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The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole
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1983
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THE INTERACTIONBETWEEN MICROTUBULES AND INTERMEDIATEFILAMENTS IN CULTUREDCELLS TREATEDWITH TAmL AND NOCODAZOLE G. Geuens, M. de Brabander, R. Nuydens and J. De Mey Laboratory of Oncology, Janssen Pharmaceutics Research Laboratories, B-2340 Beerse, Belgium ABSIXACT Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MP) and intermediate filaments (IF) in MO cells treated with various combinaiions of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MC bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated is cells a close association between these two filamentous systems with nocodazole followed by addition observed. In cells pretreated to induce the bundles of free MT, the preexisting IF of taxol, coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MC determine the spatial distribution of IF which most probably involves some kind of physicochemical link. INTRODUCTION A close association between MT and IF of the vimentin or desmin type has been observed in many cells using different techdescribed the parallel alignment of niques. Goldman (1969) first MT and IF at the ultrastructural level in BBK cells. By means of double-label immunofluorescence , Geiger and Singer (1980) and Ball and Singer (1981) showed that in whole interphase fibroblasts but not complete superposition there was an extensive of the labelling patterns of the two filamentous systems. Microtubule disintegration by specific inhibitors results in loss of cell polarity, disordered organelle topography and arrest of saltatory motions (Freed and Lebowits, 1970; De Brabander et al., 1975; Moskalewski et al., 1975). Invariably, this also induces IF to bundles or caps (Wang and Goldman, aggregate into perinuclear 1978; De Brabander, 1975). These observations suggested that 60th types of fibers are normally engaged in some kind of structural
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interaotion and that they serve as a functional unit in the determination of cell polarity and cytoplasmic organisation. However, some doubt is raised by experiments in which the IF distribution is profoundly altered without apparently affecting the MT system. Ball and Singer (1981) showed that the association can be disrupted upon transformation by a temperature-sensitive mutant of ROUS Sarcoma virus. Hynes (1978) described that NIL-8 cells showed an altered organisation of IF after HSV transformation, while MT remained normal. Sharpe et al. (1980) examined by immunofluorescence the effect of diphtheria toxin, exotoxin A and cycloheximide on CV-1 cell cytoskeletons and found that these agents specifically disrupted the vimentin filament system without any effect on MT. Wang et al. (1981) reported the effect of vanadate on virus-induced syncytia and infected cells. They found with sodium vanadate an altered distribution of IF, whereas MT. orgaBy means of microinjection of Gerbil finisation was unaltered. broma cells with a monoclonal antibody against components of the IF system, Lin and Feramisco (1981) showed by double label immunofluorescence and electron microscopy that the vimentin containing IF of the antibody-injected cells redistributed, while MT were Klymkowsky (1981) found similar results in 3T3 mouse undisturbed. Both authors found no changes in cell morphology and fibroblasts. polarity and the antibody-injected cells showed normal saltatory It can thus not be excluded that the apmovements of organelles. parent codistribution of MT and IF is fortuitous, both systems occupying the same cytoplasmic channels. More information on the putative interaction and its role in cytoplasmic organisation may be obtained in experiments where the MT distribution is altered without completely dissolving it. Recently such experiments have become possible by the use of taxol (Schiff, 1980). Treatment of cells with taxol produces a disorganised network of free MT, which 1981; tend to form bundles to various degrees (De Brabander, is altered as with clasSchiff, 1980). Cell shape and motility (De Brasical microtubule inhibitors (colchicine or nocodazole) bander, 1976, 1981a). We have investigated, using double label immunofluorescence microscopy and electron microscopy, the interaction of MT and IF in cells treated with taxol and nocodazole in We see that in cells treated with tax01 IF various combinations. do not form coils but become rearranged so as to codistribute partially with the free MT. Furthermore we show that in cells first treated with nocodazole (2.5 x 10m6 M) and then further incubated with taxol (10B4 M) and nocodazole the IF leave the coils and associate with the disordered arrays of MT. In cells treated with nocodazole (2.5 x 10m5 M) and taxol (5.10B6 M), containing only short MT connected to the centrosomes, the IF coils persist. MATERIALSAND METM)DS Cell onal
lines: MO (De Brabander et al., 3T3-type cell line with epitheloid
1976), a C3H mouse embrycharacter was used. The
Cell Biology
Fig.
1.
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of the antidesmin
antibody.
la MO control cell fixed with methanol and incubated with an affinity purified antibody against pig stomach desmin. The stained filaments are marked with gold particles (5 nm) coupled to goat Note the parallel anti rabbit Ig. Single IF are densely labelled. alignment between single IF (arrow) and MT (arrowhead) (x 43 200). lb MO cell treated with nocodazole (2.5 x 10B6 M 24 hrs). The nocodazole induced IF coils are marked with 5 nm gold particles. (x 37 400). cultures were grown in Eagle's minimal essential medium, supplemented with non-essential amino acids and 10 % fetal bovine serum and incubated in a humidified atmosphere of 5 % CC2 in air at 37O c. Immunofluorescence: MO cell cultures were seeded on glass coverslips. After treatment, cells were fixed with methanol for 10 min at -20° C and washed with acetone at -20' C. The cells were sequentially stained with a mixture of anti-intermediate filament antibody and monoclonal antitubulin (Kilmartin, 1982) and then a mixture of TRITC-coupled goat anti-rat immunoglobulins (Nordic) and FITC-coupled goat anti-rabbit immunoglobulin (Nordic). The nuclei were stained with propidium iodide (Rarni et al., 1981). After washing, coverslips were mounted in gelvatol and viewed with a Leitz orthoplan microscope equipped with @ase contrast and fluorescence optics.
38
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MO cell cultures were seeded in plastic Electron microscopy: cells were fixed with glutaraldedishes (Lux). After treatment, hyde 3 % for 1 hour at room temperature and osmificated with 2 % 0~04 for 1 hour at 4O C. They were then impregnated with 0.5 % uranyl acetate in 70 % ethanol supplemented with 1 % phosphoimmunogold label experiment tungstic acid. For the ultrastructural the cells were incubated with goat anti-rabbit coupled to colloidal gold particles (De Mey et al., 1981) (GA&G5: 5 nm gold particles: Janssen Life Science Products Division, Beerse, Belgium). Dehydration was done in a series of ethanol and embedding in epon 812. Cells were selected and ultrathin sections were viewed with a Philips PI 300 or EM 201. Experiments: Cells were seeded either on glass coverslips or plastic petri dishes. After 24 or 48 hours, cells were treated with nocodazole (2.5 x lo6 M or 2.5 x lo-5 M) for 24 hours, then taxol (low4 M or 5 x 10e6 M) was added in the presence of Control culnocodazole. Cells were fixed 4 and 24 hours later. tures were treated with nocodazole and taxol only, or the appropriate solvent (DMSO) which did not affect the cells at the concentrations used. Microtubules were labelled with a monoclonal antibody Antibodies: (MC AB/YL1/2) to yeast tubulin (gift of J.V. Kilmartin, 1982). Intermediate filaments were stained with a rabbit antibody raised against and affinity purified on pig stomach desmin. This antibody decorates the IF-network of MO cells in a rather complete fashion as documented with immunofluorescence and immuno gold staining (Fig. la). The coils induced by nocodazole are also entirely labelled (fig. lb). The antibody did not stain at all MT, microprekeratin filaments in PTK2 cells or any other orfilaments, ganelles. Using the antigen spot test (Herbrink et al., 1982) the antibody reacted at the same dilution as that used for immuno+ 2 pg protein per ml) with purified pig fluorescence (l/50; stomach desmin (gift Gf V. Small and A. Sobieszek) down to 0.5 ng and with purified Ehrlich Ascites tumor cell vimentin (gift of Fig. 2-4 Double label stained with antitubulin,
immunofluorescence. those at the right
Cells at the left with antidesmin.
are
2a-b Untreated MO cells: Bundles of MT and IF and individual fibers (arrowhead) show extensive overlapping. The IF do not extend as far to the periphery as the MT' (x 500). 3a-b MO cell, treated with nocodazole (2.5 x low6 M 24 hrs). The MI network has completely disappeared, while the IF are coiled. The nuclei are stained with propidium iodide (x 500). 4a-b MO cell, treated with tax01 10m4 M for 24 hrs. The MT network has been replaced by free MC and abundant MT bundles. Note the IF in parallel alignment (arrow) with MT bundles and IF staining between fascicles (arrowhead) (x 500).
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W. J. Nelson, 1982) down to 50 ng. We are currently investigating which type of intermediate filaments predominates in MO cells. For the present paper the outcome is of minor importance since both types of fibers show a similar distribution in untreated cultered cells and react in the same way to nocodazole or colchicine. FUZSULTS In control MO cells, MT and IF extend throughout the cells into the peri@ery, although the extension of IF is not always as far (Fig. 2a,b) as described previously for similar cells (Geiger & Singer, 1980; Ball and Singer, 1981). Treatment with nocodazole causes MT dissolution, and IF coils (Fig. 3a,b). These coils are mostly limited to the organelle rich central cytoplasmic dome and excluded from thin peripheral lamellae (Fig. 3a,b). Treatment with tax01 (Fig. 4) results in cell shape change, identical as with nocodazole (Fig. 3) (De Brabander, 1976, 1981a). Long MT radiating from the perinuclear vicinity are replaced by multiple short free MT (De Brabander, 1981) (Fig. 9) within 4 hours. Between 4-24 hrs they show an increased tendency to form parallel bundles (De Brabander, 1981) (Fig. 4a, 9, 11). Microtubules are usually excluded from peripheral lamellae (De Brabander, 1981; Schiff, 1980). FLUorescent staining of IF after tax01 treatment is seen in three one can observe fluorescent staining in a disdomains. Firstly organized fashion in regions with a mesh of free MT. Secondly staining is observed as parallel alignment along MT bundles (Fig. intensity of these IF fascicles is much less 4). The staining bright than that of the nocodazole induced coils. Thirdly, very often accumulation of stain is seen at the ends of MT bundles, where small IF bundles are arranged at various angles to microThis is particularly prominent where two fascicles of tubules. microtubules are opposed with an empty region between them (Fig. 4). After 4 hours of incubation with taxol still many cells with a rather normal IF network are observed , while MT are redistributed. This shows that the IF rearrangement lags behind MT rearrangement. These observations with double-label fluorescence microscopy are confirmed at the ultrastructural level (Fig. 9, 10, 11). Whithin 4 cells with multiple free MT contain free hrs after taxol addition, IF in the same region in a disorganised way (Fig. 9). With longer taxol treatment, one can see loose bundles of MT intermingled with IF running in the same direction (Fig. 10). With dense MT bundles one can see a number of IF along these bundles (Fig. 11). In telophase cells that underwent abortive mitosis during taxol treatment one can observe midbody like structures consisting of MT fanning Their formation and structure will out from osmiophylic material. No IF are seen between the MT of these be described separately. of the centrosomes of taxol structures (Fig. 12). In the vicinity treated cells as well as in control cells some radiating IF can be observed. Ultrastructural observations of taxol treated cells show also that other cytoplasmic organelles such as the golgi apparatus
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Fig. 5-6 Double label stained with antitubulin,
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immunofluorescence. those at the right
1983
Cells at the left with antidesmin.
are
treated with nocodazole (2.5 x 10m6 M) and then 5a-b MO cell, further treated with taxol 1O'4 M for 24 hrs in the presence of the cytoplasm. The IF coils nocodazole. Free MT and bundles fill formed after nocodazole, have disappeared. IF align along MT bundles (arrow) and accumulate in a disordered fashion between two fascicles (arrowhead) (x 500). treated with nocodazole (2.5 x 10'5 M) and then 6a-b MO cell, further incubated with taxol 5.10m6 M in the presence of nocoof MT is seen (arrowhead). IF dazole. A small aster consisting coils persist (x 750).
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and mitochondria are randomly distributed as is the case in nocodazole treated cells. None of these organelles appeared to engage in any consistent way in a specific interaction with the free MT untreated cells, however, their topoor MT bundles. In control graphy is clearly related to the organized pattern of the cytoplasmic microtubule complex. The above data indicate that rearrangement of MT is followed by between both rearrangement of IF, and show that the interaction systems is independent of the normal cytoplasmic organisation. In the next set of experiments we investigate what happens with the normal MT-IF interaction pattern, when first coils are induced by nocodazole and then taxol is added. Cells are first treated with (low4 M) nocodazole (2.5 x lo+ M) for 24 hrs and then taxol is added (Fig. 5). With this combination one can see free MT as in cells treated with taxol only (De Brabander, 1981). They appear virtually immediately after addition of taxol. IF bundles, formed after nocodasole treatment (Fig. 3b, 8), dissolve gradually after many IF coils are addition of taxol 10S4 M. After 4 hrs still seen but the cytoplasm is also filled with a fine mesh of disperAfter 24 hrs all the coils have disappeared and a sed fibers. clear interaction between MT and IF is again seen, identical to treated with a cells treated with taxol only. When cells are first higher concentration of nocodazole (2.5 x low5 M), and then tax01 (5.10W6 M) is added (Fig. 6), only a small pericentrosomal aster of MT is observed (Fig. 6a). Most of the cytoplasm remains The preformed IF coils persist essentially without microtubules. between the MT aster and IF coils is (Fig. 6b) and no relation observed. Fig.
7-12
Ultrastructural
Fig. 7 Control (x 41 250).
MO cell.
distribution
of MT and IF.
IF and MT are in close association.
8 MO cell treated for 24 hrs with IF are redistributed in bundles, while dispersed Golgi organelles (x 12 120).
Fig. Ml
l
nocodazole (2.5 x 10m5 MT are absent. Note the
Fig. 9 MO cell treated with tax01 low4 M. Note free and numerous IF (arrowhead) (x 36 000).
MT
(arrow)
treated with taxol 10e4 M. MT form a loose Fig. 10 MO cell bundle. IF (arowhead) in between are aligned in parallel (x 26 200). treated with taxol 10e4 M. MT form a dense Fig. 11 MO cell bundle. Note the IF (arrowhead) along the bundle (x 37 400). Fig. 12 MO cell treated with a combination of nocodazole (2.5 x cells midbodylO+j M) and taxol (10m4 M). In multinucleated like microtubule structures are observed. Between these MT no IF are seen (x 27 500).
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DISCUSSION with Previous investigations electron microscopy and immunofluorescence documented a partial codistribution of IF and MT in normal interphase cells and coiling of IF after MT dissolution (Ball and Singer, 1981; Geiger and Singer, 1980; Goldman, 1969; De Brabander et al., 1975). Although suggestive these data do not prove the existence of a physico-chemical link between both fibers. One alternative explanation is that an organized MT network directs cytoplasmic flow and thus defines channels in which IF are also distributed. Other organelles such as mitochondria assume a similar distribution (Ball and Singer, 1982). Our experiments with tax01 may shed some light on the nature of the interaction. The compound induces a disappearance of organised MT which are replaced by multiple free MT and MT bundles (De Brabander et Q., 1981). We have previously argued that this may be due to the lowering of the critical tubulin concentration in the cytoplasm (De Brabander et al., 1981; Schiff and Horwitz, 1980). Taxol induces changes in cell shape and organisation of the cytoplasm identical to those induced by colchicine or nocodazole (De Brabander et al., 1976). Organelles such as the golgi cisternae, mitochondria and lysosomes are randomly distributed. Preliminary with double-label immunofluorescence and electron experiments microscopy show no specific interaction between these organelles and the disorganized MT, which means that the cytoplasmic organisation is not dependent upon the mere presence of MT, but on the presence of an organised MT network. In taxol treated cells the interaction between IF and MT is still seen both at the lightmicroscopical and ultrastructural level. The interaction is similar to that in untreated cells: partial codistribution and parallel alignment. This is not merely the result of fragmentation of filaments first induced preexisting MT-IF complexes. Intermediate redistribute to form IF-MT complexes when MT assembly is to coil, induced by taxol in the continued presence of nocodazole. We do not know whether this is due to unraveling of IF coils or disassembly and reassembly of subunits in connection with MT. Anyway it indicates that the interaction between MT and IF has a higher affinity than the interaction between IF and IF. The present data thus provide strong evidence that the distribution of IF is deterof the normal ormined by MT, and not vice versa, independently ganisation and functioning of the MT system. The interaction thus most probably involves some kind of specific physicochemical link. the lack of complete codistribution inAs suggested, previously, dicates that this putative crosslink is rather dispersed along the fibers and probably of a labile character (Geiger and Singer, substantiated by the observation that 1980). This is further rearrangement of the IF always follows with some lag in time the rearrangement of the MT system. Recently Virtanen et al. (1980) showed that in cells incubated with immunofluorat low temperature MT were no longer visible a normal distribution. From this they escence, but IF retained concluded that the distribution of IF is independent of MT. We have also seen the absence of IF coiling in cold treated cells treated with nocodazole or colchicine at low even in cells
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We see a similar inhibition of IF coiling in cells temperature. treated with nocodazole or colcbicine in the presence of metabolic inhibitors such as sodium azide (unpublished observation). However, both cold treatment and ATP depletion not only leave a sub1981; Bershadsky, stantial number of MT intact (De Brabander, 1981) they also inhibit the disassembly induced by microtubule inhibitors. Moreover, both cold and sodium azide, probably by ATP arrest all signs of cellular motility including saltadepletion, tory motions and cytoplasmic mass flow which is most prominent after MT disintegration. These data can thus not be used as evidence against a structural interaction between MT and IF. Instead we concluded that an energy dependent process such as e.g. cytoplasmic flow is instrumental in collecting IF into coils after dissolution of the MT system. ACENOWLEDXMENT Tax01 was a gift from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. We also thank L. Leijssen and G. Jacobs for the photographical work, H. Vanhove for reviewing and C. Verellen for typing the This research was supported by a grant from the manuscript. Onderzoek in Nijverheid Instituut voor Wetenschappelijk en Landbouw, Brussels, Belgium. REFERENCES S.J. (1981) Association of microtubules E.J. and Singer, and intermediate filaments in normal fibroblasts and its disruption upon transformation by a temperature-sensitive mutant of Rous Sarcoma virus. Proceedings of the National Academy of Science 78: 69866990. (1982) Mitochondria are associated Ball, E.J. and Singer, Sz. with microtubules and not with intermediate filaments in cultured fibroblasts. Proceedings of the National Academy of Science 79: 123-126. Barni, S., De PiGis Polver, Gerzeli, G. and Hano, R. (1981) Propidium Iodide as a probe for the study of chromatin thermal denaturation in situ. Histochemical Journal 13: 781-791. Bershadsky, A.D. and Gelfand, V.I. (1981) ATP-degndent regulalation of cytoplasmic microtubule disassembly. Proceedings of the National Academy of Science 78, 3610-3613. Bershadsky, A.D., Gelfand, V.I., Svitkina, T.M. and Tint, I.S. (1979) Cold-stable microtubules in the cytoplasm of mouse embryo fibroblasts. Cell Biology International Reports 3: 45-50. De Brabander, M., Aerts, F., Van de Veire, R. and Borgers, M. (1975) Evidence against interconversion of microtubules and filaments. Nature 253: 119-120. Ball,
Cell Biology International
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De Brabander, M., Van de Veire, R., Aerts, R., Borgers, M. and Janssen, P. (1976) The effects of methyl[5-(2-Thienylcarbonyl)-lH-benzimidazol-2-yl)carbamate (R 17 934, NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer ,Research 36: 905-916. De Brabander, M., Geuens, G., Nuydens, R., Willebrords, R. and Tax01 induces the assembly of free De Mey, J. (1981a) in living cells and blocks the organizing microtubules capacity of the centrosomes and kinetochores. Proceedings of the National Academy of Science 78: 5608-5612. De Brabander, M., Geuens, G., NuydeE, R., Willebrords, R. and De Mey, J. (1981b) Microtubule assembly in living cells after release from nocodazole block: The effects of metabolic inhibitors, Tax01 and PH. Cell Biology International Reports 2: 913-920. De Brabander, M., Geuens, G., Nuydens, R., Willebrords, R. and Microtubule stability and assembly in De Mey, J. (1982) The influence of metabolic inhibitors, taxol living cells: and pH. Cold spring harbor symposia on quantitative biology: 46, 227-240. De Mey, J., Moeremans, M., Geuens, G., Nuydens, R. and M. De Braresolution High light electron bander (1981) and microscopic localization of tubulin with the IGS (immuno Reports, gold staining) method. Cell Biology International 2: 889-899. of a class Freed, J.J. and Lebowits, M.M. (1970) The association of saltatory movements with microtubules in cultured cells. Journal of Cell Biology, 45: 334-354. (1980) Association of microtubules Geiger, G. and Singer, S.J. filaments in chicken gizzard cells as and intermediate detected by double immunofluorescence. Proceedings of the National Academy of Science 77: 4769-4773. Goldman, R.D. and Follett (1969) The structure of the major cell processes of isolated BHK 21 fibroblasts. Experimental Cell Research -57: 263-276. Herbrink, P., Van Bussel, F. and Warmaar, S. (1982) The antigen assay for the detection spot test (AST): a highly sensitive of antibodies. Journal of Immunological Methods 48: 293-298. normal Hynes, R.O. and Destree, A.T. (1978) 10 nm filaments-in and transformed cells. Cell 13: 151-163 (1982) Rat monoclonal Kilmartin, J.V., Wright, B. and CrMilstein antitubulin antibodies derived by using a new nonsecreting rat cell line. Journal of cell Biology, 93, 576-582. filaments in 3T3 cells colKlymkowsky, M.W. (1981) Intermediate collapse after injection of a monoclonal anti-intermediate filament antibody. Nature 291: 249-251. Disruption of the in ViVO Lin, J.J.C. and Feramisco, J.R. (1981) distribution of the intermediate filaments in fibroblasts of a specific monoclonal through the microinjection antibody. Cell -24: 185-193.
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Moskalewski, S., Thyberg, J., Lohmander, S., Friberg, V. (1975) Influence of colchicine and vinblastine on the golgi complex and matrix deposition in chondrocyte aggregates. Experimental Cell Research 95: 440-454. Nelson, W.J., Vergias, C.E. and Tranb, P. (1982) A rapid method for the large scale purification of the intermediate filament protein vimentin by single-stranded DNA-cellular affinity chromatography, Biochemical and Biophysical Research Communications 106: 1141-1147. Schiff, P.B., Horwitz, S.B. (1980) Tax01 stabilizes microtubules in mouse fibroblast cells. Proceedings of the National Academy of Science 77: 1561-1565. Sharper A.H., Chen, L.B.7 Murphy, J.R. Fields, B.N. (1980) Specific disruption of vimentin filament organization in monkey kidney CV-1 cells by diphtheria toxin, exotoxin A and cycloheximide. Proceedings of the National Academy of Science -77: 7267-7271. Virtanen, I., Lehto, V.P., Lehtonen, E. (1980) Organization of intermediate filaments in cultured fibroblasts upon disruption of microtubules by cold treatment. European Journal of Cell Biology 23: 80-84. Wang, E. and Goldman, R.D. (lE8) Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells. European Journal of Cell Biology 79: 708-726. (1981) Effect of vanadate on intracelluWang, E. and Choppin,P. lar distribution and function of lo-nm filaments. Proceedings of the National Academy of Science -78: 2363-2367.
Received:
18th
October
1982
Accepted:
8th November 1982
International
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Vol. 7, No. 1, January
1983
35
THE INTERACTIONBETWEEN MICROTUBULES AND INTERMEDIATEFILAMENTS IN CULTUREDCELLS TREATEDWITH TAmL AND NOCODAZOLE G. Geuens, M. de Brabander, R. Nuydens and J. De Mey Laboratory of Oncology, Janssen Pharmaceutics Research Laboratories, B-2340 Beerse, Belgium ABSIXACT Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MP) and intermediate filaments (IF) in MO cells treated with various combinaiions of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MC bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated is cells a close association between these two filamentous systems with nocodazole followed by addition observed. In cells pretreated to induce the bundles of free MT, the preexisting IF of taxol, coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MC determine the spatial distribution of IF which most probably involves some kind of physicochemical link. INTRODUCTION A close association between MT and IF of the vimentin or desmin type has been observed in many cells using different techdescribed the parallel alignment of niques. Goldman (1969) first MT and IF at the ultrastructural level in BBK cells. By means of double-label immunofluorescence , Geiger and Singer (1980) and Ball and Singer (1981) showed that in whole interphase fibroblasts but not complete superposition there was an extensive of the labelling patterns of the two filamentous systems. Microtubule disintegration by specific inhibitors results in loss of cell polarity, disordered organelle topography and arrest of saltatory motions (Freed and Lebowits, 1970; De Brabander et al., 1975; Moskalewski et al., 1975). Invariably, this also induces IF to bundles or caps (Wang and Goldman, aggregate into perinuclear 1978; De Brabander, 1975). These observations suggested that 60th types of fibers are normally engaged in some kind of structural
0309-1651/83/010035-13l$O3.00/0
@ 1983 Academic
Press Inc. (London)
Ltd.
36
Cell Biology
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Reports,
Vol. 7, No. 1, January
1983
interaotion and that they serve as a functional unit in the determination of cell polarity and cytoplasmic organisation. However, some doubt is raised by experiments in which the IF distribution is profoundly altered without apparently affecting the MT system. Ball and Singer (1981) showed that the association can be disrupted upon transformation by a temperature-sensitive mutant of ROUS Sarcoma virus. Hynes (1978) described that NIL-8 cells showed an altered organisation of IF after HSV transformation, while MT remained normal. Sharpe et al. (1980) examined by immunofluorescence the effect of diphtheria toxin, exotoxin A and cycloheximide on CV-1 cell cytoskeletons and found that these agents specifically disrupted the vimentin filament system without any effect on MT. Wang et al. (1981) reported the effect of vanadate on virus-induced syncytia and infected cells. They found with sodium vanadate an altered distribution of IF, whereas MT. orgaBy means of microinjection of Gerbil finisation was unaltered. broma cells with a monoclonal antibody against components of the IF system, Lin and Feramisco (1981) showed by double label immunofluorescence and electron microscopy that the vimentin containing IF of the antibody-injected cells redistributed, while MT were Klymkowsky (1981) found similar results in 3T3 mouse undisturbed. Both authors found no changes in cell morphology and fibroblasts. polarity and the antibody-injected cells showed normal saltatory It can thus not be excluded that the apmovements of organelles. parent codistribution of MT and IF is fortuitous, both systems occupying the same cytoplasmic channels. More information on the putative interaction and its role in cytoplasmic organisation may be obtained in experiments where the MT distribution is altered without completely dissolving it. Recently such experiments have become possible by the use of taxol (Schiff, 1980). Treatment of cells with taxol produces a disorganised network of free MT, which 1981; tend to form bundles to various degrees (De Brabander, is altered as with clasSchiff, 1980). Cell shape and motility (De Brasical microtubule inhibitors (colchicine or nocodazole) bander, 1976, 1981a). We have investigated, using double label immunofluorescence microscopy and electron microscopy, the interaction of MT and IF in cells treated with taxol and nocodazole in We see that in cells treated with tax01 IF various combinations. do not form coils but become rearranged so as to codistribute partially with the free MT. Furthermore we show that in cells first treated with nocodazole (2.5 x 10m6 M) and then further incubated with taxol (10B4 M) and nocodazole the IF leave the coils and associate with the disordered arrays of MT. In cells treated with nocodazole (2.5 x 10m5 M) and taxol (5.10B6 M), containing only short MT connected to the centrosomes, the IF coils persist. MATERIALSAND METM)DS Cell onal
lines: MO (De Brabander et al., 3T3-type cell line with epitheloid
1976), a C3H mouse embrycharacter was used. The
Cell Biology
Fig.
1.
International
Ultrastructural
Reports,
Vol. 7, No. 1, January
localization
1983
of the antidesmin
antibody.
la MO control cell fixed with methanol and incubated with an affinity purified antibody against pig stomach desmin. The stained filaments are marked with gold particles (5 nm) coupled to goat Note the parallel anti rabbit Ig. Single IF are densely labelled. alignment between single IF (arrow) and MT (arrowhead) (x 43 200). lb MO cell treated with nocodazole (2.5 x 10B6 M 24 hrs). The nocodazole induced IF coils are marked with 5 nm gold particles. (x 37 400). cultures were grown in Eagle's minimal essential medium, supplemented with non-essential amino acids and 10 % fetal bovine serum and incubated in a humidified atmosphere of 5 % CC2 in air at 37O c. Immunofluorescence: MO cell cultures were seeded on glass coverslips. After treatment, cells were fixed with methanol for 10 min at -20° C and washed with acetone at -20' C. The cells were sequentially stained with a mixture of anti-intermediate filament antibody and monoclonal antitubulin (Kilmartin, 1982) and then a mixture of TRITC-coupled goat anti-rat immunoglobulins (Nordic) and FITC-coupled goat anti-rabbit immunoglobulin (Nordic). The nuclei were stained with propidium iodide (Rarni et al., 1981). After washing, coverslips were mounted in gelvatol and viewed with a Leitz orthoplan microscope equipped with @ase contrast and fluorescence optics.
38
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MO cell cultures were seeded in plastic Electron microscopy: cells were fixed with glutaraldedishes (Lux). After treatment, hyde 3 % for 1 hour at room temperature and osmificated with 2 % 0~04 for 1 hour at 4O C. They were then impregnated with 0.5 % uranyl acetate in 70 % ethanol supplemented with 1 % phosphoimmunogold label experiment tungstic acid. For the ultrastructural the cells were incubated with goat anti-rabbit coupled to colloidal gold particles (De Mey et al., 1981) (GA&G5: 5 nm gold particles: Janssen Life Science Products Division, Beerse, Belgium). Dehydration was done in a series of ethanol and embedding in epon 812. Cells were selected and ultrathin sections were viewed with a Philips PI 300 or EM 201. Experiments: Cells were seeded either on glass coverslips or plastic petri dishes. After 24 or 48 hours, cells were treated with nocodazole (2.5 x lo6 M or 2.5 x lo-5 M) for 24 hours, then taxol (low4 M or 5 x 10e6 M) was added in the presence of Control culnocodazole. Cells were fixed 4 and 24 hours later. tures were treated with nocodazole and taxol only, or the appropriate solvent (DMSO) which did not affect the cells at the concentrations used. Microtubules were labelled with a monoclonal antibody Antibodies: (MC AB/YL1/2) to yeast tubulin (gift of J.V. Kilmartin, 1982). Intermediate filaments were stained with a rabbit antibody raised against and affinity purified on pig stomach desmin. This antibody decorates the IF-network of MO cells in a rather complete fashion as documented with immunofluorescence and immuno gold staining (Fig. la). The coils induced by nocodazole are also entirely labelled (fig. lb). The antibody did not stain at all MT, microprekeratin filaments in PTK2 cells or any other orfilaments, ganelles. Using the antigen spot test (Herbrink et al., 1982) the antibody reacted at the same dilution as that used for immuno+ 2 pg protein per ml) with purified pig fluorescence (l/50; stomach desmin (gift Gf V. Small and A. Sobieszek) down to 0.5 ng and with purified Ehrlich Ascites tumor cell vimentin (gift of Fig. 2-4 Double label stained with antitubulin,
immunofluorescence. those at the right
Cells at the left with antidesmin.
are
2a-b Untreated MO cells: Bundles of MT and IF and individual fibers (arrowhead) show extensive overlapping. The IF do not extend as far to the periphery as the MT' (x 500). 3a-b MO cell, treated with nocodazole (2.5 x low6 M 24 hrs). The MI network has completely disappeared, while the IF are coiled. The nuclei are stained with propidium iodide (x 500). 4a-b MO cell, treated with tax01 10m4 M for 24 hrs. The MT network has been replaced by free MC and abundant MT bundles. Note the IF in parallel alignment (arrow) with MT bundles and IF staining between fascicles (arrowhead) (x 500).
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W. J. Nelson, 1982) down to 50 ng. We are currently investigating which type of intermediate filaments predominates in MO cells. For the present paper the outcome is of minor importance since both types of fibers show a similar distribution in untreated cultered cells and react in the same way to nocodazole or colchicine. FUZSULTS In control MO cells, MT and IF extend throughout the cells into the peri@ery, although the extension of IF is not always as far (Fig. 2a,b) as described previously for similar cells (Geiger & Singer, 1980; Ball and Singer, 1981). Treatment with nocodazole causes MT dissolution, and IF coils (Fig. 3a,b). These coils are mostly limited to the organelle rich central cytoplasmic dome and excluded from thin peripheral lamellae (Fig. 3a,b). Treatment with tax01 (Fig. 4) results in cell shape change, identical as with nocodazole (Fig. 3) (De Brabander, 1976, 1981a). Long MT radiating from the perinuclear vicinity are replaced by multiple short free MT (De Brabander, 1981) (Fig. 9) within 4 hours. Between 4-24 hrs they show an increased tendency to form parallel bundles (De Brabander, 1981) (Fig. 4a, 9, 11). Microtubules are usually excluded from peripheral lamellae (De Brabander, 1981; Schiff, 1980). FLUorescent staining of IF after tax01 treatment is seen in three one can observe fluorescent staining in a disdomains. Firstly organized fashion in regions with a mesh of free MT. Secondly staining is observed as parallel alignment along MT bundles (Fig. intensity of these IF fascicles is much less 4). The staining bright than that of the nocodazole induced coils. Thirdly, very often accumulation of stain is seen at the ends of MT bundles, where small IF bundles are arranged at various angles to microThis is particularly prominent where two fascicles of tubules. microtubules are opposed with an empty region between them (Fig. 4). After 4 hours of incubation with taxol still many cells with a rather normal IF network are observed , while MT are redistributed. This shows that the IF rearrangement lags behind MT rearrangement. These observations with double-label fluorescence microscopy are confirmed at the ultrastructural level (Fig. 9, 10, 11). Whithin 4 cells with multiple free MT contain free hrs after taxol addition, IF in the same region in a disorganised way (Fig. 9). With longer taxol treatment, one can see loose bundles of MT intermingled with IF running in the same direction (Fig. 10). With dense MT bundles one can see a number of IF along these bundles (Fig. 11). In telophase cells that underwent abortive mitosis during taxol treatment one can observe midbody like structures consisting of MT fanning Their formation and structure will out from osmiophylic material. No IF are seen between the MT of these be described separately. of the centrosomes of taxol structures (Fig. 12). In the vicinity treated cells as well as in control cells some radiating IF can be observed. Ultrastructural observations of taxol treated cells show also that other cytoplasmic organelles such as the golgi apparatus
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Fig. 5-6 Double label stained with antitubulin,
Reports,
Vol. 7, No. 1, January
immunofluorescence. those at the right
1983
Cells at the left with antidesmin.
are
treated with nocodazole (2.5 x 10m6 M) and then 5a-b MO cell, further treated with taxol 1O'4 M for 24 hrs in the presence of the cytoplasm. The IF coils nocodazole. Free MT and bundles fill formed after nocodazole, have disappeared. IF align along MT bundles (arrow) and accumulate in a disordered fashion between two fascicles (arrowhead) (x 500). treated with nocodazole (2.5 x 10'5 M) and then 6a-b MO cell, further incubated with taxol 5.10m6 M in the presence of nocoof MT is seen (arrowhead). IF dazole. A small aster consisting coils persist (x 750).
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and mitochondria are randomly distributed as is the case in nocodazole treated cells. None of these organelles appeared to engage in any consistent way in a specific interaction with the free MT untreated cells, however, their topoor MT bundles. In control graphy is clearly related to the organized pattern of the cytoplasmic microtubule complex. The above data indicate that rearrangement of MT is followed by between both rearrangement of IF, and show that the interaction systems is independent of the normal cytoplasmic organisation. In the next set of experiments we investigate what happens with the normal MT-IF interaction pattern, when first coils are induced by nocodazole and then taxol is added. Cells are first treated with (low4 M) nocodazole (2.5 x lo+ M) for 24 hrs and then taxol is added (Fig. 5). With this combination one can see free MT as in cells treated with taxol only (De Brabander, 1981). They appear virtually immediately after addition of taxol. IF bundles, formed after nocodasole treatment (Fig. 3b, 8), dissolve gradually after many IF coils are addition of taxol 10S4 M. After 4 hrs still seen but the cytoplasm is also filled with a fine mesh of disperAfter 24 hrs all the coils have disappeared and a sed fibers. clear interaction between MT and IF is again seen, identical to treated with a cells treated with taxol only. When cells are first higher concentration of nocodazole (2.5 x low5 M), and then tax01 (5.10W6 M) is added (Fig. 6), only a small pericentrosomal aster of MT is observed (Fig. 6a). Most of the cytoplasm remains The preformed IF coils persist essentially without microtubules. between the MT aster and IF coils is (Fig. 6b) and no relation observed. Fig.
7-12
Ultrastructural
Fig. 7 Control (x 41 250).
MO cell.
distribution
of MT and IF.
IF and MT are in close association.
8 MO cell treated for 24 hrs with IF are redistributed in bundles, while dispersed Golgi organelles (x 12 120).
Fig. Ml
l
nocodazole (2.5 x 10m5 MT are absent. Note the
Fig. 9 MO cell treated with tax01 low4 M. Note free and numerous IF (arrowhead) (x 36 000).
MT
(arrow)
treated with taxol 10e4 M. MT form a loose Fig. 10 MO cell bundle. IF (arowhead) in between are aligned in parallel (x 26 200). treated with taxol 10e4 M. MT form a dense Fig. 11 MO cell bundle. Note the IF (arrowhead) along the bundle (x 37 400). Fig. 12 MO cell treated with a combination of nocodazole (2.5 x cells midbodylO+j M) and taxol (10m4 M). In multinucleated like microtubule structures are observed. Between these MT no IF are seen (x 27 500).
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DISCUSSION with Previous investigations electron microscopy and immunofluorescence documented a partial codistribution of IF and MT in normal interphase cells and coiling of IF after MT dissolution (Ball and Singer, 1981; Geiger and Singer, 1980; Goldman, 1969; De Brabander et al., 1975). Although suggestive these data do not prove the existence of a physico-chemical link between both fibers. One alternative explanation is that an organized MT network directs cytoplasmic flow and thus defines channels in which IF are also distributed. Other organelles such as mitochondria assume a similar distribution (Ball and Singer, 1982). Our experiments with tax01 may shed some light on the nature of the interaction. The compound induces a disappearance of organised MT which are replaced by multiple free MT and MT bundles (De Brabander et Q., 1981). We have previously argued that this may be due to the lowering of the critical tubulin concentration in the cytoplasm (De Brabander et al., 1981; Schiff and Horwitz, 1980). Taxol induces changes in cell shape and organisation of the cytoplasm identical to those induced by colchicine or nocodazole (De Brabander et al., 1976). Organelles such as the golgi cisternae, mitochondria and lysosomes are randomly distributed. Preliminary with double-label immunofluorescence and electron experiments microscopy show no specific interaction between these organelles and the disorganized MT, which means that the cytoplasmic organisation is not dependent upon the mere presence of MT, but on the presence of an organised MT network. In taxol treated cells the interaction between IF and MT is still seen both at the lightmicroscopical and ultrastructural level. The interaction is similar to that in untreated cells: partial codistribution and parallel alignment. This is not merely the result of fragmentation of filaments first induced preexisting MT-IF complexes. Intermediate redistribute to form IF-MT complexes when MT assembly is to coil, induced by taxol in the continued presence of nocodazole. We do not know whether this is due to unraveling of IF coils or disassembly and reassembly of subunits in connection with MT. Anyway it indicates that the interaction between MT and IF has a higher affinity than the interaction between IF and IF. The present data thus provide strong evidence that the distribution of IF is deterof the normal ormined by MT, and not vice versa, independently ganisation and functioning of the MT system. The interaction thus most probably involves some kind of specific physicochemical link. the lack of complete codistribution inAs suggested, previously, dicates that this putative crosslink is rather dispersed along the fibers and probably of a labile character (Geiger and Singer, substantiated by the observation that 1980). This is further rearrangement of the IF always follows with some lag in time the rearrangement of the MT system. Recently Virtanen et al. (1980) showed that in cells incubated with immunofluorat low temperature MT were no longer visible a normal distribution. From this they escence, but IF retained concluded that the distribution of IF is independent of MT. We have also seen the absence of IF coiling in cold treated cells treated with nocodazole or colchicine at low even in cells
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We see a similar inhibition of IF coiling in cells temperature. treated with nocodazole or colcbicine in the presence of metabolic inhibitors such as sodium azide (unpublished observation). However, both cold treatment and ATP depletion not only leave a sub1981; Bershadsky, stantial number of MT intact (De Brabander, 1981) they also inhibit the disassembly induced by microtubule inhibitors. Moreover, both cold and sodium azide, probably by ATP arrest all signs of cellular motility including saltadepletion, tory motions and cytoplasmic mass flow which is most prominent after MT disintegration. These data can thus not be used as evidence against a structural interaction between MT and IF. Instead we concluded that an energy dependent process such as e.g. cytoplasmic flow is instrumental in collecting IF into coils after dissolution of the MT system. ACENOWLEDXMENT Tax01 was a gift from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. We also thank L. Leijssen and G. Jacobs for the photographical work, H. Vanhove for reviewing and C. Verellen for typing the This research was supported by a grant from the manuscript. Onderzoek in Nijverheid Instituut voor Wetenschappelijk en Landbouw, Brussels, Belgium. REFERENCES S.J. (1981) Association of microtubules E.J. and Singer, and intermediate filaments in normal fibroblasts and its disruption upon transformation by a temperature-sensitive mutant of Rous Sarcoma virus. Proceedings of the National Academy of Science 78: 69866990. (1982) Mitochondria are associated Ball, E.J. and Singer, Sz. with microtubules and not with intermediate filaments in cultured fibroblasts. Proceedings of the National Academy of Science 79: 123-126. Barni, S., De PiGis Polver, Gerzeli, G. and Hano, R. (1981) Propidium Iodide as a probe for the study of chromatin thermal denaturation in situ. Histochemical Journal 13: 781-791. Bershadsky, A.D. and Gelfand, V.I. (1981) ATP-degndent regulalation of cytoplasmic microtubule disassembly. Proceedings of the National Academy of Science 78, 3610-3613. Bershadsky, A.D., Gelfand, V.I., Svitkina, T.M. and Tint, I.S. (1979) Cold-stable microtubules in the cytoplasm of mouse embryo fibroblasts. Cell Biology International Reports 3: 45-50. De Brabander, M., Aerts, F., Van de Veire, R. and Borgers, M. (1975) Evidence against interconversion of microtubules and filaments. Nature 253: 119-120. Ball,
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De Brabander, M., Van de Veire, R., Aerts, R., Borgers, M. and Janssen, P. (1976) The effects of methyl[5-(2-Thienylcarbonyl)-lH-benzimidazol-2-yl)carbamate (R 17 934, NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer ,Research 36: 905-916. De Brabander, M., Geuens, G., Nuydens, R., Willebrords, R. and Tax01 induces the assembly of free De Mey, J. (1981a) in living cells and blocks the organizing microtubules capacity of the centrosomes and kinetochores. Proceedings of the National Academy of Science 78: 5608-5612. De Brabander, M., Geuens, G., NuydeE, R., Willebrords, R. and De Mey, J. (1981b) Microtubule assembly in living cells after release from nocodazole block: The effects of metabolic inhibitors, Tax01 and PH. Cell Biology International Reports 2: 913-920. De Brabander, M., Geuens, G., Nuydens, R., Willebrords, R. and Microtubule stability and assembly in De Mey, J. (1982) The influence of metabolic inhibitors, taxol living cells: and pH. Cold spring harbor symposia on quantitative biology: 46, 227-240. De Mey, J., Moeremans, M., Geuens, G., Nuydens, R. and M. De Braresolution High light electron bander (1981) and microscopic localization of tubulin with the IGS (immuno Reports, gold staining) method. Cell Biology International 2: 889-899. of a class Freed, J.J. and Lebowits, M.M. (1970) The association of saltatory movements with microtubules in cultured cells. Journal of Cell Biology, 45: 334-354. (1980) Association of microtubules Geiger, G. and Singer, S.J. filaments in chicken gizzard cells as and intermediate detected by double immunofluorescence. Proceedings of the National Academy of Science 77: 4769-4773. Goldman, R.D. and Follett (1969) The structure of the major cell processes of isolated BHK 21 fibroblasts. Experimental Cell Research -57: 263-276. Herbrink, P., Van Bussel, F. and Warmaar, S. (1982) The antigen assay for the detection spot test (AST): a highly sensitive of antibodies. Journal of Immunological Methods 48: 293-298. normal Hynes, R.O. and Destree, A.T. (1978) 10 nm filaments-in and transformed cells. Cell 13: 151-163 (1982) Rat monoclonal Kilmartin, J.V., Wright, B. and CrMilstein antitubulin antibodies derived by using a new nonsecreting rat cell line. Journal of cell Biology, 93, 576-582. filaments in 3T3 cells colKlymkowsky, M.W. (1981) Intermediate collapse after injection of a monoclonal anti-intermediate filament antibody. Nature 291: 249-251. Disruption of the in ViVO Lin, J.J.C. and Feramisco, J.R. (1981) distribution of the intermediate filaments in fibroblasts of a specific monoclonal through the microinjection antibody. Cell -24: 185-193.
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1983
Moskalewski, S., Thyberg, J., Lohmander, S., Friberg, V. (1975) Influence of colchicine and vinblastine on the golgi complex and matrix deposition in chondrocyte aggregates. Experimental Cell Research 95: 440-454. Nelson, W.J., Vergias, C.E. and Tranb, P. (1982) A rapid method for the large scale purification of the intermediate filament protein vimentin by single-stranded DNA-cellular affinity chromatography, Biochemical and Biophysical Research Communications 106: 1141-1147. Schiff, P.B., Horwitz, S.B. (1980) Tax01 stabilizes microtubules in mouse fibroblast cells. Proceedings of the National Academy of Science 77: 1561-1565. Sharper A.H., Chen, L.B.7 Murphy, J.R. Fields, B.N. (1980) Specific disruption of vimentin filament organization in monkey kidney CV-1 cells by diphtheria toxin, exotoxin A and cycloheximide. Proceedings of the National Academy of Science -77: 7267-7271. Virtanen, I., Lehto, V.P., Lehtonen, E. (1980) Organization of intermediate filaments in cultured fibroblasts upon disruption of microtubules by cold treatment. European Journal of Cell Biology 23: 80-84. Wang, E. and Goldman, R.D. (lE8) Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells. European Journal of Cell Biology 79: 708-726. (1981) Effect of vanadate on intracelluWang, E. and Choppin,P. lar distribution and function of lo-nm filaments. Proceedings of the National Academy of Science -78: 2363-2367.
Received:
18th
October
1982
Accepted:
8th November 1982