Distribution of F-actin, α-actinin, tropomyosin, tubulin and organelles in Euglena gracilis by immunofluorescence microscopy

Tissue & Cell, 1998 30 (5) 545-553 © 1998 Harcourt Brace & Co. Ltd

Distribution of F-actin, -actinin, tropomyosin, tubulin and organelles in Euglena gracilis by immunofluorescence microscopy C. S. Mermelstein 1, A. P. M. Rodrigues 1, M. Einicker-Lamas 2, R. E. de B. Navarrete 2, M. Farina 3, M. L. Costa 1

Abstract. Euglena gracilis, a unicellular flagellated alga, can display numerous shape changes. These changes are most probably caused by a pellicle and an internal cytoskeleton. In this paper we studied the distribution of the cytoskeletal proteins actin, c~-actinin, tropomyosin and tubulin in dark-adapted Euglena, using immunofluorescence microscopy. We found that F-actin, o~-actinin, tropomyosin and tubulin have a distribution that is coincident in the plasma membrane and, in addition, (z-actinin and tropomyosin are seen in small patches in the cytoplasm, and tubulin in the flagella. We have also studied the distribution of the endoplasmic reticulum, nucleus, and Golgi apparatus of these cells, using fluorescent probes. Both the endoplasmic reticulum and the Golgi apparatus have a meshwork pattern distributed throughout the cytoplasm, and the nucleus has a chromatin evenly distributed in the nucleoplasm.

K e y w o r d s : Euglena gracilis, cytoskeleton, fluorescence microscopy

Introduction Euglenoids are unicellular organisms, with lengths varying from <10 ~tm to >500 gin, that have acquired chloroplasts in the course of evolution. The euglenoids have both animal and plant characteristics. Euglena display the structural complexity characteristic of protists in which all functions and activities of the organisms are manifest in a single cell. Euglena gracilis lives in still water and can display numerous shape changes. The swimming movements and rapid cell-shape changes are widely known. Several investi'Departamento de Histologia e Embriologia, Instituto de Ci~ncias Biom6dicas ~lnstituto de Biofisica Carlos Chagas Filho 3Departamento de Anatomia, Instituto de Ci~ncias Biom~dicas, Centro de Ciencias da Sa0de, Universidade Federal do Rio de Janeiro, Ilha do FundAo, Rio de Janeiro, RJ, 21949-590, Brazil.

Received 17 March 1998 Accepted 9 June 1998 Correspondence to: Claudia dos Santos Mermelstein, Tel.: +55 21 507 8545; Fax: +55 21 590 1841

gators have reported cell rounding in response to chemical or physical perturbations, including the lowering of extracellular cation concentration (Lonergan, 1984), exposure to ethanol or chemical fixatives (Lonergan, 1983, 1984), and exposure to bright illumination during microscopic examination (Murray, 1981). Euglenoid movement was first noted by Harris in 1969. The protozoan swims by means of a single long flagellum which is attached to the anterior end of the cell. This flagellum wraps around the cell pointing rearward. The cell is driven forward by helical waves moving from the base of the flagellum to its tip. As a consequence, the organism performs a complex pattern of movement characterized by a gyrating pathway through the water in which the anterior end sweeps through a larger radius than the posterior (Bray, 1992). The Euglena cell is delimited by a plasma membrane, in which there is a series of flexible strips, made of proteins and with a helicoidal disposition. These strips, together with the plasma membrane, form a structure called pellicle. In 545

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contrast to the rigid cell wall of plant cells, the flexible pellicle allow Euglena cells to change their shape, promoting an alternative way of locomotion (Raven et al., 1992). Although the molecular basis of euglenoid movement is still unknown, many authors have postulated that the pellicular strips and subpellicular microtubules, which are linked at the cytoplasmic side of the membrane (Grain, 1986), play an active role in the process (Lachney & Lonergan, 1985; Murray, 1981). The surface of these euglenoids consists of alternating ridges and grooves that shift in orientation during cell deformation. The ridges are relatively immobile and the sliding associated with surface motility takes place between, but not within, the surface ri'dges (Buetow, 1982; Hofmann & Bouck, 1976; Leedale, 1967; Mignot, 1966). The cytology and ultrastructure of the nucleus in Euglena were reviewed by Leedale (1968) with reference to interphase, mitosis, amitosis, and meiosis. A twomembraned nuclear envelope surrounds permanently condensed chromosomes evenly distributed in granular nucleoplasm around a central endosome (nucleolus) (Leedale, 1982). The endoplasmic reticulum (ER) constitutes the peripheral complex of bodies, various tubular (smooth) and vesiculated (rough) components in the cell and a canal sheet. Additionally, elements of the tubular ER are associated with, or directly connected to the Golgi bodies and the nuclear envelope (Leedale, 1967). There is no apparent connection between ER and the chloroplast membranes (Gibbs, 1978). Euglena contain from a few to many Golgi bodies (dictyosomes), the number increasing with cell size. Each Golgi body is a large structure, consisting of 15-30 cistemae more than 1 gm in diameter; vesicles 25-80 gm in diameter are proliferated in large numbers from the fenestrated edges of the cisternae (Leedale, 1982). Euglena has two flagella inserted on the dorsal wall reservoir; one emerges from the canal as the organelle of swimming, and the other ends within the reservoir. In addition to the usual 9 + 2 microtubule arrangements of the axoneme, euglenoids are generally characterized by a prominent paraflagella rod that lies parallel and is attached to at least two microtubule doublets (Leedale, 1982). An immunofluorescence analysis of the pellicle-associated microtubules has also previously been reported (Lachney & Lonergan, 1985). Actin and tubulin have been studied by immunofluorescence in E. gracilis (Lonergan, 1985), showing a high degree of coincidence and visualized as lines running parallel to the pellicle strips beneath the plasma membrane. All the organelles of Euglena are supposedly in close association with cytoskeletal proteins, such as tubulin and actin. Moreover, actin-binding proteins are responsible for the regulation of the assembly dynamics of microfilaments in non-muscle cells, through cross-linking filaments into networks or bundles, strengthenning filaments, and several other functions (Korn, 1982). We can expect that

actin-binding proteins, such as c~-actinin and tropomyosin, could play a role in the regulation of cell shape and movement in EugIena, by associations between microfilaments and organelles, microtubules and the epiplasm. In this paper we analyzed by immunofluorescence microscopy, the distribution of the cytoskeletal proteins actin, a-actinin, tropomyosin and tubulin in Euglena cells. Furthermore, to compare the organization of the cytoskeleton with other organelles, we have studied the distribution of the ER, nucleus, and Golgi apparatus of these cells using fluorescence probes. Dark adapted Euglena, which have no chloroplasts, were used in this work in order to prevent the interference of natural fluorescence of chlorophyll.

Materials and methods Cultured Euglena cells and interference contrast microscopy The unicellular organisms Euglena gracE& strain Z were grown in the following complete growth medium: 2 g/1 tryptose, 2 g/1 yeast extract, 0.1 g/1 liver infuse and 10 g/1 sucrose. EugIenacells were grown in flasks, in two different conditions: complete dark and complete light. Some cells were grown in light conditions for interference contrast microscopy, in order to compare the general morphologies. Cells were inoculated with fresh medium once every 3 days. Interference contrast microscopic images were obtained using an Axioplan Microscope (Carl Zeiss, Germany). Images of live cells were acquired with a Pasecon (Grundig electronic) high definition video camera. After the analog processing (ACE-Zeiss), the video signal was digitized using the IBAS (Kontron-Zeiss) Image Processing System. As a result, sequences of 48 image frames, including a background reference image, were obtained and could be processed for the background subtraction, shading correction and enhancement of contours. Photographs of the best processed image frames were made directly from the monitor screen. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis were done as previously reported by Laemmli (1970), in a 8% running gel. Cells were lysed on ice with sample buffer (PBS, Triton X-100 0.5%, EDTA 3 raM, PMSF 1 raM, Benzamidine 10 mM) and sonicated for 30s. Pellets were collected by centrifugation, resuspended in lysis buffer (SDS 2%, 2-mercaptoethanol 5%, glycerol 10%, Bromophenol Blue 0.001%, Tris 6.25 mM pH 6.8) and heated for 5 rain at 100°C. The amount of protein in each sample was determined according to Bradford (1976), using bovine serum albumin, as a standard. Samples were applied into the gel at a concentration of 120 gg of protein per lane. The following molecular weight standards were used: fructose-6-phosphate kinase (84 KDa) from Sigma Chemical Co.; glyceraldehyde3-phosphate dehydrogenase (36 KDa) from Sigma Chemical Co.; and a purified fraction of tubulin (55 KDa) from mouse

CYTOSKELETAL PROTEINS IN EUGLENA GRACILIS

brain. Electrophoresis were run at 12 mA. Proteins were fixed and stained by Coomassie Brilliant Blue (Sigma Chemical Co., St. Louis, MO, USA) and dried (Juang et al., 1984).

Antibodies and fluorescent probes Polyclonal antibody against chicken gizzard tropomyosin, monoclonal antibody against ~-tubulin (clone 2-28-33, immunogen: sea urchin sperm axonemes) and PhalloidinTRITC labelled were purchased from Sigma Chemical Co. (St. Louis, MO). Polyclonal antibody against human blood platelets t~-actinin was a gift from Dr Y. Gache and Dr A. Olomucki (Gache et al., 1984). DAPI, NBD hexanoic ceramide and rhodamine B were purchased from Molecular Probes (Eugene, OR).

Immunoblotting For the western blotting, Euglena cells were gently washed twice in PBS and re-suspended in a concentration of 107 cells/ml; 1 ml of this suspension was centrifuged and the pellet was homogeneized with PBS containing 0.5% Triton X-100, 3raM EDTA, 10gg/ml Leupeptin, 0.1mM Phenylmethyl sulfonyl fluoride. The suspensions were centrifuged at 16 000 x g for 10 rain, and the detergent insoluble pellet, containing the cytoskeletal fraction, was treated with SDS-sample buffer (Laemmli, 1970). The amount of protein in each sample was determined according to Bradford (1976), using bovine serum albumin, as a standard. Samples (150 gg of protein per lane) were applied in a polyacrylamide gel electrophoresis (Laemmii, 1970), in a 8% running gel for the o~-actinin blotting and in a 12% for the tropomyosin and tubulin blottings. The following molecular weight markers (Amersham Int. plc) were used: lysozyme (14.3 KDa), trypsin inhibitor (21.5 KDa), carbonic anhydrase (30 KDa), ovalbumin (46 KDa), bovine serum albumin (66 KDa), phosphorylase b (97.4 KDa) and myosin (220 KDa). Electrophoresis were run at 60 V for 2.5 h in a Mini-V 8.10 Vertical Gel Electrophoresis Apparatus (Gibco BRL) and proteins were transferred to a nitrocellulose sheet (HybondTM, ECL, Amersham Int. plc, England), according to Towbin et al. (1979). The nitrocellulose sheet was treated following the ECL Western Blotting Protocol (Amersham Int. plc) and for the chimioluminescence detection a Hyperfilm ECL (Amersham Int. plc) was used. Reversible staining with Ponceau Red was used to ensure that an efficient transfer had taken place. Antibodies against ct-actinin, tropomyosin and tubulin were used at the following dilutions: 1:100, 1:40 and 1:800, respectively. Peroxidase labelled antirabbit and anti-mouse antibodies (Amersham Int. plc) were used in a 1:10000 dilution in PBS containing 1% Tween 20.

Immunofluorescence microscopy For immunofluorescence microscopy, dark adapted cells were fixed in suspension at room temperature by slowly adding equal volumes of 4% (w/v) paraformaldehyde in Phosphate Buffer Solution (PBS) to the cell suspension while gently mixing on a vortex mixer, resulting in a final

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2% (w/v) paraformaldehyde solution. Cells were collected by centrifugation in a clinical centrifuge at maximum setting for 30 s and gently resuspended in a second fixative consisting of 2% (w/v) paraformaldehyde in PBS for 10 min. They were then centrifuged and resuspended in PBS with 0.5% Triton-X 100 (Sigma Chemical Co., St. Louis, MO), and extracted for 15 min while agitating in a gyratory shaker. This PBS-Triton solution was also used for all subsequent antibody washing steps. All subsequent centrifugations were at maximum setting for 30 s in a clinical centrifuge. After extraction, cells were centrifuged and re-suspended in 5 ml 80% (v/v) acetone for 5 min while agitating in a gyratory shaker to remove remaining chlorophyll, which causes an interfering red fluorescence. Cells were then centrifuged and re-suspended twice for 5 min, while agitating in 5 ml of PBS-Triton. Cells were incubated with 100gl of 3% Bovine Serum Albumin (Sigma Chemical Co., St. Louis, MO) for 10 min at 37°C, in order to block non-specific binding, and then centrifuged and resuspended twice for 5 rain in PBS-Triton. All primary antibodies were used at appropriated dilutions for 1 h at 37°C and cells were washed three times for 10 rain each. All secondary goat antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were affinity purified and tagged with either rhodamine or fluorescein and used at 1:100 dilution for 1 h at 37°C and cells were washed three times for 10 rain each. Control experiments with no primary antibody showed no staining (not shown). Controls using a preimmune serum from animals from which the primary antibody against a-actinin had been raised, showed only a faint background staining evenly distributed throughout the cells (not shown). Light-adapted cells were fixed in the same conditions as dark-adapted cells to indicate how chloroplast fluorescence interferes with studying the system. Some light and dark-adapted cells were submitted to a Phalloidin staining in which these cells were washed twice in PBS (after the acetone step) before an incubation of Phalloidin-TRITC labelled for 20 rain at 37°C. After incubation cells were centrifuged and re-suspended twice for 5 rain in PBS. The nuclear dye, DAPI (4, 6-Diamidino-2-phenylindole dihydrochloride, Polysciences) was used at 0.2 mg/ml in 0.9% NaC1 for 5 min. The Golgi apparatus dye, NBD hexanoic ceramide (Molecular Probes, Eugene, OR) was used at 10 gg/ml for 5 rain. The ER dye, rhodamine B (Molecular Probes, Eugene, OR) was used at 10 pg/ml for 5 rain. The F-actin dye, Phalloidin-TRITC labelled (Sigma Chemical Co., St. Louis, MO) was used at a 1:100 dilution. Specimens were mounted in glycerol containing, by weight, 5% n-propyl gallate (Sigma Chemical Co., St. Louis, MO), 0.25% DABCO (1, 4-Diazabicyclo (2, 2, 2) octane, Sigma Chemical Co., St. Louis, MO) and 0.0025% para-phenylenediamine (Sigma Chemical Co., St. Louis, MO), according to Giloh and Sedat (1982), and Johnson et al. (1982). Cells were examined with an epifluorescence inverted microscope Axiovert 100 (Carl Zeiss, Germany),

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Fig. 1 Interference contrast microscopy followed by image processing of Euglena gracilis. A Grown in the presence of the light. B Grown in dark conditions. Pellicular strips can be seen along the cell membrane in both conditions, but chloroplasts are seen only in cells grown in the presence of light. Bar, 5 ~l,m.

using filter sets which were selective for rhodamine, fluorescein, or the blue wavelength channel. Photographs were taken on 400 ASA TMAX film (Eastman Kodak Co.).

Results

Euglena can grow both in light and dark conditions; in the last case without chloroplasts. Since the chlorophyll present in chloroplasts can interfere with fluorescent studies, we decided to grow cells used for immunofluorescence microscopy in the absence of light. Images of both Euglena gracilis grown in light (Fig. 1A) and in dark conditions (Fig. 1B) were acquired with an Axioplan Microscope (Carl Zeiss, Germany) and electronically processed as described in materials and methods. The pellicular strips along the cell membrane could be easily seen after digital contrast enhancement, with the characteristic helical pattern presenting ca. 60 turns in both light and dark-grown types of cells. Chloroplasts are seen in cells grown in the presence of light and not seen in dark conditions. These Euglena cells grown in the dark retain equal abundance of major cytoskeletal components. As seen in the SDS-PAGE (shown in Fig. 2), we can infer the presence of protein bands that correspond to the molecular weight of tubulin (55 KDa) and actin (43 KDa). This figure shows a polyacrylamide gel electrophoresis of Euglena cells grown in light (Fig. 2, lane B) and dark conditions (Fig. 2, lane C). Molecular weight markers: purified mouse brain tubulin in lane A, fructose-6-phosphate kinase (84 KDa) from Sigma Chemical Co. and glyceraldehyde-3-phosphate dehydroge-

nase (36 KDa) from Sigma Chemical Co. in lane D. The cytoskeletal proteins tubulin and actin are pointed (from top to bottom) with asterisks. The immunological identification of c~-actinin in cytoskeletal fractions of Euglena showed a positive reaction in a band with a molecular weight of approximately 95 KDa (Fig. 3A). The immunological identification of tropomyosin in the same cytoskeletal fractions of Euglena showed a positive reaction in a band of approximately 35 KDa (Fig. 3B) and the immunological identification of tubulin in these cytoskeletal fractions showed one major positive band with molecular weight of approximately 56 KDa (Fig. 3C), corresponding to ~-tubulin. Filamentous actin (F-actin) was revealed by Phalloidin, showing that this cytoskeletal element is equally visible in both light- and dark-adapted cells (Fig. 4A and 4B). F-actin staining is apparently at the plasma membrane of these cells. The presence of c~-actinin, tropomyosin and tubulin was demonstrated by indirect immunofluorescence. The immunofluorescence patterns for c~-actinin, tropomyosin and tubulin (Fig. 5A-D and Fig. 6C) shows a high degree of coincidence and are visualized beneath the plasma membrane. Besides that, o~-actinin and tropomyosin are both distributed in small patches in the cytoplasm. Tubulin is also present, as expected, in the flagella of these cells. The organelles Golgi apparatus, ER and nucleus were analyzed by indirect immunofluorescence. Staining of the Golgi apparatus with NBD hexanoic ceramide is shown in Figure 6A. Golgi bodies are distributed throughout the cytoplasm of the cell, similar to that described by Pagano (1989). ER, stained with rhodamine B, is shown in Figure 6B. It is possible to see a complex of bodies and vesicles.

CYTOSKELETAL PROTEINS IN EUGLENA GRAC1LIS 549

A B

C

antibody against (z-actinin had been raised showed no staining (not shown). Light-adapted cells fixed in the same way as darkadapted cells showed how chloroplast fluorescence interferes with studying the system (Fig. 7A and 7B), since only the light-adapted cells have an intense autofluorescence.

D

Discussion

2 Fig. 2 Polyacrylamide gel electrophoresis of protein extract of Euglena gracilis grown in light (B) and dark (C) conditions. Asterisks point to molecular weight standards (see materials and methods) and arrows point (from top to bottom) to tubulin and actin. Cells grown in the dark retain equal abundance of major cytoskeletal components.

The ER staining pattern appears to be similar to the Golgi apparatus, indicating a possible connection between both organelles, as described by Leedale (1982). The nuclear staining with DAPI shows two interphase nuclei, with chromatin evenly distributed in the nucleoplasm (Fig. 6D). The external integrity and internal structural arrangements were maintained after all washing and incubating steps in the preparation of cells for immunofluorescence microscopy, as shown by nuclei stainmg with DAPI probe (Fig. 6D) and in tubulin staining in the flagella (Fig. 6C). Control experiments with no primary antibody or using a preimmune serum from animals from which the primary

Several authors have studied, by electron microscopy, the unicellular flagellated algae Euglena gracilis, and it is, therefore, quite well known (Buetow, 1982; Graves et al, 1971). On the other hand, the optical microscopy associated with immunofluorescence has been less studied (Lachney & Lonergan, 1985; Lonergan, 1985). This paper focuses on the presence and distribution of cytoskeletal proteins and organelles in these cells. Euglena grown in light have chloroplasts, which have autofluorescence; but it is possible to use dark-grown cells in immunofluorescence study, since they keep their structural integrity including their helicoidal arrangement and, more importantly, they loose chloroplasts and, therefore, loose autofluorescence. Moreover, dark-grown cells keeps their major cytoskeletal components, such as microtubules and microfilaments. So, this model seems to be very useful for studying the organization of cytoskeletal components, such as tubulin, actin and actin-associated proteins such as o~-actinin and tropomyosin. These two proteins were found underneath the membrane skeletordpellicle of the Euglena (as shown in this paper), which is expected since they are actin-binding proteins and actin is present associated with the Euglena's pellicle (Lonergan, 1985; and as shown in this paper). They might have a role in the generation of movement and cell-shape changes, by associations with actin and other proteins from the cytoskeleton and the epiplasm. In addition, c~-actinin and tropomyosin were found in the cytoplasm, probably associated with the membrane of vesicles. (z-actinin and tropomyosin shows a high degree of coincidence in the immunofluorescence and are visualized beneath the plasma membrane and distributed in small patches in the cytoplasm. These aggregates might be a pool of small polymers of the two proteins, that could be used to form long strings of filaments. The immunological identification of (~-actinin in SDSPAGE using cytoskeletal fractions of Euglena showed a positive reaction in a band of approximately 95 KDa (Fig. 3A). This molecular weight is in accordance with the expected, as described by Blanchard and colleagues (Blanchard et al., 1989). The immunological identification of tropomyosin in the same cytoskeletal fractions of Euglena showed a positive band of approximately 35 KDa (Fig. 3B), as expected by the molecular weight of this protein from other sources (Paulin et al., 1979). The immunological identification of tubulin in these cytoskeletal fractions showed one positive band with molecular weights of approximately 56 KDa (Fig. 3C), corresponding to

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A

B

C

3 Fig. 3 Immunoblottings using antibodies against c~-actinin (A), tropomyosin (B) and tubulin (C), after SDS-PAGE of cytoskeletal fractions of Euglena cells grown in dark conditions. The immunoblottings confirms the presence of c~-actinin, tropomyosin and tubulin (indicated by an arrowhead in each case) in dark-adapted cells.

B

4 Fig. 4 Immunofluorescence microscopy of cultured Euglena cells stained with Phalloidin-TRITC labelled. A Grown in the presence of light. B Grown in dark conditions. F-actin is equally visible in both light- and dark-adapted cells. Bar, 10 gm.

l]-tubulin and in accordance with the well described molecular weight of this protein isoform. It is important to note that the antibodies against c~-actinin, tropomyosin and tubulin used in this work (in protein extracts and whole cells), showed an immunoreactivity between the e~-actinin from Euglenaand that from human blood platelets, and between the tropomyosin from Euglena and that from chicken gizzard, and finally between the tubulin from Euglena and that from sea urchin sperm axonemes. It seems that this structural homology was

maintained in the course of evolution. Nevertheless, immunoblots against the above proteins show other minor bands reacting with each antibody (Fig. 3A, 3B and 3C). This fact might be explained by the use of antibodies that were made against other cell types and may have some lowspecific affinities for other proteins epitopes. Tubulin was localized in the membrane and in the flagellum of the Euglena, as expected (Lachney & Lonergan, 1985; Leedale, 1982). The punctuated pattern of tubulin in the flagellum might be explained if the

CYTOSKELETAL PROTEINS IN EUGLENA GRACIL1S 551

Fig. 5 Immunofluorescence microscopy of cultured Euglena cells (grown in dark conditions) stained with (A,B) anti-c~-actininand (C,D) anti-tropomyosin, c~-actininand tropomyosin are visualized beneath the plasma membrane and distributed in small patches in the cytoplasm. Bar, 10 gm.

Fig. 6 Immunofluorescence microscopy of cultured Euglena cells (grown in dark conditions) incubated with (A) NBD-ceramide, (B) rhodamine B (C) anti-tubulin and (D) 4',6-diamidino-2-phenylindole dihidrochloride (DAPI). Golgi bodies and ER appears as a complex of bodies and vesicles in the cytoplasm. Tubulin is visualized beneath the plasma membrane and in the flagella. Nuclear staining shows interphase nuclei, with chromatin evenly distributed in the nucleoplasm. Bar, 10 gm.

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B

7 Fig. 7 Immunofluorescencemicroscopy of cultured Euglenacells. A Grown in the presence of light. B Grown in dark conditions. Cells were fixed as described in materials and methods. Chloroplast fluorescence in light-adapted cells interferes with studying the system. Bar, 10 gm.

monoclonal antibody used was specific for a subtype of [3-tubulin, which is not found in the whole structure. This finding needs more detailed immuno-biochemical studies. The immunofluorescence staining with NBD-ceramide and rhodamine B shows that dictyosomes and ER vesicles are distributed in the whole cytoplasm of the Euglena, suggesting a dynamic organization of these organelles. It will be interesting to study 'in vivo' and 'in vitro' the dynamics of what seems to be an interconnected system of membranes by means of a video-enhanced microscopy system. The nucleus staining with the fluorescent probe DAPI, shows a chromatin evenly distributed in the nucleoplasm. It seems important to make a complete study of this organism during different cell division phases as well as in interphase, using this probe that turned out to be suitable to these cells. It can also be used to study the organization of chromossomes in Euglena. It will be interesting to continue this work studying different cell shapes of Euglena in order to correlate these forms with the organization of the cytoskeletal components and organelles, which probably change their dynamics to follow these cellular movements. Actin and tubulin are major cytoskeletal proteins of all eukaryotic cells, and their presence in EugIena have been already shown by others (Lachney & Lonergan, 1985; Lonergan, 1985). On the other hand, c~-actinin and tropomyosin have been less studied. The nature of the mechanism for altering cell shape in Euglena is unknown. More detailed biochemical and electron microscopic studies will be necessary before a precise model can be described to explain how these proteins are structurally arranged in these cells, and how they might participate in movement and cellshape changes in Euglena.

ACKNOWLEDGEMENTS The authors would like to thank Dr Mdcia Maria de Oliveira (Instituto de Biofisica Carlos Chagas Filho, RJ, Brasil) for kindly giving EugIena cells and Yugo de L i m a Brand~o Murakami for photography assistance. This work was supported by grants from the Brazilian agencies: Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq), Conselho de Ensino de Pds-Graduag~o e Pesquisa da Universidade Federal do Rio de Janeiro (CEPG-UFRJ), Funda~fio de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Financiadora de Estudos e Projetos (FINEP), Fundaqfio Universitfiria Jos6 Bonifficio da Universidade Federal do Rio de Janeiro (FUJB-UFRJ) and PRONEX. REFERENCES Blanchard, A., Ohanian, V. and Critchley, D. 1989. The structure and function of c~-actinin.J. Muscle Res. Cell Motil., 10, 280-289. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254. Bray, D. 1992. Cell movements. Garland Publishing, Inc., New York & London, 10. Buetow, D.E. 1982. The biology of Euglena.Academic Press, New York, vol III. Gache, Y., Landon, F. and Olomucki, A. 1984. Polymorphism of ~actinin from human blood platelets. Eur. J. Biochem., 141, 57-61. Gibbs, S.P. 1978. The chloroplasts of Euglenamay have evolved from symbiontic green algae. Can. J. Bot., 56, 2883-2889. Giloh, H. and Sedat, J.W. 1982. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by NPropyl-Gallate.Science, 217, 1252-1255. Grain, J. 1986. The cytoskeletonin protists: nature, structure and functions. Int. Ver. Cytol., 104, 153-249. Graves, L.B. Jr, Hanzeley, L. and Trelease, R.N. 1971. The occurrence and fine structural characterization of microbodies in Euglena gracilis. Protoplasma, 72, 141-152. Harris, J. 1969. Microscopical observations of vast numbers of animals seen in water. Philos. Trans. R. Soc. London Ser. B., 19, 254-259. Hofmann, C. and Bouck, G.B. 1976. Immunological and structural

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evidence for patterned intussusceptive surface growth in a unicellular organism. J. Cell Biol., 69, 693-715. Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D. and Holborow, E.J. 1982. Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J. Immunol. Methods, 55, 231-242. Juang, R.H., Chang, Y.D., Sung, H.Y. and Su, J.C. 1984. Overdrying method for polyacrylamide gel slab packed in cellophane sandwich. Anal. Biochem., 141,348-350. Korn, E.D. 1982. Actin polymerization and its regulation by proteins from nonmuscle cells. Physiol. Rev., 62, 672-737. Lachney, C.L. and Lonergan, T.A. 1985. Regulation of cell shape in Euglena gracilis. III. Involvement of stable microtubules. J. Cell Sci., 74, 219-237. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Leedale, G.F. 1967. Euglenoid flagellate. Prentice Hall, Euglewood Cliffs. Leedale, G.F. 1968. The nucleus in Euglena. In: The biology of Euglena (ed. Buetow, D.E.) Academic Press New York, vol I, 185-242. Leedale, G.F. 1982. Ultrastructure. In: The biology ofEuglena (ed. Buetow, D.E.) Academic Press New York, vol III (Physiology), 1-27. Lonergan, T.A. 1983. Regulation of cell shape in Euglena gracilis I. Involvement of the biological clock, respiration, photosyntesis and cytoskeleton. P1. Physiol., 71,719-730.

Lonergan, T.A. 1984. Regulation of cell shape in Euglena gracilis II. The effects of altered extra and intracellular Ca+2concentrations and effect of calmodulin antagonists. J. Cell Sci., 71, 37-50. Lonergan, T.A. 1985. Regulation of cell shape in Euglena gracilis. IV. Localization of actin, myosin and calmodulin. J. Cell Sci., 77, 197-208. Mignot, J.-P. 1966. Structure et ultrastructure de quelques Eugl6nomonadines. Protistologica, 2, 51-117. Murray, J.M. 1981. Control of cell shape by calcium in Euglenophyceae. J. Cell Sci., 49, 99-117. Pagano, R.E. 1989. A fluorescent derivative of ceramide: physical properties and use in studying the Golgi apparatus of animal cells. In: Fluorescence microscopy in living cells in culture (eds. Wang, Y.-L. and Taylor, D.L.) Academic Press New York, Part A, Fluorescent Analogs, Labeling Cells, and Basic Microscopy, 75-85. Panlin, D., Perreau, J., Jakob, H., Jacob, F. and Yaniv, M. 1979. Tropomyosin synthesis accompanies formation of actin filaments in embryonal carcinoma cells induced by hexamethylene bisacetamide. Proc. Natl. Acad. Sci. USA., 76, 1891-1895. Raven, P.H., Evert, R.F. and Eichhorn, S.E. 1992. Biology of Plants. Worth Publishers, Inc., New York. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA., 76, 4350-4354.