Intermediate filament collapse is an ATP-dependent and actin-dependent process P. J. HOLLENBECK1'*, A. D. BERSHADSKY2, O. Y. PLETJUSHKINA3, I. S. TINT3 and J. M. VASILIEV2'3 ^Medical Research Council Cell Biophysics Unit, 26 Dntry Lane, London WC2B 5RL, England 2AII-Union Cancer Research Centre of the Academy of Medical Science, 24 Kashirskoje Schosse, 115478 Moscotv, USSR 3Moscoui State University, 119899 Moscow, USSR •Author for correspondence at: Department of Anatomy and Cellular Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA Summary In this study, we have investigated the properties of collapse was revealed by treating cells with cyto- intermediate filament rearrangements using exper- chalasin D: the formation of intermediate filament imentally induced collapse of vimentin intermedi- cables still occurs after disruption of the actin ate filaments in mouse fibroblasts. In these cells, filament system by cytochalasin, but the sub- depolymerizing microtubules by colchicine or vin- sequent coiling of cables to form a perinuclear mass blastine treatment at 37 °C results in a two-stage is strongly inhibited by these conditions, and can be collapse of intermediate filaments. First, the vimen- reversed by applying cytochalasin to cells in which tin filaments aggregate into large cables; then, the intermediate filaments have already undergone cables coil into a dense mass surrounding the complete collapse. We propose that the formation nucleus. By using inhibitors of oxidative phos- of vimentin cables involves a phosphorylation phorylation along with glucose deprivation to lower event, while the coiling of cables into a perinuclear intracellular ATP levels by 95 %, we have found that mass relies on interaction of intermediate filaments both stages of intermediate filament collapse re- with a component of the actin cortex. quire ATP. However, once collapse has occurred, only the second stage can be reversed in the absence of microtubules by lowering ATP levels. An ad- Key words: intermediate filaments, microtubules, actin, ATP ditional difference between the two stages of depletion. Introduction vimentin (Klymkowsky, 1981; Gawlitta et al. 1981), against an IF-associated protein (Lin & Feramisco, Vimentin-containing intermediate filaments (IFs) form 1981), or against tubulin (Blose et al. 1984). In addition, extended networks in the cytoplasm of cultured fibro- other less-specific agents, such as acrylamide (Durham et blasts. It is well known that the morphology of these al. 1983; Eckert, 1985), vanadate ion (Wang & Choppin, networks is microtubule-dependent. IFs are colocalized 1981) and heat shock (Thomas et al. 1981), have been with microtubules (Goldman & Follett, 1969; Geiger & shown to cause IF collapse in some cell types. Singer, 1980; Geuens et al. 1983; Traub, 1985), and in Very little is known about the mechanism of this many cell types the depolymerization of cytoplasmic process or its relationship to other regulatory events in microtubules prior to mitosis is accompanied by the the cell cycle. In this paper, we describe experiments collapse of the IF array into a dense mass near to or showing that the IF collapse induced by microtubule surrounding the nucleus (e.g. see Aubin et al. 1980; depolymerization can be inhibited by agents that lower Zieve et al. 1980). A similar collapse results from intracellular ATP levels; in addition, we demonstrate that experimentally induced depolymerization of micro- the final stage of collapse, perinuclear coiling of IF tubules (e.g. see Goldman & Knipe, 1972; Croop & cables, is inhibited and reversed by cytochalasin D. Holtzer, 1975; Blose & Chako, 1976; Osborn et al. 1977; These results suggest that IF collapse is an active, ATP- Franked al. 1978; Osborn et al. 1980; Forry-Schaudies dependent process, and that its final stages require the et al. 1986) or after microinjection of antibodies against presence of a normal actin microfilament system. Journal of Cell Science 92, 621-631 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 621 Materials and methods minor fraction of the total vimentin staining. Actin staining with rhodamine-conjugated phalloidin revealed a Materials well-developed system of stress fibres in these cells. One Colcemid (demecolcine), vinblastine, 2,4-dinitrophenol or several groups of parallel fibres filled the whole (DNP), and all dye-conjugated secondary antibodies were cytoplasm uniformly, without appreciable gaps obtained from Sigma (St Louis, MO); cytochalasin D and (Fig. 1C,D). carbonylcyanide-3-chlorophenylhydrazone (CCCP) were ob- Treatment of cells with the microtubule-depolymeriz- tained from Aldrich (Gillingham, UK); oligomycin was ob- ing drugs colcemid (10/!gml~') or vinblastine tained from Serva (Heidelberg, FRG). Mouse monoclonal anti- (50^gml~ ) caused almost complete disappearance of vimentin ('clone 30') has been characterized elsewhere (Troya- novsky et al. 1985). Rabbit monospecific antibody against microtubules in 45 min; only a few residual microtubules bovine brain tubulin was prepared by V. I. Rodionov (Moscow remained. Vinblastine treatment led in addition to the State University) according to published procedures (Fuller et formation of numerous vinblastine 'paracrystals' of tubu- al. 1975). Rhodamine-conjugated phalloidin was a generous gift lin. The loss of microtubules was accompanied by a from Dr T. Wieland (Heidelberg, FRG). profound reorganization of the system of IFs. This reorganization, referred to as 'collapse', took place in two Cell culture and expeiimental treatments relatively distinct phases. First, the numerous thin Secondary cultures of mouse embryonic fibroblasts were grown vimentin filaments were condensed into a small number on glass coverslips at a density of approximately 104 cells cm" of thick cables, leaving a considerable part of the cyto- in a 1:1 (v/v) mixture of Eagle's basal medium and 05 % plasm devoid of vimentin staining. Then, the cables lactalbumin hydrolysate supplemented with 10% bovine became coiled into a dense perinuclear mass, with little serum. For cold treatment, coverslips were transferred to pre- vimentin staining remaining in the cell periphery. Col- cooled L-15 medium with 10% bovine serum and incubated on 1 lapse was apparent 45-75 min after application of either an ice bath. Stock solutions of colcemid (lmgml" ) and 1 vinblastine (5 mgml" ) were prepared in water. For short-term drug, and reached a maximum after 24 h in l^gml" treatment, these were added to the growth medium at final colcemid. At that time more than 60% of the cells had concentrations of lO^gml"1 colcemid and 50jUgml~' vinblast- concentrated their vimentin in a few large cables coiled ine; for long-term treatment, colcemid was added at 1 /<gml~'. around the nucleus (Fig. 2A,B). The system of actin Cytochalasin D was dissolved in dimethyl sulphoxide (DMSO) stress fibres underwent a concurrent reorganization: after at 10mgml" , diluted in Dulbecco's phosphate-buffered saline IF collapse, the stress fibres as a rule no longer penetrated to IQjlgmV , and used at a final concentration of the region in which vimentin staining was concentrated. 1 0-3-1-O^gmP . Incubation of cells with inhibitors of oxidative Instead, they assumed a circumferential distribution, phosphorylation was performed in Dulbecco's phosphate- leaving the perinuclear area largely devoid of actin buffered saline solution. Stock solutions of oligomycin staining (Fig. 2A,B). (1 mgml"1), DNP (100 mM) and CCCP (5 mM) were prepared in ethanol; sodium azide was dissolved in the incubation buffer immediately before use. Final concentrations of the inhibitors Prevention of intermediate filament collapse by ATP and their mechanisms of action are summarized in Table 1. Cell deprivation fixation, permeabilization, double indirect immunofluorescent To study the possible ATP-dependence of IF collapse, it staining and epifluorescence microscopy were performed as was necessary to obtain microtubule-free cells with sig- described (Bershadsky et al. 1987). nificantly reduced ATP levels. We have shown previously that the reduction of intracellular ATP levels makes microtubules resistant to drug-induced depolymerization Results (Bershadsky et al. 1980); thus, it was necessary to depolymerize microtubules prior to ATP deprivation and Collapse of vimentin intermediate filame>its after without disrupting the IF network. To satisfy this microtubule depolymerization requirement, we adopted the cell-cooling procedure of Immunofluorescent staining with anti-vimentin revealed Virtanen et al. (1980), and confirmed that incubation of a network of numerous thin filaments filling the cyto- mouse fibroblasts at 4°C caused nearly complete loss of plasm of untreated control fibroblasts. The distribution microtubules but did not induce reorganization of IFs. of IFs was similar but not identical to that of micro- We then combined cold treatment, colcemid (or vinblast- tubules (Fig. 1A-B). Thick vimentin-positive bundles ine) treatment, and treatment with inhibitors of oxidative were seen in some cells, but these accounted for only a phosphorylation according to the following experimental schedule. First, we incubated cells at 0°C in growth Table 1. Metabolic inhibitors used in these medium for a total of 2h, with colcemid (lO^gml"1) or 1 experiments vinblastine (30 jig ml" ) included in the medium for the last 20-30min. Cells treated in this way showed almost Inhibitor Mechanism of action Concentration complete loss of microtubules, while