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 ofintermediate filaments in cultured melanophoresof two teleost species - Gymnocorymbus ternetziand Pterophyllum scalare - were studied by im-munofluorescence staining and immunoblottingtechniques. The immunofluorescence staining ofthe melanophores with monoclonal and poly-clonal antibodies to the intermediate filamentprotein vimentin revealed a system of fibrilsradiating from the cell centre. These fibrils wereresistant to 0-6M-KC1 and nocodazole treatments

as has been found in other cell types. Trans-mission electron microscopy confirmed the pres-ence of intermediate filaments in melanophores.Immunoblotting experiments showed the pres-ence of the intermediate filament protein vimen-tin in melanophore lysates. Therefore, teleostmelanophores possess a developed radial systemof vimentin intermediate filaments.

Key words: melanophore, intermediate filaments,vimentin.

Introduction

Melanophores are highly specialized cells, containing alot of pigment granules known as melanosomes.Teleost melanophores can aggregate melanosomes tothe cell centre or disperse them throughout the cyto-plasm. These melanosome movements, which deter-mine the colour changes of animals, are governed byneurohumoral stimuli. Cultured melanophores can beinduced to aggregate pigment particles by adrenalinetreatment and to disperse them by caffeine treatment.Melanosome movement is easily observable using lightmicroscopy, and that is why melanophores are aconvenient system in which the mechanisms of intra-cellular movements can be studied (reviewed bySchliwa, 1981; Stearns, 1984; McNiven & Porter,1984).

It is well known that the movement of particles in thecytoplasm depends on cytoskeletal structures - micro-tubules, actin microfilaments and intermediate fila-ments. In many types of animal cells the intracellularmovement was shown to depend on microtubules (seeSchliwa, 1984), though two other structures may also

Journal of Cell Science 88, 649-655 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

be involved. Microtubules form a well-developed radialpattern in fish melanophores and their disruptioninhibits pigment granule movement (Schliwa, 1981;Stearns, 1984). In contrast to microtubules, the systemof actin microfilaments in melanophores is poorlydeveloped. Sparse microfilaments have been found inthe cell cortex and in the cell surface microvilli(Schliwa et al. 1981). Very little is known about thethird component of the melanophore cytoskelcton -intermediate filaments. Only recently Murphy &Grasser (1984) found 10 nm filaments in black tetramelanophores. However, their distribution has not yetbeen studied and their chemical composition remainsundetermined. In this paper we describe an immuno-fluorescence and immunoblotting study of intermedi-ate filaments in fish melanophores.

Materials and methods

Tissue culturesPrimary cultures of fish melanophores were obtained essen-tially according to Schliwa el al. (1978) and Luby & Porter

649

(1980). The scales of aquarium fishes Gymnocorymbus ter-netzi and Pterophyllum scalare were placed in a Ringersolution (103mM-NaCl, l-8mM-KCl, 0-8mM-NaHCO3,2mM-CaCl2, 5mM-Tris-HCl, pH7-3), containing 1 mgml"1

collagenase (Fluka), lmgml" ' hyaluronidase (Type I,Sigma Chemical Co.) and 5mgml~' bovine serum albumin(Fraction V, Sigma Chemical Co.). After incubation for30-60 min at 30-35 °C the melanophores were removed fromthe scales by pipetting and washed three times in Ringersolution by transferring from one Petri dish to another.Finally, the melanophores were placed for attachment ontocarbon-coated coverslips and after incubation for 30-60 minat 30°C in the Ringer solution were covered with a tissueculture medium (Dulbecco's modified Eagle's mediumbuffered with Hepes and supplemented with 20 % foetal calfserum - all from Flow Labs). After overnight incubation at30°C, the cells were used in experiments.

Pigment aggregation in melanophores was induced by10~4M-adrenaline. For pigment dispersion the coverslipswith spread cells were transferred into Ringer solutioncontaining 5 mM-caffeine.

Bovine tracheal epithelial cells (FBT line, Machatkova &Pospisil, 1975) were grown in a mixture of Eagle's minimumessential medium (45%), 0-5% lactalbumin hydrolysate(45 %), bovine serum (9%) and foetal calf serum (1 %).

Immunoflnorescence staining

Cells were extracted with Triton X-100, fixed and stainedwith antibodies, as described earlier (Rodionov et al. 1985).Extraction was performed in 0-1 % Triton X-100 solution inbuffer M, containing SOmM-imidazole, 50mM-KCl, 0 5 mM-MgCI2, lmM-EGTA, OlmM-EDTA and 1 mM-2-mercapto-ethanol and supplemented with 4% poly(ethyleneglycol)40000. Formaldehyde (4%) in phosphate-buffered salinewas used as a fixative.

The monospecific antibody to bovine brain tubulin and themonoclonal antibody to vimentin, clone NT30, were charac-terized elsewhere (Bershadsky et al. 1978; Troyanovsky et al.1985). Monoclonal antibodies to 4O(XlO3)Mr, 49(XlO3)Mr

and 55 (X 103)A/r rat cytokeratins were a generous gift fromDr G. A. Bannikov (Cancer Research Centre, Moscow).Monoclonal antibody against 210(Xl03)Mr neurofilamentprotein was obtained as described earlier (Rodionov et al.1985).

Antiserum to porcine lens vimentin was obtained byimmunization of rabbits with vimentin, prepared accordingto Geisler & Weber (1981) and additionally purified by slabSDS-gel electrophoresis. Each rabbit was immunized with2mg of protein in Freund's complete adjuvant, boosted 30days later with an additional 2 mg, and bled within 8-12 daysafter the boost. Antiserum specificity was checked by irarau-noblotting against mouse embryo fibroblast lysate where itreacted with only one band having an electrophoretic mo-bility identical to porcine vimentin. To check whether thisantiserum reacted with other intermediate filament proteinswe stained frozen sections of rat tongue. In these sections theantiserum reacted only with cells of the lamina propria as wellas the cells in blood vessels, thus showing the absence ofcross-reactivity with desmin, prekeratins and neurofilamentproteins.

Secondary antibodies conjugated with fluorescein andrhodamine (Sigma Chemical Co.) were used in a dilution of1:100.

Polyacrylamide gel electrophoresis andimmunoblotting

Electrophoresis was performed in polyacrylamide-SDS slabgels (Laemmli, 1970). The method of Towbin et al. (1979)was used for immunoblotting. Peroxidase-conjugated second-ary antibodies were purchased from Sigma Chemical Co. andused in a dilution of 1:200.

Electron microscopy

For transmission electron microscopy the cells were fixedwith 2 % glutaraldehyde, buffered with 0-1 M-sodium cacody-late at pH 7-2 and post-fixed with 1 % OsO4. The fixed cellswere embedded in Epon after ethanol-acetone dehydration.Ultrathin sections of cells were cut and stained with aqueousuranyl acetate and lead citrate according to Reynolds (1963).The sections were examined and photographed in a HitachiHU-12 electron microscope, operated at 75 kV.

Microinjection

A monoclonal antibody to vimentin (clone NT30) wasprecipitated from ascites fluid with 50 % ammonium sulphateand dialysed against microinjection buffer (Klymkowsky,1981). The antibody solution was clarified by centrifugationat lOOOOO f̂or 1 h, diluted with the microinjection buffer to afinal concentration of 3mgml~' and used within 8h aftercentrifugation. Microinjection was performed essentially asdescribed by Graessmann & Graessmann (1976).

Results

Cultured melanophores of black tetra are large, well-spread cells of round or stellate morphology. Melano-somes are uniformly distributed throughout the cyto-plasm of each cell kept in the tissue culture medium,except for a small zone in the cell centre, which isusually free of pigment (Fig. 1). Adrenaline treatmentinduced the aggregation of melanosomes to the cellcentre within 3-5 min.

To visualize cytoskeletal structures in cultured mel-anophores we used indirect immunofluorescence stain-ing with antibodies against tubulin and vimentin. Onlymelanophores with aggregated pigment were stainedbecause opaque melanosomes interfered with the cyto-skeletal images. For immunofluorescence staining, thecells were first treated with the solution of TritonX-100 to extract the plasma membrane and solubleproteins, then fixed with formaldehyde and finallystained with antibodies. The results of immunofluor-escence staining of melanophores with monoclonalantibody NT30 against vimentin are shown in Fig. 2A.This antibody revealed a dense net of fibrils, radiatingfrom the cell centre. Some of these fibrils were curvedand the ends of most of them bent to follow the cellmargins (Fig. 2A).

650 F. K. Gyoeva et al.

Fig. 1. Adrenaline-induced aggregation of nielanosomes in mclanophores of black tetra. A, melanophore with dispersedmelanosomes; 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 thevimentin antibody were really intermediate filaments,the resistance of intermediate filaments to high salttreatment was used. Melanophores were first extractedwith 0 1 % Triton X-100, and then treated with 0-6 M-KC1 in buffer M, fixed and stained. It could be clearlyseen (Fig. 3) that in KCl-treated cytoskeletons thevimentin antibody stained fibrils having the samedistribution as in the untreated cells.

To test whether intermediate filaments were reallypresent in the melanophores, thin sections of cells wereexamined by transmission electron microscopy. Fig. 4shows a section of a melanophore with aggregatedmelanosomes. A dense mesh work of filaments is visiblein the cytoplasm. Between these filaments 25 nmmicrotubules are clearly identifiable. In addition to themicrotubules, filaments with a diameter of about 10 nmcan be seen. Thus, electron microscopy reveals fila-ments in the cytoplasm of melanophores which are

indistinguishable from the intermediate filaments bymorphological criteria.

The melanophores were not stained with monoclonalanticytokeratin antibodies or with an antibody against210 (X 10') Mr neurofilamcnt protein. So it seems likelythat both cytokeratin intermediate filaments and neuro-filaments are not expressed in melanophores.

The NT30 antibody used in this study interacts onlywith vimentin in mammalian cell extracts (Troya-novsky el al. 1()85). However, it is well known thatmonoclonal antibodies may recognize the same epitopeon different proteins and a possibility remains that infish melanophores NT30 antibody interacts with aprotein different from vimentin. To confirm thatintermediate filaments of melanophores were reallycomposed of vimentin, the rabbit antiserum to porcinelens vimentin containing the mixture of antibodies todifferent vimentin epitopes was used for melanophore

Inlennediatc filaments in fish nielanophores 651

"mt

if

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) andintermediate filaments (if) are indicated. Bar, O'lfim.

staining. It was clear from double-labelling exper-iments that both monoclonal and polvclonal antibodiesstained the same set of fibrils in melanophores. As wehave demonstrated above (see Materials and methods),the antiserum used reacted only with vimentin, butdid not cross-react with other intermediate filamentproteins of rat tongue. These data suggest that inter-mediate filaments of fish melanophores reacted withvimentin antibodies.

To confirm these results we used immunoblotting.Up to 1500-2000 melanophores were manually isolatedfrom the scales of black tetra and lysed in 1 % SDS.Proteins of the lysate were separated by SDS-gelelectrophoresis, transferred to nitrocellulose andstained with vimentin antiserum. Fig. 5 lane b showsthe electrophoresis of total melanophore proteins.Lane d shows that vimentin antiserum reacts in thelysate with a protein of the same molecular weight asvimentin. Thus both immunoblotting and immuno-fluorescence data show the presence of vimentinintermediate filaments in melanophores.

To study whether intermediate filaments are presentin melanophores of more than one teleost species, theNT30 antibody was used to stain melanophores ofangelfish. These cells, too, had radially arranged fila-ments which bound the vimentin antibody (notshown). So, the vimentin intermediate filaments arenot unique for black tetra melanophores, but arepresent in melanophores of other teleost species.

In most cells the distribution of intermediatefilaments is very similar to the distribution of micro-tubules. The arrangement of microtubules and inter-mediate filaments in melanophores was directlycompared by double immunofluorescent labelling.Figs 2A and 2B show that both microtubules and

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Fig. 5. Identification of the protein which reacts withpolvclonal anti-vimentin antiserum in melanophore lysateby immunoblotting. a,b, Polyacrylamide gel stained withCoomassie Blue R-250; c,d, immunoblotting stained byvimentin antiserum; a,c, purified vimentin; b,d,melanophore lysate; positions of the molecular weightmarkers are shown at the left side of the gel.

intermediate filaments are radially arranged, originat-ing from the geometrical centre of a melanophore.

In all the cells studied so far, the distribution ofvimentin-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 disruptionof microtubules intermediate filaments collapse to forma bundle at the cell centre, so we studied the rearrange-ment of intermediate filaments after disruption ofmicrotubules in the melanophores with aggregatedpigment granules. Immunofluorescent staining by theantibody to tubulin showed that treatment of themelanophore with lOjUgml"1 nocodazole for 5h dis-rupted microtubules completely. These cells oftenretracted their cytoplasm, sometimes leaving severalbranched processes attached to the substrate (Fig. 6A).Intermediate filaments in nocodazole-treated cellsformed bundles or aggregates of variable width(Fig. 6B). However, intermediate filaments did notaggregate to the cell centre. This result indicates thatthe distribution of intermediate filaments in melano-phores similarly to that in other cell types depends onthe microtubules, but microtubule disruption does notinduce collapse of intermediate filaments to the cellcentre. Similar results were obtained if microtubuleswere disrupted by colchicine (not shown).

To study whether intermediate filaments participatein pigment transport, the NT30 antibody wasmicroinjected into melanophores. Immunofluorescentstaining with fluorescein-labelled anti-mouse immuno-globulins clearly showed that NT30 antibody binds tointermediate filaments in injected cells (Fig. 7). Incontrast to the results in other cell types microinjectionof NT30 antibody did not change intermediate fila-ment distribution in melanophores. Microinjection alsohad no influence on the melanosome movement. In thecells which had not been damaged by microinjection ofNT30 antibody, both adrenaline- and caffeine-inducedmovements seem to be normal. Approximately 60 % of

Fig. 7. Effect of the microinjection of monoclonalantibody NT30 into melanophores. The melanophoreshown in this figure was microinjected by the vimentinantibody, treated withTriton X-100, fixed and stained withfluorescein-conjugated goat antibody against mouseimmunoglobulins.

the cells retained their ability to move melanosomesafter microinjection of NT30 antibody, bovine serumalbumin or microinjection buffer. The NT30 antibody,however, was able to induce collapse of intermediatefilaments in cultured FBT cells (Fig. 8). Therefore theabsence of intermediate filament redistribution in mel-anophores after microinjection can be explained bysome properties of these cells rather than by someproperties of antibody.

Intermediate filaments in fish melanophores 653

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

Discussion

The composition and spatial organization of the inter-mediate filaments in the melanophores of the twoteleost species, Gymnocorymbus ternetzi and Ptero-phyllum scalare, were studied in this paper by immu-nofluorescent staining and immunoblotting tech-niques. The immunofluorescent staining with bothmonoclonal and polyclonal anti-vimentin antibodies(but not with antibodies against cytokeratins or neuro-filament proteins) revealed a fibrillar network in themelanophore cytoplasm. These fibrils were identical tointermediate filaments in their properties: they weresalt-insoluble and did not depolymerize after nocod-azole treatment. Immunoblotting experimentsdetected vimentin in melanophore lysates. All theseresults, taken together, demonstrate the presence ofvimentin intermediate filaments in the melanophorecytoplasm.

Intermediate filaments in fish pigment cells have notyet been studied extensively and their protein compo-sition has not been determined. Walker et al. (1985)recently purified intermediate filaments from trans-formed goldfish xanthophores. These fibrils were com-posed of four protein species with molecular weights of45, 51, 56 and 6O(XlO3)jV/r. Thus, they differed fromall the known intermediate filaments, at least in proteincomposition. Murphy & Grasser (1984) describedintermediate filaments in fish melanophores, but theydid not determine their composition.

The distribution of intermediate filaments in mel-anophores differs significantly from their distributionin other cell types. Vimentin filaments usually form a

dense irregular network, while in melanophores theyshow a precise radial organization.

However, as in other cells, the distribution ofintermediate filaments in melanophores is identical tothat of microtubules. As both types of cytoskeletalstructures are oriented along the melanosome path-ways, it seems probable that they participate inintracellular transport. The role of microtubules andintermediate filaments in intracellular organelle trans-port has already been suggested by Wang et al. (1979)and Wang & Goldman (1978). In this work we initiatedthe study of the role of intermediate filaments inmelanosome movement by microinjection of a vimen-tin antibody into melanophores. NT30 antibody usedin these experiments bound to intermediate filamentsafter microinjection but failed to block melanosomeaggregation and dispersion. Our results are consistentwith the data of Klymkowsky et al. (1983) and Eckert(1986). They induced a collapse of intermediate fila-ments in cultured cells by antibody microinjection(Klymkowsky et al. 1983) or by acrylamide treatment(Eckert, 1986). Because the particles continued tomove in the peripheral cytoplasm where intermediatefilaments were absent, it is clear that intermediatefilaments are not essential for organelle movement.Nevertheless, it cannot be definitely concluded fromour experiments that intermediate filaments do notparticipate in aggregation and/or dispersion of melano-somes. It is possible that the NT30 antibody is directedagainst the vimentin epitope not essential for inter-mediate filament functions. If intermediate filamentsare really the components of the cytoskeleton partici-pating in melanosome movement, possibly some other

654 F. K. Gvoeva et al.

anti-vimentin antibody would inhibit the pigmentgranule transport.

One more possibility is that instead of being involvedin pigment transport, intermediate filaments are purelystructural components. The indirect evidence in sup-port of this view is based on the 'rigidity' of themelanophore intermediate filament system. While inother cells the distribution of intermediate filamentsdepends closely on the distribution of mierotubules, inmelanophores depolymerization of mierotubules doesnot cause aggregation of intermediate filaments to thecell centre. Intermediate filaments failed to aggregateeven after microinjection of antibodies, which cause theformation of the perinuclear intermediate filament coilsin cultured mammalian cells (Klymkowsky, 1981;Klymkowsky et al. 1983).

It is clear in any case that there is a well-developedradial system of vimentin intermediate filaments in fishmelanophores. Further studies of this system canelucidate its role not only in melanophores but also inother cells.

The authors thank Dr O. B. Solovjanova for thin section-ing and Dr S. A. Kuznetsov for help in immunoblottingexperiments.

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