In the present paper a method of immunocytochemical visualization of microtubular structures in Paramecium caudatum is described and the specifity of reaction between monoclonal antibody and tubulin in this species is discussed.
Material and Methods a) Model objects and culture conditions Cells of Paramecium caudatum were isolated from a local source and cultivated in a medium with wheat grains according to VILLANEUVE-BRACHON (1940). The culture in Petri dishes was regularly fed on Klebsiella pneumoniae.
b) Antibody To visualize tubulin, a monoclonal TV-OI antibody described by VIKLlCKY et al. (1982) and produced by the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague was used. Indirect fluorescence was based on a secondary antibody SwAM-FITC (Institute for Sera and Vaccines, Prague).
c) Preparation of specimens Essentially, two techniques were used. Cells were exposed to antibody either in suspension or in a layer adhering to a slide. Advantages and disadvantages of each approach are discussed in the section "Results and Discussion" .
The slide method Paramecium cells were rinsed in phosphate buffer solution (PBS - 0.8g NaCl, 0.2 g KCI, 1.15 g NA2 HP04 , 0.2 g KH 2 P04 in 1,000 mr H2 0, pH 7.4) for 10 min. A drop with infusoria was spread onto a slide over an area corresponding in size to that of a cover slip and allowed to dry. Shortly before the layer dried completely, a drop of fixative was added. This consisted of2 % glutaraldehyde and 0.5 % Triton X- 100 in PBS. The cells were then treated with cold methanol for 8 min and with acetone for 30 s and exposed to three reduction procedures in 0.5 % NaBH4 lasting 5, 5, and 10 min, respectively, at 4°C. This was followed by washing the cell layer in PBS containing 2 %
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bovine serum albumin (BSA). The cells were then exposed to TU-Ol monoclonal antibody (at a dilution of 0.1 mg/ml) for 30 min at 37°C. After three washes with PBS the cells were treated with the secondary antibody SwAM-FITC (concentration 0.4 mg/ml) for 45 min at 3rC and then washed with PBS and mounted in 90% glycerol.
The suspension method The procedure was based on a modified method by COHEN et al. (1982). After preceding deciliation with two rinses in 10 mM MnCh, the cells were subjected to permeabilization treatment with 0.5 % Triton X-IOO in a buffer of the following composition: 60 mM PIPES, 25 mM EGTA and 2 mM MgCl 2 (SCHLIWA & VAN BLERKOM 1981). This buffer, further referred to as PHEM, was then used to dilute antibodies and wash specimens. After 20-30 min of permeabilization, the cells were rinsed 3 times 5 min in PHEM and incubated in TU-Ol antibody with 2 % BSA for 30 min. Then the cells were rinsed 3 times 5 min in PHEM and incubated ion SwAM-FITC antibody followed by mounting in 90 % glycerol. All procedures were carried out in small centrifuge tubes at low centrifugation speed.
d) Fluorescence microscopy and microphotography The specimens were examined and photographed with a lenalumar fluorescence microscope at 500 or 1,000 X magnification using Foma 21 DIN negative material.
Results and Discussion The presence of microtubules in cilia and kinetosomes, and of postoral microtubular bundles originating from the oral apparatus was demonstrated in paramecium cells with both methods of cell preparation described in the paper. Ciliary microtubules were stained with TU-Ol antibody along the whole length of the cilium (Figs. 1, 2). No difference in labelling was found between cilia from different surface regions. The cilia of the oral apparatus, i.c. the quadrulus and peniculi, however, failed visualization (Fig.
3,4).
Fig. 1. A cell of Paramecium caudatum treated with TU-Ol and SwAM-FlTC in suspension without deciliation.
Tubulin in the ciliary axonemes reacts with antibody along the whole length of the cilium. Focussed on the middle plane of the cell. Fig. 2. The same preparation as in Fig. 1 but after focussing on the upper plane of the cell. Fig. 3. A region of the oral apparatus of a Paramecium caudatum cell prepared in the same way as in Fig. l. Flourescent strips correspond to the quadrulus and peniculi. Single kinetosomes can not be recognized due to their high density. A small area of the somatic cortex with kinetosomes adjoins these structures. Cilia are labelled only in the somatic region. Fig. 4. A region of the oral apparatus of a paramecium cell adhering to a slide. The strips of kinetosomes (quadrulus and peniculi) are labelled but the cilia are not. Flourescence of intracellular structures interferes with the labelled structures. Fig. 5. A part of the dorsal surface of Paramecium caudatum treated with TU-OI and SwAM-FITC in suspension after deciliation. Kinetosomes are seen as dots alTanged in parallel rows. Fig. 6. A part of the ventral surface of a paramecium cell prepared by the same technique as in Fig. 5 but without deciliation. The tlourescent dots correspond to pairs of kinetosomes with adjoining transverse and postciliary microtubules. Figs. 7 and 8. A fragment of the paramecium cortex with an isolated oral apparatus. The object was prepared in suspension. Besides the quadrulus and peniculi there are bundles of postoral microtubules. Each figure shows a different focal plane in the same object. 21*
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Kinetosomal microtubules were seen as bright fluorescent dots corresponding to the whole kinetosomes (Figs. 5, 6). Both the kinetosomes of the somatic cilia and the kinetosomes of the oral apparatus showed the same intensity of fluorescence (Fig. 3). Fluorescence staining was particularly intense in the bundles of postoral microtubules originating from the oral apparatus and running through the endoplasm of the posterior end towards the cytoproct. In whole cells it is difficult to identify the postoral microtubules due to the fluorescence of tubulin from the interior of the cell. However, postoral microtubules usually remain attached to the oral apparatus on occasional fragmentation of cells during permeabilization treatment with Triton X-lOa (Figs. 5,7,8). Within the oral skeleton, however, it was not possible to determine exactly which other microtubular structures, apart from those described above, were visualized by immunofluorescence. Nor was it possible to detect the short bundles of transverse and postciliary microtubules identified by electron microscopy near all somatic kinetosomes. Cells processed on a slide provide a good insight into the details of mcirotubular structures in various regions of the cortex. However, they are not suitable for studying the overall pattern of the cortical cytoskeleton for two reasons: the cells are considerably deformed during the drying procedure, and there is often a nonspecific fluorescence of the intracellular structures due to the glutaraldehyde fixation. Cells prepared in suspension, on the other hand, give a comprehensive picture of the overall distribution of microtubular structures in the cortex of an infusorian. The detection of microtubular structures in Paramecium tetraurelia by means of immunofluorescence was first reported by COHEN et al. (1982). Using a specific antiserum produced against tubulin isolated from axonemes of paramecia, they demonstrated the presence of the tubulin in the cytospindle during nuclear division, and in postoral fibrils at the time of interphase. Monoclonal antibodies against tubulin have so far been produced in many laboratories; they differ mostly in their reactivity with alpha or beta subunits of the porcine brain tubulin. Besides, even antibody against one of the subunits shows varying reactivity with different antigenic determinants of this subunit. The TU-Ol antibody was prepared by VIKLICKY et al. (1982) as a specific monoclonal antibody to the alpha subunit of the porcine brain tubulin. It has been demonstrated that the antibody binds to a tubulin epitope present in higher eukaryote, such as mammals, birds, and amphibians, as well as in fungi, echinodermata, worms and slime molds. In protozoa, the reactivity of tubulin antibody with microtubules has been shown in Trichomonas vaginalis (DRABER et al. 1985). Apparently, antigenic determinants for this antibody are either missing completely in many eukaryotic microorganisms or are present only in some of the microtubules. Of the Flagellata so far tested, i.e. Herpetomonas, Leishmania and Trichomonas, positive results were obtained only for some microtubular structures of Trichomonas, namely, axostyles, mitotic spindle, distal parts of the flagellum. TU-Ol antibody bound to ciliary tubulin was first reported in Paramecium caudatum by JANISCH & HASEK (1985). Although this immunocytochemical procedure was affected by imperfect methods of extraction and permeabilization, the reaction of TU-Ol with tubulin of cilia was clearly demonstrated and the specifity of the reaction was confirmed by SDS-polyacrylamide gel electrophoresis and immunoblotting. With the use of TU-Ol antibody, tubulin in Paramecium caudatum has been visualized only in some of the known microtubular structures. TU-Ol binds to axonemes along the whole length of the cilium. The absence of tubulin labelling in ciliary axonemes of the oral apparatus suggests a different tubulin epitope in these structures. Some of the cilia in the oral region have been reported to resist deciliating agents (KUZNICKI 1963; OGURA 1981) or immobilizing substances (SIKORA & WASIK 1978) for a long time. DRABER et al. (1985) demonstrated tubulin staining only in distal parts of the flagellum in Trichomonas vaginalis. In this organism, no TU-Ol labelling was recorded in microtubules of flagellar kinetosomes. In Paramecium caudatum, TU-Ol bound to tubulin of all kinetosomes of the somatic and oral regions of the cortex. In comparison with the results of COHEN et al. (1982) TU-Ol failed to label bundles of short transverse and postciliary microtubules situated near kinetosomes. This can be explained by either a lower resolution of the method used or a different specifity of the antibody. COHEN et al. (1982) reported immunofluorescence staining of all the microtubular structures consisting of at least 3~4 microtubules.
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OSBORN et al. (1978) described visualization of single microtubules by indirect immunofluorescence even in flattened fibroblasts spread on a slide during incubation. Even after a complete extraction, a paramecium cell is still a very thick object, in which the strong diffuse fluorescence of cytoplasmic microtubules diminishes the contrast of fluorescence shown by cortical microtubules and thus hinders the discernment of details. The resolution of the indirect immunofluorescence method applied to whole cells is related to the degree of extraction of Triton X-lOO. Only optimal extraction and permeabilization will permit the tubulin antibody to penetrate through the cortical membranes and will avoid nonspecific fluorescence shown by intracellular structures. For this reason cells processed in suspension are more suitable objects, since they retain their shape, i.e., an unchanged pattern of the cortical cytoskeleton. Extraction is more thorough than in those attached to a slide. In a certain proportion of cells (about 10%) treatment with Triton X-IOO results in their disruption, which enables one to study cortical fragments, such as isolated oral apparatus with attached cytoskeletal components. The method of extraction and fixation of cells adhering to a slide proved useful in many small organisms e.g. yeast cells or their protoplasts (HASEK et al. 1986). In larger cells like paramecia, attachment to a slide results in preservation of incompletely extracted parts in the cell which interfere with visualization of the cortical cytoskeleton. Immunocytochemical observations in infusoria allow us to gain insight into the overall morphology and microtubular patterning of the cortical cytoskeleton together with its changes due to morphological mutations or shape abnormalities. Using indirect immunofluorescence, WILLIAMS & HONTS (1987) were able to demonstrate abnormalities in the development of the oral cytoskeleton in a temperature sensitive mutant of Tetrahymena thermophila. This technique may also prove a useful tool in studying changes in the cytoskeleton during repair processes of mechanically injured cells.
Conclusions 1. The TU-O I monoclonal antibody binds to microtubules of cilia in the somatic region, to kinetosomes of the whole surface including the oral apparatus and to postoral microtubules in Paramecium caudatum. 2. The processing of cells adhering to a slide, including fixation and permeabilization keeps the cell undisturbed but results in defonnation in shape. Extraction is less thorough, rendering the method less suitable for indirect immunofluorescence staining in large cells such as paramecia. 3. Treatment of organisms in suspension without fixation leads to better extraction but produces about 10% of disrupted cells. However, fragments of their cortex are useful for a detailed study of microtubular arrays. 4. The technique of indirect immunofluorescence based on a TU-01 monoclonal antibody is a suitable tool in investigation into the cortical structures and their changes in Paramecium.
Literature ALLEN, R. D. (1971): Fine structure of membranous and microfibrillar systems in the cortex of Paramecium caudatum. 1. Cell. BioI. 49: 1-20. - (1974): Food vacuole membrane growth with microtubule-associated membrane transport in Paramecium. 1. Cell BioI. 63: 904-922. COHEN, J., ADOUTTE, A., GRANDCHAMP, S., HOUDEBlNE, L.-M., & BEISSON, 1. (1982): Immunocytochemical study of microtubular structures throughout the cell cycle of Paramecium. BioI. Cell 44: 35-44. - DE LOUBRESSE, N. G., & BEISSON, J. (1984): Actin microfilaments in Paramecium: Localization and role in intracellular movements. Cell Moti!. 4: 443-468. DR.\BER, P., RUBINO, S., DRABEROVA, E., VIKLlCKY V., & CAPPUCCINELLI, P. (1985): A broad spectrum monoclonal antibody to alpha-tubulin does not recognize all protozoan tubulins. Protoplasma 128: 201-207. EHRET, C. F., & MAC ARDLE, E. W. (1974): The structure of Paramecium as viewed from its constituent levels of organization. In: WAGTENDONG, W. J. VAN (ed.), Paramecium. A current survey, pp. 263-338. New York.
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GRANDCHAMP, S., & BEISSON, J. (1981): Positional control of nuclear differentiation in Paramecium. Develop. BioI. 81: 336- 34l. HASEK, J., SVOBODA, J., & STREIBLOV A, E., (1986): Immunofluorescence of the microtubular skeleton in growing and drug-treated yeast protoplasts. Europ. J. Cell BioI. 41: 150-156. JANISCH, R. (1987): Biomembranes in the life and regeneration of Paramecium. Bmo. - & HASEK, J. (1985): An immunocytochemical study of Paramecium microtubular structures. J. Protozool. 31: 183. KERKSEN, H., VILMART-SEUWEN, J., MOMAYEZI, M., & PLATTNER, H. (l986a): Filamentous actin in Paramecium cells: Mapping by phalloidin affinity labeling in vivo and in vitro. J. Histochem. Cytochem. 34: 443-454. MOMAYEZI, M., BRAUN, C., & PLATTNER, H. (I986b): Filamentous actin in Paramecium: Functional and structural changes correlated with phalloidin affinity labelling in vivo. J. Histochem. Cytochem. 34: 455-465. KUZNICKI, 1. (1963): Recovery in Paramecium caudatum immobilized by chloralhydrate treatment. Acta Protozool. 1: 177-185. LUND, E. E. (1933): A correlation of the silverline and neuromotor systems of Paramecium. Univ. Calif. Publ. Zool. 39: 35-76. OGURA, A. (1981): Deciliation and reciliation in Paramecium after treatment with ethanol. Cell Struct. Funct. 6: 43-50. OSBORN, M., WEBSTER, R. E., & WEBER, K. (1978): Individual microtubules viewed by immunofluorescence and electron microscopy in the same PfK2 cell. J. Cell BioI. 77: R27-R34. PiTELKA, D. R. (1969): Fibrillar systems in protozoa. In: CHEN, T. T. (ed.), Research in Protozoology 3, pp. 279-388. New York. SCHLIWA, M., & BLERKOM, J. VAN (1981): Structural interaction of cytoskeletal components. J. Cell BioI. 90: 222-235. SIKORA, J., & WASIK, A. (1978): Cytoplasmic streaming within nickel ion immobilized Paramecium aurelia. Acta Protozool. 17: 389-397. SUNDARARAMAN, V., & HANSON, E. D. (1976): Longitudinal microtubules and their functions during asexual reproduction in Paramecium tetraurelia. Genet. Res. Camb. 27: 205-211. TUCKER, J. B. (1979): Spatial organization of microtubules. In: ROBERTS, K. R., & HYAMS, J. S. (eds.), Microtubules, pp. 315-357. New York, London. - BEISSON, J., ROCHE, D. 1. J., & COHEN, J. (1980): Microtubules and control of macronuclear "amitosis" in Paramecium. J. Cell Sci. 44: 135-15l. VIKLICKY, V., DRABER, P., HASEK, J., & BARTEK, J. (1982). Production and characterization of a monoclonal antitubulin antibody. Cell BioI. Int. Rep. 6: 726-731. VILLANEUVE-BRACHON, S. (1940): Recherches sur les cilies heterotriches. Arch. Zool. Exp. Gen. 82: 1-100. WILLIAMS, N. E., & HONTS, J. E. (1987): The assembly and positioning of cytoskeletal elements in Tetrahymena. Development 100: 20- 30. Author's address: Dr. ROMAN JANISCH, Department of Biology, Medical Faculty, Purkyne University, CS 66243 Bmo, Czechoslovakia.
Department of Biology, Medical Faculty, Purkyne University, Brno, Czechoslovakia
Immunofluorescence Detection of Microtubular Structures in Paramecium caudatum by Means of Monoclonal Antibody TU-Ol By ROMAN JANISCH With 8 Figures Key words: Immunofluorescence; Microtubules; Paramecium caudiltum
Summary Microtubular structures of the cortical cytoskeleton in Paramecium caudatum were identified by means of indirect immunofluorescence employing the monoclonal antibody TU-OJ. Specimens for observation were fixed with glutaraldehyde in a layer attached to a slide or made into a suspension and without fixation, permeabilized with Triton X-lOO. The antibody TU-Ol reacted with kinetosomes, bundles of postoral microtubules and microtubules of somatic cilia along their whole length. The cells on a slide were well preserved though often deformed in shape and poorly extracted. The cells in suspension, on the other hand, were better extracted but about 10 % of them became disrupted. The resultant fragments were used with advantage for studying organization of microtubular structures. Immunofluorescence labelling with TV-Ol is a useful tool in the study of the cortical cytoskeleton in, for instance, morphological mutants or abnormal cells of paramecia, and in investigations into the behaviour of the cytoskeleton during repair processes occurring in mechanically injured cells.
Introduction Visualization of various cellular structures by means of immunofluorescence techniques is a valuable and exact approach to research in cytology. These methods have also found broad application in investigation of the cell cytoskeleton, its structure and function. Among many model objects used in studying the cytoskeleton, infusoria of the genus Paramecium play an important role. Their advantages include a relatively large size and specific arrangement of the cortical cytoskeleton. On the other hand, the numerous microtubular structures present in the cytoplasm of a large cell interfere with immunofluorescent staining of the cortical structures. The cortex of Paramecium has been studied extensively and its ultrastructure has been described in great detail in many papers (PITELKA 1969; ALLEN 1971; EHRET & MAC ARDLE 1974). The model makes it possible to study changes in structure of the cortical cytoskeleton during cell reproduction (COHEN et a1. 1982), following treatment with various inhibitors of metabolism (COHEN et a!. 1984; KERKSEN et al. 1986a, b) or toxic substances, or after mechanical injury (JANISCH 1987). The cortical cytoskeleton of Paramecium consists of many microfilamentous and microtubular components. The latter include ciliary axonemes, kinetosomal microtubules, bundles of short postciliary and transverse microtubules (ALLEN 1971) and suprakinetodesmal microtubules, "a cytospindle" present at cytodieresis (EHRET & MAC ARDLE ]974; SUNDARARAMANN & HANSON 1976) and disappearing afterwards. The oral cytoskeleton is characterized mainly by bundles of microtubules originating from the right posterior wall of the buccal cavity and reaching the posterior cell pole (ALLEN 1974), referred to as postoral microtubules or earlier, postesophageal fibrils (LUND 1933). Electron microscopic studies have demonstrated two more units of 21
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microtubular components belonging to the oral apparatus, namely, bundles of 2 -4 microtubules of the right cytosome lip found below the right ribbed wall of the buccal cavity, and flat bands composed of 10-12 microtubules running along the left cytostome lip. The part of the buccal cavity shows 3 quadruple rows of cilia; one is calculated the quadrulus, the other two peniculi. During karyokinesis, microtubules have also been seen in the nucleus. Micronuclear division, which occurs without disruption of the nuclear envelope, involves successively two types of spindles: an orthodox mitotic spindle, and then a long separation spindl~ which pushes the two daughter micronuclei apart to the opposite poles of the cell (TUCKER 1979; GRANDCHAMP & BEISSON 1981). Segregation of genomes in the macronucleus is accompanied by the appearance of microtubule bundles inside the nucleus (TUCKER et al. 1980). These are apparently involved in the process of macronuclear elongation. All the structures described here have been identified by electron microscopy. Apart from them, the cytoplasm of Paramecium contains many more transient microtubular structures. The first attempt at detecting microtubular structures in Paramecium by means of immunofluorescence with the use of polyclonal antibody was published by COHEN et al. (1982). The authors were able to visualize some of the cortical microtubules and, for the first time demonstrate with the immunofluorescence technique dynamic changes in the microtubular system during the cell cycle in Paramecium. Preliminary results on the reaction of TU-OI monoclonal antibody with tubulin bands in Paramecium caudatum were published as an abstract in the proceedings of a conference (JANISCH & HASEK 1985). In the present paper a method of immunocytochemical visualization of microtubular structures in Paramecium caudatum is described and the specifity of reaction between monoclonal antibody and tubulin in this species is discussed.
Material and Methods a) Model objects and culture conditions Cells of Paramecium caudatum were isolated from a local source and cultivated in a medium with wheat grains according to VILLANEUVE-BRACHON (1940). The culture in Petri dishes was regularly fed on Klebsiella pneumoniae.
b) Antibody To visualize tubulin, a monoclonal TV-OI antibody described by VIKLlCKY et al. (1982) and produced by the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague was used. Indirect fluorescence was based on a secondary antibody SwAM-FITC (Institute for Sera and Vaccines, Prague).
c) Preparation of specimens Essentially, two techniques were used. Cells were exposed to antibody either in suspension or in a layer adhering to a slide. Advantages and disadvantages of each approach are discussed in the section "Results and Discussion" .
The slide method Paramecium cells were rinsed in phosphate buffer solution (PBS - 0.8g NaCl, 0.2 g KCI, 1.15 g NA2 HP04 , 0.2 g KH 2 P04 in 1,000 mr H2 0, pH 7.4) for 10 min. A drop with infusoria was spread onto a slide over an area corresponding in size to that of a cover slip and allowed to dry. Shortly before the layer dried completely, a drop of fixative was added. This consisted of2 % glutaraldehyde and 0.5 % Triton X- 100 in PBS. The cells were then treated with cold methanol for 8 min and with acetone for 30 s and exposed to three reduction procedures in 0.5 % NaBH4 lasting 5, 5, and 10 min, respectively, at 4°C. This was followed by washing the cell layer in PBS containing 2 %
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bovine serum albumin (BSA). The cells were then exposed to TU-Ol monoclonal antibody (at a dilution of 0.1 mg/ml) for 30 min at 37°C. After three washes with PBS the cells were treated with the secondary antibody SwAM-FITC (concentration 0.4 mg/ml) for 45 min at 3rC and then washed with PBS and mounted in 90% glycerol.
The suspension method The procedure was based on a modified method by COHEN et al. (1982). After preceding deciliation with two rinses in 10 mM MnCh, the cells were subjected to permeabilization treatment with 0.5 % Triton X-IOO in a buffer of the following composition: 60 mM PIPES, 25 mM EGTA and 2 mM MgCl 2 (SCHLIWA & VAN BLERKOM 1981). This buffer, further referred to as PHEM, was then used to dilute antibodies and wash specimens. After 20-30 min of permeabilization, the cells were rinsed 3 times 5 min in PHEM and incubated in TU-Ol antibody with 2 % BSA for 30 min. Then the cells were rinsed 3 times 5 min in PHEM and incubated ion SwAM-FITC antibody followed by mounting in 90 % glycerol. All procedures were carried out in small centrifuge tubes at low centrifugation speed.
d) Fluorescence microscopy and microphotography The specimens were examined and photographed with a lenalumar fluorescence microscope at 500 or 1,000 X magnification using Foma 21 DIN negative material.
Results and Discussion The presence of microtubules in cilia and kinetosomes, and of postoral microtubular bundles originating from the oral apparatus was demonstrated in paramecium cells with both methods of cell preparation described in the paper. Ciliary microtubules were stained with TU-Ol antibody along the whole length of the cilium (Figs. 1, 2). No difference in labelling was found between cilia from different surface regions. The cilia of the oral apparatus, i.c. the quadrulus and peniculi, however, failed visualization (Fig.
3,4).
Fig. 1. A cell of Paramecium caudatum treated with TU-Ol and SwAM-FlTC in suspension without deciliation.
Tubulin in the ciliary axonemes reacts with antibody along the whole length of the cilium. Focussed on the middle plane of the cell. Fig. 2. The same preparation as in Fig. 1 but after focussing on the upper plane of the cell. Fig. 3. A region of the oral apparatus of a Paramecium caudatum cell prepared in the same way as in Fig. l. Flourescent strips correspond to the quadrulus and peniculi. Single kinetosomes can not be recognized due to their high density. A small area of the somatic cortex with kinetosomes adjoins these structures. Cilia are labelled only in the somatic region. Fig. 4. A region of the oral apparatus of a paramecium cell adhering to a slide. The strips of kinetosomes (quadrulus and peniculi) are labelled but the cilia are not. Flourescence of intracellular structures interferes with the labelled structures. Fig. 5. A part of the dorsal surface of Paramecium caudatum treated with TU-OI and SwAM-FITC in suspension after deciliation. Kinetosomes are seen as dots alTanged in parallel rows. Fig. 6. A part of the ventral surface of a paramecium cell prepared by the same technique as in Fig. 5 but without deciliation. The tlourescent dots correspond to pairs of kinetosomes with adjoining transverse and postciliary microtubules. Figs. 7 and 8. A fragment of the paramecium cortex with an isolated oral apparatus. The object was prepared in suspension. Besides the quadrulus and peniculi there are bundles of postoral microtubules. Each figure shows a different focal plane in the same object. 21*
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Kinetosomal microtubules were seen as bright fluorescent dots corresponding to the whole kinetosomes (Figs. 5, 6). Both the kinetosomes of the somatic cilia and the kinetosomes of the oral apparatus showed the same intensity of fluorescence (Fig. 3). Fluorescence staining was particularly intense in the bundles of postoral microtubules originating from the oral apparatus and running through the endoplasm of the posterior end towards the cytoproct. In whole cells it is difficult to identify the postoral microtubules due to the fluorescence of tubulin from the interior of the cell. However, postoral microtubules usually remain attached to the oral apparatus on occasional fragmentation of cells during permeabilization treatment with Triton X-lOa (Figs. 5,7,8). Within the oral skeleton, however, it was not possible to determine exactly which other microtubular structures, apart from those described above, were visualized by immunofluorescence. Nor was it possible to detect the short bundles of transverse and postciliary microtubules identified by electron microscopy near all somatic kinetosomes. Cells processed on a slide provide a good insight into the details of mcirotubular structures in various regions of the cortex. However, they are not suitable for studying the overall pattern of the cortical cytoskeleton for two reasons: the cells are considerably deformed during the drying procedure, and there is often a nonspecific fluorescence of the intracellular structures due to the glutaraldehyde fixation. Cells prepared in suspension, on the other hand, give a comprehensive picture of the overall distribution of microtubular structures in the cortex of an infusorian. The detection of microtubular structures in Paramecium tetraurelia by means of immunofluorescence was first reported by COHEN et al. (1982). Using a specific antiserum produced against tubulin isolated from axonemes of paramecia, they demonstrated the presence of the tubulin in the cytospindle during nuclear division, and in postoral fibrils at the time of interphase. Monoclonal antibodies against tubulin have so far been produced in many laboratories; they differ mostly in their reactivity with alpha or beta subunits of the porcine brain tubulin. Besides, even antibody against one of the subunits shows varying reactivity with different antigenic determinants of this subunit. The TU-Ol antibody was prepared by VIKLICKY et al. (1982) as a specific monoclonal antibody to the alpha subunit of the porcine brain tubulin. It has been demonstrated that the antibody binds to a tubulin epitope present in higher eukaryote, such as mammals, birds, and amphibians, as well as in fungi, echinodermata, worms and slime molds. In protozoa, the reactivity of tubulin antibody with microtubules has been shown in Trichomonas vaginalis (DRABER et al. 1985). Apparently, antigenic determinants for this antibody are either missing completely in many eukaryotic microorganisms or are present only in some of the microtubules. Of the Flagellata so far tested, i.e. Herpetomonas, Leishmania and Trichomonas, positive results were obtained only for some microtubular structures of Trichomonas, namely, axostyles, mitotic spindle, distal parts of the flagellum. TU-Ol antibody bound to ciliary tubulin was first reported in Paramecium caudatum by JANISCH & HASEK (1985). Although this immunocytochemical procedure was affected by imperfect methods of extraction and permeabilization, the reaction of TU-Ol with tubulin of cilia was clearly demonstrated and the specifity of the reaction was confirmed by SDS-polyacrylamide gel electrophoresis and immunoblotting. With the use of TU-Ol antibody, tubulin in Paramecium caudatum has been visualized only in some of the known microtubular structures. TU-Ol binds to axonemes along the whole length of the cilium. The absence of tubulin labelling in ciliary axonemes of the oral apparatus suggests a different tubulin epitope in these structures. Some of the cilia in the oral region have been reported to resist deciliating agents (KUZNICKI 1963; OGURA 1981) or immobilizing substances (SIKORA & WASIK 1978) for a long time. DRABER et al. (1985) demonstrated tubulin staining only in distal parts of the flagellum in Trichomonas vaginalis. In this organism, no TU-Ol labelling was recorded in microtubules of flagellar kinetosomes. In Paramecium caudatum, TU-Ol bound to tubulin of all kinetosomes of the somatic and oral regions of the cortex. In comparison with the results of COHEN et al. (1982) TU-Ol failed to label bundles of short transverse and postciliary microtubules situated near kinetosomes. This can be explained by either a lower resolution of the method used or a different specifity of the antibody. COHEN et al. (1982) reported immunofluorescence staining of all the microtubular structures consisting of at least 3~4 microtubules.
Microtubular Structures in Paramecium caudatum
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OSBORN et al. (1978) described visualization of single microtubules by indirect immunofluorescence even in flattened fibroblasts spread on a slide during incubation. Even after a complete extraction, a paramecium cell is still a very thick object, in which the strong diffuse fluorescence of cytoplasmic microtubules diminishes the contrast of fluorescence shown by cortical microtubules and thus hinders the discernment of details. The resolution of the indirect immunofluorescence method applied to whole cells is related to the degree of extraction of Triton X-lOO. Only optimal extraction and permeabilization will permit the tubulin antibody to penetrate through the cortical membranes and will avoid nonspecific fluorescence shown by intracellular structures. For this reason cells processed in suspension are more suitable objects, since they retain their shape, i.e., an unchanged pattern of the cortical cytoskeleton. Extraction is more thorough than in those attached to a slide. In a certain proportion of cells (about 10%) treatment with Triton X-IOO results in their disruption, which enables one to study cortical fragments, such as isolated oral apparatus with attached cytoskeletal components. The method of extraction and fixation of cells adhering to a slide proved useful in many small organisms e.g. yeast cells or their protoplasts (HASEK et al. 1986). In larger cells like paramecia, attachment to a slide results in preservation of incompletely extracted parts in the cell which interfere with visualization of the cortical cytoskeleton. Immunocytochemical observations in infusoria allow us to gain insight into the overall morphology and microtubular patterning of the cortical cytoskeleton together with its changes due to morphological mutations or shape abnormalities. Using indirect immunofluorescence, WILLIAMS & HONTS (1987) were able to demonstrate abnormalities in the development of the oral cytoskeleton in a temperature sensitive mutant of Tetrahymena thermophila. This technique may also prove a useful tool in studying changes in the cytoskeleton during repair processes of mechanically injured cells.
Conclusions 1. The TU-O I monoclonal antibody binds to microtubules of cilia in the somatic region, to kinetosomes of the whole surface including the oral apparatus and to postoral microtubules in Paramecium caudatum. 2. The processing of cells adhering to a slide, including fixation and permeabilization keeps the cell undisturbed but results in defonnation in shape. Extraction is less thorough, rendering the method less suitable for indirect immunofluorescence staining in large cells such as paramecia. 3. Treatment of organisms in suspension without fixation leads to better extraction but produces about 10% of disrupted cells. However, fragments of their cortex are useful for a detailed study of microtubular arrays. 4. The technique of indirect immunofluorescence based on a TU-01 monoclonal antibody is a suitable tool in investigation into the cortical structures and their changes in Paramecium.
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