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Vimentin Intermediate Filaments in Fish Melanophores

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Vimentin Intermediate Filaments in Fish Melanophores Vimentin intermediate filaments in fish 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 cell 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 intermediate filament 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. Microtubules form a well-developed radial pattern in fish melanophores and their disruption Melanophores are highly specialized cells, containing a inhibits pigment granule 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 microfilaments 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 animals, 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 cytoplasm depends on cytoskeletal structures - micro- Materials and methods tubules, actin microfilaments and intermediate fila- ments. In many types of animal 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 fishes 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
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