Vimentin intermediate filaments in melanophores

F. K. GYOEVA

Institute of I'mlein Research, Academy of Scienc of the USSR, 142292 I'ushchino, Moscmv Region, USSR E. V. LEONOVA, V. I. RODIONOV and V. I. GELFAND*

A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscmv State University, 119S99 Moscmv, USSR

* Author for correspondence

Summary

The distribution and chemical composition of as has been found in other types. Trans- intermediate filaments in cultured melanophores mission electron microscopy confirmed the pres- of two teleost species - Gymnocorymbus ternetzi ence of intermediate filaments in melanophores. and Pterophyllum scalare - were studied by im- Immunoblotting experiments showed the pres- munofluorescence staining and immunoblotting ence of the protein vimen- techniques. The immunofluorescence staining of tin in melanophore lysates. Therefore, teleost the melanophores with monoclonal and poly- melanophores possess a developed radial system clonal antibodies to the intermediate filament of vimentin intermediate filaments. protein vimentin revealed a system of fibrils radiating from the cell centre. These fibrils were Key words: melanophore, intermediate filaments, resistant to 0-6M-KC1 and nocodazole treatments vimentin.

Introduction be involved. form a well-developed radial pattern in fish melanophores and their disruption Melanophores are highly specialized cells, containing a inhibits pigment movement (Schliwa, 1981; lot of pigment granules known as melanosomes. Stearns, 1984). In contrast to microtubules, the system Teleost melanophores can aggregate melanosomes to of actin in melanophores is poorly the cell centre or disperse them throughout the cyto- developed. Sparse microfilaments have been found in plasm. These melanosome movements, which deter- the cell cortex and in the cell surface microvilli mine the colour changes of , are governed by (Schliwa et al. 1981). Very little is known about the neurohumoral stimuli. Cultured melanophores can be third component of the melanophore cytoskelcton - induced to aggregate pigment particles by adrenaline intermediate filaments. Only recently Murphy & treatment and to disperse them by caffeine treatment. Grasser (1984) found 10 nm filaments in black tetra Melanosome movement is easily observable using light melanophores. However, their distribution has not yet microscopy, and that is why melanophores are a been studied and their chemical composition remains convenient system in which the mechanisms of intra- undetermined. In this paper we describe an immuno- cellular movements can be studied (reviewed by fluorescence and immunoblotting study of intermedi- Schliwa, 1981; Stearns, 1984; McNiven & Porter, ate filaments in fish melanophores. 1984). It is well known that the movement of particles in the depends on cytoskeletal structures - micro- Materials and methods tubules, actin microfilaments and intermediate fila- ments. In many types of cells the intracellular Tissue cultures movement was shown to depend on microtubules (see Primary cultures of fish melanophores were obtained essen- Schliwa, 1984), though two other structures may also tially according to Schliwa el al. (1978) and Luby & Porter Journal of Cell Science 88, 649-655 (1987) Printed in Great Britain © The Company of Biologists Limited 1987 649 (1980). The scales of aquarium Gymnocorymbus ter- Secondary antibodies conjugated with fluorescein and netzi and Pterophyllum scalare were placed in a Ringer rhodamine (Sigma Chemical Co.) were used in a dilution of solution (103mM-NaCl, l-8mM-KCl, 0-8mM-NaHCO3, 1:100. 1 2mM-CaCl2, 5mM-Tris-HCl, pH7-3), containing 1 mgml" collagenase (Fluka), lmgml"' hyaluronidase (Type I, Polyacrylamide gel electrophoresis and Sigma Chemical Co.) and 5mgml~' bovine serum albumin immunoblotting (Fraction V, Sigma Chemical Co.). After incubation for Electrophoresis was performed in polyacrylamide-SDS slab 30-60 min at 30-35 °C the melanophores were removed from gels (Laemmli, 1970). The method of Towbin et al. (1979) the scales by pipetting and washed three times in Ringer was used for immunoblotting. Peroxidase-conjugated second- solution by transferring from one Petri dish to another. ary antibodies were purchased from Sigma Chemical Co. and Finally, the melanophores were placed for attachment onto used in a dilution of 1:200. carbon-coated coverslips and after incubation for 30-60 min at 30°C in the Ringer solution were covered with a tissue Electron microscopy culture medium (Dulbecco's modified Eagle's medium For transmission electron microscopy the cells were fixed buffered with Hepes and supplemented with 20 % foetal calf with 2 % glutaraldehyde, buffered with 0-1 M-sodium cacody- serum - all from Flow Labs). After overnight incubation at late at pH 7-2 and post-fixed with 1 % OsO . The fixed cells 30°C, the cells were used in experiments. 4 were embedded in Epon after ethanol-acetone dehydration. Pigment aggregation in melanophores was induced by Ultrathin sections of cells were cut and stained with aqueous 4 10~ M-adrenaline. For pigment dispersion the coverslips uranyl acetate and lead citrate according to Reynolds (1963). with spread cells were transferred into Ringer solution The sections were examined and photographed in a Hitachi containing 5 mM-caffeine. HU-12 electron microscope, operated at 75 kV. Bovine tracheal epithelial cells (FBT line, Machatkova & Pospisil, 1975) were grown in a mixture of Eagle's minimum Microinjection essential medium (45%), 0-5% lactalbumin hydrolysate A monoclonal antibody to vimentin (clone NT30) was (45 %), bovine serum (9%) and foetal calf serum (1 %). precipitated from ascites fluid with 50 % ammonium sulphate and dialysed against microinjection buffer (Klymkowsky, 1981). The antibody solution was clarified by centrifugation Immunoflnorescence staining at lOOOOO^for 1 h, diluted with the microinjection buffer to a Cells were extracted with Triton X-100, fixed and stained final concentration of 3mgml~' and used within 8h after with antibodies, as described earlier (Rodionov et al. 1985). centrifugation. Microinjection was performed essentially as Extraction was performed in 0-1 % Triton X-100 solution in described by Graessmann & Graessmann (1976). buffer M, containing SOmM-imidazole, 50mM-KCl, 05 mM- MgCI2, lmM-EGTA, OlmM-EDTA and 1 mM-2-mercapto- ethanol and supplemented with 4% poly(ethyleneglycol) Results 40000. Formaldehyde (4%) in phosphate-buffered saline was used as a fixative. Cultured melanophores of black tetra are large, well- The monospecific antibody to bovine brain tubulin and the spread cells of round or stellate morphology. Melano- monoclonal antibody to vimentin, clone NT30, were charac- somes are uniformly distributed throughout the cyto- terized elsewhere (Bershadsky et al. 1978; Troyanovsky et al. plasm of each cell kept in the tissue culture medium, 3 3 1985). Monoclonal antibodies to 4O(XlO )Mr, 49(XlO )Mr except for a small zone in the cell centre, which is and 55 (X 103)A/ rat cytokeratins were a generous gift from r usually free of pigment (Fig. 1). Adrenaline treatment Dr G. A. Bannikov (Cancer Research Centre, Moscow). induced the aggregation of melanosomes to the cell Monoclonal antibody against 210(Xl03)M neurofilament r centre within 3-5 min. protein was obtained as described earlier (Rodionov et al. 1985). To visualize cytoskeletal structures in cultured mel- Antiserum to porcine lens vimentin was obtained by anophores we used indirect immunofluorescence stain- immunization of rabbits with vimentin, prepared according ing with antibodies against tubulin and vimentin. Only to Geisler & Weber (1981) and additionally purified by slab melanophores with aggregated pigment were stained SDS-gel electrophoresis. Each rabbit was immunized with because opaque melanosomes interfered with the cyto- 2mg of protein in Freund's complete adjuvant, boosted 30 skeletal images. For immunofluorescence staining, the days later with an additional 2 mg, and bled within 8-12 days cells were first treated with the solution of Triton after the boost. Antiserum specificity was checked by irarau- X-100 to extract the plasma membrane and soluble noblotting against mouse embryo fibroblast lysate where it proteins, then fixed with formaldehyde and finally reacted with only one band having an electrophoretic mo- stained with antibodies. The results of immunofluor- bility identical to porcine vimentin. To check whether this escence staining of melanophores with monoclonal antiserum reacted with other intermediate filament proteins we stained frozen sections of rat tongue. In these sections the antibody NT30 against vimentin are shown in Fig. 2A. antiserum reacted only with cells of the lamina propria as well This antibody revealed a dense net of fibrils, radiating as the cells in blood vessels, thus showing the absence of from the cell centre. Some of these fibrils were curved cross-reactivity with desmin, prekeratins and neurofilament and the ends of most of them bent to follow the cell proteins. margins (Fig. 2A).

650 F. K. Gyoeva et al. Fig. 1. Adrenaline-induced aggregation of nielanosomes in mclanophores of black tetra. A, melanophore with dispersed melanosomes; B, the same cell, but after 15min of adrenaline treatment. Phase-contrast. Bar, 20/ini.

Fig. 2. Double immunofluorescence staining of black tctra melanophore with monoclonal vinicntin antibody, clone NT30 (A) and rabbit antibody against tubulin (B). Bar, 20jum.

In order to cheek whether the fibrils stained with the indistinguishable from the intermediate filaments by vimentin antibody were really intermediate filaments, morphological criteria. the resistance of intermediate filaments to high salt The melanophores were not stained with monoclonal treatment was used. Melanophores were first extracted anticytokeratin antibodies or with an antibody against with 01 % Triton X-100, and then treated with 0-6 M- 210 (X 10') Mr neurofilamcnt protein. So it seems likely KC1 in buffer M, fixed and stained. It could be clearly that both cytokeratin intermediate filaments and neuro- seen (Fig. 3) that in KCl-treated the filaments are not expressed in melanophores. vimentin antibody stained fibrils having the same The NT30 antibody used in this study interacts only distribution as in the untreated cells. with vimentin in mammalian cell extracts (Troya- To test whether intermediate filaments were really novsky el al. 1()85). However, it is well known that present in the melanophores, thin sections of cells were examined by transmission electron microscopy. Fig. 4 monoclonal antibodies may recognize the same epitope shows a section of a melanophore with aggregated on different proteins and a possibility remains that in melanosomes. A dense mesh work of filaments is visible fish melanophores NT30 antibody interacts with a in the cytoplasm. Between these filaments 25 nm protein different from vimentin. To confirm that microtubules are clearly identifiable. In addition to the intermediate filaments of melanophores were really microtubules, filaments with a diameter of about 10 nm composed of vimentin, the rabbit antiserum to porcine can be seen. Thus, electron microscopy reveals fila- lens vimentin containing the mixture of antibodies to ments in the cytoplasm of melanophores which are different vimentin epitopes was used for melanophore

Inlennediatc filaments in fish nielanophores 651 "mt

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Fig. 3. Iniinunofluorescence staining of 0-6M-KCl-extracted melanophore with vimentin antibody (NT30). Bar, 20fim. Fig. 4. Transmission electron microscopy of melanophore with aggregated melanosomes. Microtubules (ml) and intermediate filaments (if) are indicated. Bar, O'lfim. staining. It was clear from double-labelling exper- iments that both monoclonal and polvclonal antibodies stained the same set of fibrils in melanophores. As we have demonstrated above (see Materials and methods), the antiserum used reacted only with vimentin, but 205- did not cross-react with other intermediate filament proteins of rat tongue. These data suggest that inter- 116- mediate filaments of fish melanophores reacted with 97- Z. vimentin antibodies. To confirm these results we used immunoblotting. Up to 1500-2000 melanophores were manually isolated 66- _ from the scales of black tetra and lysed in 1 % SDS. Proteins of the lysate were separated by SDS-gel electrophoresis, transferred to nitrocellulose and stained with vimentin antiserum. Fig. 5 lane b shows 45- the electrophoresis of total melanophore proteins. Lane d shows that vimentin antiserum reacts in the lysate with a protein of the same molecular weight as vimentin. Thus both immunoblotting and immuno- 29- fluorescence data show the presence of vimentin intermediate filaments in melanophores. To study whether intermediate filaments are present in melanophores of more than one teleost species, the NT30 antibody was used to stain melanophores of angelfish. These cells, too, had radially arranged fila- Fig. 5. Identification of the protein which reacts with ments which bound the vimentin antibody (not polvclonal anti-vimentin antiserum in melanophore lysate shown). So, the vimentin intermediate filaments are by immunoblotting. a,b, Polyacrylamide gel stained with Coomassie Blue R-250; c,d, immunoblotting stained by not unique for black tetra melanophores, but are vimentin antiserum; a,c, purified vimentin; b,d, present in melanophores of other teleost species. melanophore lysate; positions of the molecular weight In most cells the distribution of intermediate markers are shown at the left side of the gel. filaments is very similar to the distribution of micro- tubules. The arrangement of microtubules and inter- intermediate filaments are radially arranged, originat- mediate filaments in melanophores was directly ing from the geometrical centre of a melanophore. compared by double immunofluorescent labelling. In all the cells studied so far, the distribution of Figs 2A and 2B show that both microtubules and vimentin-type intermediate filaments depends on the

652 F. K. Gvoeva et al. Fig. 6. Distribution of vimentin intermediate filaments in nocodazole-treated melanophore. A. Phase contrast; B, immunofluorescence with NT30 antibody. Bar, lOjiim.

integrity of cytoplasmic microtubules. After disruption of microtubules intermediate filaments collapse to form a bundle at the cell centre, so we studied the rearrange- ment of intermediate filaments after disruption of microtubules in the melanophores with aggregated pigment granules. Immunofluorescent staining by the antibody to tubulin showed that treatment of the melanophore with lOjUgml"1 nocodazole for 5h dis- rupted microtubules completely. These cells often retracted their cytoplasm, sometimes leaving several branched processes attached to the substrate (Fig. 6A). Intermediate filaments in nocodazole-treated cells formed bundles or aggregates of variable width (Fig. 6B). However, intermediate filaments did not aggregate to the cell centre. This result indicates that the distribution of intermediate filaments in melano- phores similarly to that in other cell types depends on the microtubules, but disruption does not Fig. 7. Effect of the microinjection of monoclonal induce collapse of intermediate filaments to the cell antibody NT30 into melanophores. The melanophore centre. Similar results were obtained if microtubules shown in this figure was microinjected by the vimentin were disrupted by colchicine (not shown). antibody, treated withTriton X-100, fixed and stained with fluorescein-conjugated goat antibody against mouse To study whether intermediate filaments participate immunoglobulins. in pigment transport, the NT30 antibody was microinjected into melanophores. Immunofluorescent staining with fluorescein-labelled anti-mouse immuno- the cells retained their ability to move melanosomes globulins clearly showed that NT30 antibody binds to after microinjection of NT30 antibody, bovine serum intermediate filaments in injected cells (Fig. 7). In albumin or microinjection buffer. The NT30 antibody, contrast to the results in other cell types microinjection of NT30 antibody did not change intermediate fila- however, was able to induce collapse of intermediate ment distribution in melanophores. Microinjection also filaments in cultured FBT cells (Fig. 8). Therefore the had no influence on the melanosome movement. In the absence of intermediate filament redistribution in mel- cells which had not been damaged by microinjection of anophores after microinjection can be explained by NT30 antibody, both adrenaline- and caffeine-induced some properties of these cells rather than by some movements seem to be normal. Approximately 60 % of properties of antibody.

Intermediate filaments in fish melanophores 653 Fig. 8. Redistribution of intermediate filaments in cultured FBT cells after microinjection of NT30 antibody. A. Staining with FITC-labelled antibody to mouse immunoglobulins to reveal intermediate filaments in injected cells; B, staining with rabbit antibody to tubulin and rhodamine-labelled antibody to rabbit immunoglobulins to reveal microtubules in cultured cells. Microinjected cells are shown in B by arrows. Note that microtubule system in injected cells remained intact. Bar, 20/

Discussion dense irregular network, while in melanophores they show a precise radial organization. The composition and spatial organization of the inter- However, as in other cells, the distribution of mediate filaments in the melanophores of the two intermediate filaments in melanophores is identical to teleost species, Gymnocorymbus ternetzi and Ptero- that of microtubules. As both types of cytoskeletal phyllum scalare, were studied in this paper by immu- structures are oriented along the melanosome path- nofluorescent staining and immunoblotting tech- ways, it seems probable that they participate in niques. The immunofluorescent staining with both intracellular transport. The role of microtubules and monoclonal and polyclonal anti-vimentin antibodies intermediate filaments in intracellular trans- (but not with antibodies against cytokeratins or neuro- port has already been suggested by Wang et al. (1979) filament proteins) revealed a fibrillar network in the and Wang & Goldman (1978). In this work we initiated melanophore cytoplasm. These fibrils were identical to the study of the role of intermediate filaments in intermediate filaments in their properties: they were melanosome movement by microinjection of a vimen- salt-insoluble and did not depolymerize after nocod- tin antibody into melanophores. NT30 antibody used azole treatment. Immunoblotting experiments in these experiments bound to intermediate filaments detected vimentin in melanophore lysates. All these after microinjection but failed to block melanosome results, taken together, demonstrate the presence of aggregation and dispersion. Our results are consistent vimentin intermediate filaments in the melanophore with the data of Klymkowsky et al. (1983) and Eckert cytoplasm. (1986). They induced a collapse of intermediate fila- Intermediate filaments in fish pigment cells have not ments in cultured cells by antibody microinjection yet been studied extensively and their protein compo- (Klymkowsky et al. 1983) or by acrylamide treatment sition has not been determined. Walker et al. (1985) (Eckert, 1986). Because the particles continued to recently purified intermediate filaments from trans- move in the peripheral cytoplasm where intermediate formed goldfish xanthophores. These fibrils were com- filaments were absent, it is clear that intermediate posed of four protein species with molecular weights of filaments are not essential for organelle movement. 3 Nevertheless, it cannot be definitely concluded from 45, 51, 56 and 6O(XlO )jV/r. Thus, they differed from all the known intermediate filaments, at least in protein our experiments that intermediate filaments do not composition. Murphy & Grasser (1984) described participate in aggregation and/or dispersion of melano- intermediate filaments in fish melanophores, but they somes. It is possible that the NT30 antibody is directed did not determine their composition. against the vimentin epitope not essential for inter- mediate filament functions. If intermediate filaments The distribution of intermediate filaments in mel- are really the components of the partici- anophores differs significantly from their distribution pating in melanosome movement, possibly some other in other cell types. Vimentin filaments usually form a

654 F. K. Gvoeva et al. anti-vimentin antibody would inhibit the pigment MACHATKOVA, M. & POSPISIL, Z. (1975). Biological granule transport. characteristics of cell lines derived from respiratory tract One more possibility is that instead of being involved of a bovine foetus (growth characteristics). Folia in pigment transport, intermediate filaments are purely biologica (Praha) 21, 117-121. structural components. The indirect evidence in sup- MCNIVEN, M. & PORTER, K. R. (1984). - port of this view is based on the 'rigidity' of the models for studying cytomatrix translocations. J. Cell Biol. 99, 152s-158s. melanophore intermediate filament system. While in MURPHY, D. B. & GRASSER, W. A. (1984). Intermediate other cells the distribution of intermediate filaments filaments in the cytoskeletons of fish chromatophores. J. depends closely on the distribution of mierotubules, in Cell Sci. 66, 353-366. melanophores depolymerization of mierotubules does REYNOLDS, E. S. (1963). The use of lead citrate at high not cause aggregation of intermediate filaments to the pH as an electron opaque stain in electron microscopy. J. cell centre. Intermediate filaments failed to aggregate Cell Biol. 17, 208-212. even after microinjection of antibodies, which cause the RODIONOV, V. I., NADEZHDINA, E. S., LEONOVA, E. V., formation of the perinuclear intermediate filament coils VAISBERG, E. A., KUZNETSOV, S. A. & GELFAND, V. I. in cultured mammalian cells (Klymkowsky, 1981; (1985). Identification of a 100 kD protein associated with Klymkowsky et al. 1983). mierotubules, intermediate filaments and coated vesicles It is clear in any case that there is a well-developed in cultured cells. Expl Cell Res. 159, 377-387. radial system of vimentin intermediate filaments in fish SCHLIWA, M. (1981). Microtubule-dependent intracellular melanophores. Further studies of this system can transport in chromatophores. 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