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Architecture and polypeptide composition of HeLa cytoskeletons
J. Nol.
Biol. (1982) 154, 121-143
Architecture Modification
and Polypeptide Composition HeLa Cytoskeletons
of Cytoarchitectural
Polypeptides
of
during Mitosis
RODRIGO BRAVO'. ,J. VICTOR SMALL'. STEPHEN
LDitGion of Hio&uctrrro~l Chetnistr.y . l)epartment of Chemistry .+Inrhus 1‘tt&ersity . DK-X000 A arhus (‘. Denmark ‘Jnstit,ute oj’ Molecular Biology of the -4 ustrian Academy of Sciences, Wzburg, Austria (Received
3 =1pril 1981, and in revised form
I4 July
1981)
Substrate-attached asynchronous HeLa eells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton rytoskeletons showed actin filament bundles, microtubules. intermediate filaments, and actin networks in the substrate-associated lamellae, and cont,ained around 90 pol,y.peptides (48 basic, 42 acidic; 52“~ of total artin. 99”” of viment,in. 41°, of T-aetmm and 30?<;,of p-tubulin). Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a t,otal of 19 polypeptides (13 acidic!, 6 basic). Of these, 1%corresponded to abundant acidic proteins in the 47,000 to 70,000 ill, region as determined by staining with Coomassie blue and labelling with a mixture of 14C-labelled amino acids. ITsing 1,-H-L extracted cytoplasts, and employing an a&in depolymerising protein from slime moulds, seven abundant acidic IEFf polypeptides were shown to be present in these intermediate filament-enriched, substrat,e-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 111,)and six polypeptides (IEF 12, 68,000 112~:IEF 24. 56.000 M,: IEF 31. 50,000 Nr; IEF 35, 49,000 Mr: IEF 36. 48,509 *)I, and IEF 46, 43.500 W,) not previously reported as present in cytoskeletons. I’eptide analysis showed that these were not related as products of modification or proteolysis. JAabelling of mitotie and interphase cells with [3SS]methionine followed by onedimensional peptide map analysis showed that IEF 24,26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37. and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-I, cytoplast polypeptides may be important in the reorganization of @oskeletal elements that takes place during cell division.
1. Introduction \l’hile the exist,enee of different’ types of cytoplasmic filaments, i.e. actin filaments. intermediat,e-sized filaments and mirrotubules. has long been established (for t Author to whom all correspondence 1 IEP. isoelectric focusiug.
should be addressed 121
(M)22-4836/82/010121-23
$02.00/0
0 1982 Academic Press Inc. (London) Ltd.
reviews, see Roberts. 1971: Lazarides, 1980: Small et ~1.. 1!380), their general and mutual abundance in a wide variety of tissues and cultured cells has only recentI> recscived genera.1 attention. In our laboratories we are interested in determining the role of cytoskeletal proteins in regulating cell growth and proliferation (Celis et uZ.. 1978a,b ; Bravo & Celis, 1980a,b) as well as in cell motility (Small & Celis, 1978a,b; Small et al.. 1980). In earlier studies we developed methods, similar to those adopted independently by other investigators (Brown et al., 1976; Edds. 1977: Webster et al., 1978), for revealing the different components of cultured cell cytoskeletons directly in the electron microscope (Small & Celis, 1978a,b; Small et ul., 1980). Utilizing relatively mild extraction procedures with the non-ionic detergent Triton X-100: all three filameut systems could he demonstrated. together wit.h the delicate meshwork of actin filaments that occurs at, the spreading of locomoting regions of the cell edge. Additional extractiotls of these cytoskeletons with solutions of high and low salt were further shown to remove the microt’ubules and t’he bulk of act’in, leaving cytoskeletons composed primarily of the cell nucleus and the intermediate filaments (Small &, Celis. 19785). Such high salt extraction procedures, adapted from those used in previous studies of smooth muscle (Cooke, 1976: Small & Sobieszek, 1977; Lazarides $ Hubbard. 1976), are now commonly used to achieve an enrichment of this latter filament species (Starger 8r Goldman. 1977 : Bennett et al., 1978; Franke et al., 1978a,b; Gard et al.. 1979). In this paper we present’ a parallel study of the ultrastruct’ure and polypeptide composition of HeLa cells extracted using various procedures, which retain different
e1ernent.s
of t,he csytoskeletcrn.
on the most insoluble thr cell q&.
polypeptides.
Spec:ifically.
wt
have
focused
some of which show interesting
2. Materials
our
attent,ion
changes during
and Methods
(a) (‘rll and cdl .sync~hrorr?J HeLa cells free of mycoplasmas Culture Collection, catalogue number Dulberco’s modified Eagle’s medium (penicillin 100 units/ml. streptomycin detachment essentially as described
(GIBCO Rio-(Jult Ltd, st)ock source, ,imerican Type H3002) were grown routinely as monolayer cultures in containing I 0°, (v/v) fet,al calf serum and antibiotics 50 pg/ml). Mitotic cells were obtained by mechanical by Terasima & Tolmach (1963). Normally. 2 flasks
were used. The distribut.ion of phases (250 ml) containing about lo6 t,o 2 x 106 cells/flask among mit.otic population was determined by phase contrast microscopy. About. 6S”/, of the
cell population correspond to metaphases (Bravo & Celis, 19X&). Cells for electron mirroscopy were grown on silver hexagonal electron microscope grids (150 mesh : Teepe Brandsma. Hilversum, Holland) coated with a plastic carbon support film (Small & Celis, 1978u). (b) Labrllirq
qf nsytlrhronous
cullx with [ 35Q jmethioninr
(‘ells to be treated with Triton alone or to be further extracted with buffers of low and high (Bellco, U.S.A.) placed in 0.25 ml ionic strength were grown attached to 9 mm’ coverslips flat-bottomed microtiter plates (NIJNC, Denmark) containing 02 ml of DMEMt. To label t Abbreviations used : I)MEM, Dulbecco’s isoelectric focusing: SFPHGE. non-equilibrium bis(p-aminoethyl ether)S.~~‘-tetraacetic wid:
modified Eagle’s minimal essential pH gradient electrophoresis; EGTA, SDS. sodium dodecyl sulphate.
medium : I EF. ethyleneglycol-
MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
123
the ~11s (logarithmically growing culture). the normal medium was removed with the aid of a drawn-out Pasteur pipette and was replaced by 0.1 ml of home-made DMEM lacking fetal calf serum (Flow). methionine (1 g NaHCO,/I) and containing 1O0b (v/v) dialysed 100 &i of 135S]methionine (Amersham S-1204, England) and a final concentration of 1 mg rold methionine/l (Celis et al., 1980; Bravo & Celis, 198Ou; Celis & Bravo, 1981). The cells were usually labelled for 20 h at 37”C, during which time the microtiter plates were wrapped in Saranwrap to avoid evaporation. At the end of the labelling period, the slides were washed twice by dipping in wells containing 0.25 ml of Ca’+, Mg * +-free Hank’s buffer. Under these conditions, 5000 to 6000 cts/min were incorporat,ed per cell. Excess liquid was drained from the coverslips and the samples were dissolved immediately in 20 ~1 of lysis buffer (O’Farrell. 1975) or treated furt,her, as described below. For analysis of the polypeptides synthesized by whole cells. the cells were grown and labelled directly in the microtiter wells. For the production of labelled cytoplasts, the cells t’o be enucleated were grown on 2.5 cm discs cut, from Petri dishes and were labelled as above in 3 cm Petri dishes containing 3 ml of labelling medium with 100 &i of [ 35S]methionine/ml (Bravo rf al.. 19816).
(r) Preparation
qf cytopla~vts
Asynchronous cells grown att.arhed to 2.5 cm 135S]methionine as indicated above were enucleated with & Celis, 19786: Bravo et aZ., 19816). Cells were t,reated Cyt,ochalasin B per ml of medium and then centrifuged 11,000 revs/min in an SS34 rotor of a Sorval centrifuge. was higher than 90”,. The enurleated rells (cytoplasts) before any further treatment.
(d) Labelling
of mitofic
plastic discs and labelled with Cytochalasin B as desrribed (Small for 45 min at 37°C with 10~~ of in the same medium for 30 min at The proportion of enucleated cells were allowed to recover for 1 to 2 h
cells with f35S]methioninr
Mitotic cells (lo4 t,o 3 x 104) harvested at 4°C and washed with Hank’s buffer were labelled for various t,imes at 37°C in 0.1 ml of DMEM minus methionine containing 1 g NaHCO,/l. 10YO (v/v) dialysed fetal calf serum, 100 &i of [ %]methionine (Amersham SJ204, England) and 1 mg cold methionine/l (Celis et a/.. 1980: Bravo & Celis, 198Ou; Celis & Bravo, 1981). At the end of the labelling period. the cells were washed in Hank’s buffer and resuspended immediately in lysis buffer or extracted with 0.1’?& (v/v) Triton as described below.
(e) Labelling
of mitotic
cells with 13’P jorthophosphute
Mitotic cells isolated as described above were labelled for 15 min at 37°C in 1 ml of phosphate-free medium containing 2 mCi of [ 32P]orthophosphate. After labelling, the cells were processed for electrophoresis as described for [ 35S]methionine-labelled samples.
(r) T&ton
cytoskeletons
After appropriate times in culture, the small coverslips (9 mm’) or centrifuge discs (2.5 cm diam. for enucleat)ion) with the [ 35S]methionine-labelled cells, or electron microscope grids carrying unlabelled cells were removed, rinsed in Hank’s buffer and transferred to O.lsO (v/v) T&on X-100 in PIPES cytoskeleton buffer (a Ca2+ -free Hank’s solution containing 2 mMMgCI,, 2 mrv-EGTA, 5 m&r-PIPES, pH 6.1). Treatment with Triton X-100 was for 60 to 90 s at room temperature. After washing in Hank’s buffer the roverslips were placed in microtest wells rontaining 20~1 of lysis buffer, and the electron microscope grids were fixed in 250/,, glutaraldehyde in PIPES cytoskeleton buffer for 10 to 30 min before processing for electron microscopy. Mitotic cells in suspension were extracted with Triton as described above but were washed by means of centrifugation in conical tubes (5 min at 4000g).
144 (g) Intcrmediale
Jilament
R. HRAVO
fC’7’ itl,
enriched
cytoskelctons:
L-H-L
cytoskeletons
After treatment with Triton, the coverslips, centrifuge discs or electron microscope grids were washed twice in PIPES buffer and dipped for 90s in each of the following ice-cold solutions (Small 8r Sobieszek, 1977; Small & Celis, 197%): (1) 60 mM-KCI. 1 m&EDTA, 2 mM-EGTA. 1 mM-cysteine, 10 rnM-ATP, 40 mM-imidazole, pH 7.1 (L); (2) 0% M-KU, 1 rnhfEDTA, 2 mM-ATP, 1 mM-cysteine, 40 mM-imidazole, pH 7.1 (H); (3) as for step (1). The coverslips and centrifuge discs were then rinsed in the PIPES buffer and transferred to lysis buffer (O’Farrell, 1975). The electron microscope grids were fixed in 25O, glut,araldehyde in PIPES buffer for 10 to 30 min before processing for electron microscopy. These skeletons, extracted in high and low salt will be referred to as L-H-L cyt,oskeletons. For the more complete extraction of actin from these cytoskeletons. an additional treatment was employed using the actin depolymrrizing protein purified from slime mould (Hinssen, 1979). The purified protein, kindly provided by Dr H. Hinssen. was diluted to between @3 and 0.4 mg/ml in PIPES csytoskeleton buffer plus 2 rnM-CaCl, and then incubated with either the Triton or L-H-L cyt,oskeletons for 30 min at room temperature. The (*ells attached to the coverslip were then rinsed in buffer and processed either for elecatron microscopy or gel electrophoresis as desc,ribrd above. (h) Tuwdimunsiowl
qrl c1rctrophorr.G
The procedures followed have been described elsewhere (C)‘Farrell, 1975; O’Farrell et a/.. 1977; Fey rt al., 1981). Special note should be made. however, that currently available ampholytes can give very variable and sometimes unsatisfactory results. It was therefore necessary t)o check ampholyt,es very carefully and to make optimal mixtures to ensure satisfac%ory and reproducible separation over the whole pH range. The first dimension separations were performed either as (1) acidic isoelectrofocusing gels cont,aining 2”,, (IEF. 18 h at 400V) in 130mmx 12 mm 4”,, (w/v) polyacrylamide ampholines (IW, pH 5-T (LKB. Sweden): 0.4”,, pH 3.5-10 (LKB)) or (2) basic noneyuilibrium pH gradient electrophoresis (NEPHGE. 4.5 h at 400V) in 130 mm x I.2 mm 4!,, (w/v) polyacrylamide gels containing 1”, ampholines (l’?(~ pH 7-9 (LKB): 19, pH 8-9.5 (LKB)). Bot.h IEF and NEPHGE gels were immersed in solution 0 (@06 M-Tris (pH 68), 4O,, and incubated at room (w/v) SDS, loo,, (v/v) mercapt,oet,hanol, dW, (v/v) glycerol) temperature for 20 min before applying to the second SDS/polyacrylamide dimension. Gels to be stored frozen were incubated for I5 min only and the incubation completed while drfrost,ing. The second dimension SDS/pol?;acr,vlamide gcxl electrophoretic separations were’ ac*complished by laying the equilibriated gels ont,o a stacking gel (4,75?, (w/r) acrylamide. O~24~~,,(w/v) bisacrylamide, OIC)b (w/r) SDS. 0-375 MmTris. HCI, pH 6%). A 14,,, (w/v) agarose blue secured the gel in gel made up in solution 0 and containing 0.01 O(, (w/v) bromophenol place. Electrophoresis was performed at, room temperat,ure for 14 h at 11 to 13 mA or until the tracking dye reached 1 cm from the bottom. The slab measured I7 rrn x I4 cm. Polypeptides (IEF, NEPHGE) were numbered from the top of the first dimension and from the highest molecular weight. To define the position of a given polypeptjide, we designate each spot, by its molecular weight followed by its relative mobility with respect) to /3-a&n (IEF) or the NEPHGE spot 9 (NEPHGE). IVe found the latter measurement easier and more reproducible than t,he actual measurement of pH. as there is great discrepancy among the reported pl values of several cnellular polypeptides (Petersen &. McConkey. 1976: Strand Hr August, 1978: Storti ef al., 1978: Krpstal rt al., 1978; Milcarek h Zahn, 1978: Bravo & Knowland, 1979: Garrels, 1979). The gels were processed for fluorograqh? as described by Laskey & Mills (1975). Approximate]\, 200,000 trichloroacetica-prec~p~table cts/min were routinely applied per gel. for 12 h in lOl$, (v/v) Bio-solv For quantitation. the spots were cut out. resuspended solubilizer BBS-3 (Beckman, USA) in toluene-based scintillator and counted for 10 min. The maximum variability for the spots quantitated in this study was determined by counting gel spots from at least 4 independent samples.
P(Tptide maps of the cytoskeletal proteins were obtained by srtbjec,ting individual spots. vut from the 2-dimensional gels. to a second SDS/polyac~rylamide gel electrophoresis in the prewnw of the Stnphylo~occ~s awws V8 prot,ease (Cleveland P/ nl.. 1977 : Fey pf al.. 1981 ). l’\~o-dinrellsional gels were run as described above using loadings of up to 40 x IO’ c.ts/min: this permitted peptide analysis of proteins romprising down to WOI”,, of tht> total wllular proteins. The 2--dim&onal gels were fixed, dried and left for autol,adioyraph\- at room temperature overnight. Appropriate spots were cut from the drietl pt4. swollen in &tilled water and mashed between 2 piwes of clear plastic foil. The samplrs wcrtb thrn taken up in load buffer (012.5 M-Tris. HCI (pH 6%). 03’?‘,, (a/v) SDS. %?P(),,(w/v) I)tlxtran T .iOOand O.Ol(!, (w/v) bromophenol blue) and boiled for !i min before appliration to a I.‘,“,, (rv,‘r) polyaerylamide gel containing SDS (1.2 mm thick, acrylamide to hisacvylamidr~ ratio. 30 : 0.8), and overlayed with a further .5~1 of load buffer containing IO pp S. C(~WPW~S VX ~)rotraw/ml. Ele&rophoresis was c*arrietl out at 15 mA for I2 h using the Laemmli buffi~r system (Lat~mmli, 1970). The gels were processed for fluorography as described abovch (Laskey k Mills. 197;5), except that the last water wash was replaced by a wash in so,, (v/r) gl!~cvrol.
(‘tills grown on silver hexagonal electron microscope grids (Small 8: Delis. 1978a.h) ww ~~ro~vss~d for elec,tron microscopy either after the above extraction procedures or just fi)llo\ving thcb initial t.reatment with Triton .KlOO. Following the fixation step. in each casv thcb gricls bvcrc’ rrmored individually from the coverslips and negatively stained in thr c~)l(l w,ith aqueous I ‘),I (\v/v) uranyl acetate or at room temperature with 3”,, (w/v) sodium qilicotutlpstatt,. Wectron rniwowop>, was carried out in a Zeiss IO.4 operating at 80 kY.
3. Results
For whole mount preparations of’ HeI,a (*ells prepared for elrvtron rniwosvop~~ (SW Jlatwi& and AMethods). the distribution of filamentous cwmponents wuld by d-rrsolvecl only in cells that had sprea.d reasondAy well on the support film. In sucah WI/S. af’tw extraction with Triton both miwotubules and actin filaments c~)ukl Iw watlily visualized. the latter mainly owurring in hundIes or sheets of’ parallel filwtnents (Triton cytoskeletons). The actin filament huntlles. or sheds. c.hilr.Rc.trristic.~ll~ formed small paralld arrays tangential to the cdl nwlew (Fig, I (a)). As in other cultured cells ((‘elis et nl.. 1HiHrr : Small Rr (‘elis. lW80: Small vt t/l.. I!)xo). the concave (~11 edges \\x’re delimited l)y bundles of actin filaments. while t hr spreacling parts of the carIt perimeter \vtw wmpos~l of’ a tarninar cliaponat meshwork of actin filaments containing variabk numbers of radiating mic*rorilli. or microspikes (Fig. 1(a) anti (b)). The microspikes \vere sometimes notic*eabty long. extending up to many mirrometres and often being associated at their tips with nvighbouring cells. ~licwdnbules ivere numerous a.nd occurred at random. curved or’ straight t~lvmrnts or in parallel arra,ys. Ihe latter sometimes ext’ending in width from close to the. cell Iwripher?~ to the perinuclear region (not shown). The microtubules showed IIO pr&~rwcl orientation to the adin filament. bundles. alt,hough the? cwuld bc*
iwoDIFrc.~TIo~
0~ (~YT~SKEI,ETA~.
PoI,YPEPTII)bX
127
found t.ogether with actin. running in parallel groups. The third component of the cytoskeleton. t’he intermediate-sized filaments, could also be recognized t’ogether with microtubules and actin filaments (not shown). However, in cytoskelet’ons extracted only with Triton, the number of these filament,s seen in different regions of the cytoplasm was extremely variable. (ii) Intermediate jilament enriched cytoskeletons: L-H-L cyto,skeletons \Z’e showed earlier (Small & Celis, 19783) that the further extraction of Triton cytoskeletons with solut)ions of high and low salt served to remove both the microtubules and the bulk of actin and, as a result, revealed the extent of the int,ermediate filament net (L-H-L cytoskelet,on). The results of such extractions with HeLa cells are shown in Figure 2(a) and (b). As in other cultured cells (for a review. see Lazarides, 1980), the intermediate-sized filaments were seen to be mainly concentrated around the cell nucleus and became progressively depleted in number towards the cell periphery. The filaments were generally coiled and frecl ends could not be detected. A residual number of act)in filaments could also be recognized in the 1,-H-l, cytoskeleton mainly occurring at or close to the cell periphery (Fig. 2(a) and (b)). These appeared to correspond in the main to remnants of the actin bundles and meshwork found in normal cytoskeletons t,o delimit the cell perimeter. The caoexistence of the two types of filament in extracted cgtoskeletons correlated with the elrctrophoretic analyses described in the following sections. Essentially the same result was obtained with 1,-H-I, evtoskeletons of enucleated cells (rytoplasts : rcbsults not, shown: Small & Celis, 19786). By the further t’reatment of the 1,-H-L cytoskeletons with t,he actin modulatw protein of slime mould (Hinssen. 1979), all visible traces of F-a&in filaments were removed, as judged by electron microscopy (Fig. 2(c) and (d)). Triton cytoskelet,ons treated directly with the actin depolymerising prot’ein were also visibly deplet,ed of’ both actin and microtubules (the latter removed by the necessary inclusion of C”a2+ in the buffer) but retained a large complement, of their ribosomes attached to the LO nm filament net,.
Figure 3(a) shows a two-dimensional gel electrophoretic pattern (IEF and XEPHGE) of total [35S]methionine-labe11ed polgpept’ides from about IO0 asynchronous HeLa cells: 855 acidic (IEF) and 314 basics (NEPHGE) polypeptides
h:. 1. (a) IClectron micrograph of Triton cytoskeleton of a well-spread HeLa cell showing actin filament bundles and peripheral lamella regions. Microtubules and intermediate filaments are also present in such cytoskeletons but are not resolvable at this magnification. Negatively stained with wanyl acetate. Magnification 2250x. (b) E:nlargement of peripheral region of HeLa cell Triton ytoskeleton showing a microspike bundle of F-actin and the associated meshwork of artin tilamrnt,s. NegativeI>- stained w-ith sodium siiic:otungstat.e. Magnification 65.OOox
118
R.
BRAVO
ET
.-II,
MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
129
can be detected in overexposed fluorograms (Bravo et al., 1981a) but of these, only a tot,al of 450 can be seen in the photograph. The films have been exposed so as to keep the intensity of the spots within the linear range of the film response. For reference purposes and to maintain continuity with our previous work, we have used the numbering system described by Bravo et al. (1981a). An important proportion of the major cellular polypeptides labelled with [ 358]methionine are clustered in a region of the gels between M, 35,000 and 70,000 (Bravo & Celis, 198Ou,6; Bravo et al., 1981a). Of these the major polypeptide is actin. which shows the normal @ (IEF 47) and y (IEF 47a) variants described by others. Spots 19 plus 19b and 25 correspond to in and j+tubulin respectively; 8 to aactinin and 26 to a major component of the intermediate filaments (vimentin, Franke et al., 1978a). The percentages and co-ordinates of some of these proteins have been determined (Bravo et al., 1981a).t Polypeptides whose numbers have been underlined (IEF 12, 24, 26 (vimentin), 31, 35, 36 and 46; termed L-H-L cytoplast polypeptides) will be followed throughout this study as they correspond to the most abundant and insoluble polypeptides present in t,he cytoskeleton (see also Table 1). (ii) Triton
cytoskeletons Figure 3(b) illustrates the composition of the cytoskeleton-associated polypeptides from asynchronous HeLa cells treated with 010, (v/v) Trit’on X-100. The Triton cptoskeleton corresponding to that illustrated in Figure 1 contained about 90 polypeptides (48 basic and 42 acidic) and about 307; of the total counts present in whole cells. Spots indicated with large arrows in Figure 3(b) correspond to polypeptides that are mainly present in cytoskeletons (600/, or more), while t’hose indicated with small arrows are present in about equal amounts in the Triton supernatant and in the cytoskeleton. Even though 300/b of IY and /3-tubulin remain in t’he cytoskeleton, they are indicated with large arrows as they are part of the known cytoarchitectural structure. About 52% of total actin and 41% of a-actinin remain in the cytoskeletons. The percentages of the L-H-L cytoplast polypeptides present in these cytoskeletons are given in Table 1. (iii) Irrtermediate $Zument enriched cytoskeletons: L-H-L cytoskezeton We have already shown (Fig. 2; Small & Celis, 1978b) that the further extraction of Triton cytoskeletons with cold buffers of high and low ionic strength under conditions in which microtubules and most of the microfilaments are dissociated leaves a cytoskeleton (L-H-L cytoskeleton) that remains attached to the substrate and that is composed of the nucleus, a prominent web of intermediate-sized VI{:. 2. (a) and (b) Triton cytoskeletons after extraction in high salt: L-H-L cytoskeletons. The main part of the cytoplasm is composed of intermediate filaments (if). Residual actin filament bundles (mf) occur at the cell periphery. Uranyl acetate staining. Magnification: (a) 2340 x ; (b) 17,000 x (c) and (d) Triton 1,-H-I, cytoskeletons treated additionally with the actin modulator protein from slime mould. Actin filament, bundles and single actin filaments are removed. and only intermediate filaments remain. I!ranyI acetate staining. Magnification: (c) 3200 x : (d) 79.000 x t The percentages of the major HeLa cells proteins labelled with a mixture of 16 [“C].labelled aminoacids has now been submitted for publication to Clinical Chemistry (R. Bravo & J. E. Celis).
130
R. BRAVO
e---
NEPHGE
IEF
ET
AL.
w
(a)
92.5 69 55 43
30
PK. 3. (a) Two-dimensional gel electrophoresis of total [ s%]methionine-labelled polypeptides from 100 asynchronous HeLa cells (IEF and NEPHGE). The gel has been exposed so as to keep the intensit> ofthe’spots within the linear range of the film response. Spots were numbered starting from the top of the first dimension and from the highest molecular weight. Only the spots indicated with large numbers hare been quantitated in this study. at. m-tubulin : j3t, j%tubulin ; v, vimentin. fl and yactin are the major polypeptides species in HeLa cells. (b) Two-dimensional gel electrophoresis of ] 358]methionine-labelled from Triton cytoskeletons from asynchronous HeLa cells.
:
0
48.5/0.74 435/090
98
90 80 90 90
34
99 90 90 89 87 74
46 75 70 60 60
32
43 35
Cytoplasts
L’nknown Vimentin (_:nknown I’nknown 1Tnknown rnknown
41
cytoplnst
54 25 30 40 40 2
65
Constant Constant Constant
I)ecreases§ Constant
Constant Constant
Relative proportion during cell cycle$
polypeptides
Karyoplsstst
O/J Distribution
Vnknown
Identit?
%, in Triton cytoskeleton Asynchronous Mitotic cells cells
of L-H-L
1
ml (unknown) m2. m3 (phosphorylations) m4. m5 (phosphorylations) m6. m7 and m8 (phosphorylations)
Modified forms during mitosis (nature of modification)
t From Bravo el al. (19816). : From Bravo 8: Celis (1978). Only polypeptides whose relative proportion changed by g-fold or more were considered as variable in that study (based on 30 min labellings). § The decrease observed could be due in part to modification. The quantitations are based on the analysis of at least 4 independent samples.
49/1.18
36 46
56/097 54/1.19 50/O%
68/0.55
31 35
24 26
12
IF’P1
Polypeptide
Molecular weight ( x 10-q/ mobility relative to fi-actin
Some properties
TABLE
132
R. BRAVO +--
NEPHGE
ET
.-LL
IEF ---+
-69
-55 -43
-30
? 0 x $
*69 .55 -43
,30
FIG. 4. Acidic (IEF) and basic (NEPHGE) [35S]methionine-labelled polypeptides present in HeLa I,H-L cytoskeletons from (a) whole cells and (b) cytoplasts. Polypeptides indicated with small arrows and small numbers are present in variable amounts in these cytoskeletons. Spots indicated with an asterisk in (a) correspond to polypeptides that are mainly present in karyoplasts.
filaments and some residual, mainly peripheral actin. Electrophoretic analysis (Fig. 4(a)), shows that several of the major [ 35S]methionine-labelled polgpeptides remain in the L-H-L cytoskeletons. Beside the 1,-H-L cy%oplast polypept,ides. these cytoskeletons contain a sizeable amount’ of actin together with IEF polypept’ides 13. 13e. 33,38, as well as NEPHGE polypept,ides 3,6,8,1ls. 2lh and 23s. There are also other acidic and basic polypeptides that are present in variable amounts. examples being indicated with small arrows and numbers in Figure 4(a). Essentially the same patterns were obtained if the gels were stained with Coomassie blue (not, shown).
>MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
133
Enucleation of asynchronous cells prelabelled for 20 hours with [ 35SJmethioninc~ (see Materials and Methods) followed by exbraction with 0.194 (v/v) Triton S-ICH) and buffers of high and low salt concentration (L-H-L) showed that polypeptides IEF 13, 13e, 33, 38 and NEPHGE 3, 6 and 9 are mainly of karyoplastic origin (indicated with an asterisk in Fig. 4(a) and (b); Bravo et al., 1981b). Finally, analysis of the L-H-L cytoskeletons depleted of actin (Fig. 2(c) and (d)) using the actin depolymerizing protein from slime mould (Hinssen, 1979) showed the almost complete removal of actin, leaving the proportion of the L-H-L cytoplast polypeptides unchanged (Fig. 5(a); see also Fig. 4(a)). This has been further supported by quantitation of the spots (not given) and analysis of the supernatant. (Fig. 5(b)). IEF
---+-
IEF -
(0)
(b)
FIG. 5. (a) Acidic (IEF) polypeptides present in HeLa L-H-L cytoskeletons treated with the a&in depolymerising protein from slime mould (Hinssen, 1979). (b) Polypeptides present in the supernatants.
the substrate-attached. intermediat,e filament enriched To summarize. +oskeletons are composed of seven abundant polypeptides. which have been termed 1,-H-L cytoplast polypeptides. The ratio of these polypept,ides to total acids actin, as determined from labelling with a mixture of 16’ “C-labelled-amino are t.he following: IEF 12, @08; IEF 24, @14; IEF 26 (vimentin), 0.195; IEF 31, 0.02 : IEF 35.0.04 ; IEF 36,002 : and IEF 46,0.0.5. There are also basic polypeptides present in these cytoskeletons but these have not been investigated further. Similar studies by Franke et al. (1979) using one-dimensional gel electrophoresis have revealed four polypeptides besides t,otal actin in HeLa cells treated with 19,, (v/v) Triton X-100 and extracted with 1.5 M-KCI. The four polypeptide bands had a,pparent molecular weights of 57,000, 52,000, 48,000 and 46,000. (iv) I’eptidr analysis of L-H-L cytoplast polypeptides Having identified the major polypeptides that are present in the intermediate filament enriched. substrate-attached cytoskeletons, it was necessary to establish if they were all distinct or were related in peptide composition. Accordingly, onedimensional pept,ide analysis of the individual [3sS]methionine labelled spots recovered from two-dimensional gels was undertaken. the results of which are 5’
13-l
R. BRAVO
ET
AL
shown in Figure 6. As can be seen from this analysis, and given the molecular weight. and charge differences, t,here is no obvious homology between IEF spots 12. 24. 26 (vimentin), 31, 35, 36 and 46. (c) ModiJication
of L-H-L
cytoplast
polypeptides
during
mitosis
Recently. we showed that the relative proportion of a few polypeptides changes signiticantly- during mitosis (Bravo & Delis. 1980a). In Figure 7 we show enlargements of selected areas of IEF gels from mit.otic HeLa cells labelled for (a)
12
242631
35364647
PK. 6. One-dimensional peptide maps of I.-H-I, cytoplast polypeptides from HeLa cells. [35Slmethionine-labelled polypeptides were digested with S. r~z(teus V8 protease as described in Mabrials and Methods. The peptide map of actin (IEF 47) has been included as a reference.
MODIFICATION IEF :: 0-J
OF (:YTOSKELETAL IEF -
a
POLYPEPTIDES
)_
x
(a)
I
- 55 -43
FIG. i. Two-dimensional gel electrophoresis (IEF) of [35S]methionine-labelled polypeptides from mitotic- HeLa cells lahelled for (a) 10. (b) 22, (c) 30. (d) 35, (e) 40. and (f) 90min after collection. Polypeptides indicated by the letter m correspond to those whose relative proportion increases preferentially during mitosis. Only a fraction of the gel is shown. at and fit indicate a and fl-tubulin. respectiveI!
IO. (b) 22. (P) 30, (d) 35, (e) 40 and (f) 90 minutes after collection. The polypeptides that appear preferentially during mitosis are indicated by the letter m, followed by a number and. for convenience. t,hey have been termed mitosis spec*ific polypeptides. All these polypeptides occur transiently and none of t’hem can be detect,ed. at least at comparable intensities. in long-term labellings: that is. when the cells have already reached G,(Fig. 7(f)). The larger number of mitosis specific polypeptides detect’ed in this study as compared to our previous work (Bravo & (‘rlis. 198)cr) is due to the shorter labelling time of t)en minutes included in this study. With the exception of m2 (also called 26e: Bravo rt nl.. 1981c), which can be tlrtected in significant amounts in synchronized 6, cells (Fig. 8: Bravo & (Delis.
IEF
-
IEF
-
FK. 8. Two-dimensional gel electrophoresis (TEF) of [‘5S]methionine-labelled polypeptides from G ,. S and G, HeLa cells labelled for 80 min. m2 has also been called IEF 26e in the text. Only a fraction of the gel is shown. at and ,9t indicate a and fl-t,ubulin. respectively.
IEF -
-30
-43
-55
-69
:
0 T s-
MOIJIF’ICATIO~
OF (:YTOSKELETAL
POLYPEPTIDES
1x
198oU) and in lesser but detectable amounts in G, and S cells (Fig. 8). the other mitosis specific polypeptides can be det,ected only at vet-y low levels in I,& frotn interphase cells (Fig. 8). Similar results are obtained if the synchronized celis are labelled for ten minutes (not shown). The relative proportion of a few polypeptides has been shown to change in mitosis and G2? and these correspond to IEF 30, rd, (IEF 32). vd3 (IEF41), vd, (IEF44i) and vd, (IEF47m: Figs 7 and 8: Bravo Kr (‘elk. 198&L; Bravo et a.Z., 1981a). Since some of the mitosis specific polypeptides exhibit the same molecular weight and migrat.e closely to some of the L-H-L cytoplast polypept.ides, we determined their one-dimensional peptide maps in order t,o search for possible homologies. As shown in Figure !I, ml is related to IEF 24; m:! is related to vimentin: and IEF 37. m6 and m7 are related to IEF 36. We have previously reported that the relative proportion of IEF 37, which occurs in significant amounts in interphase, increases during mitosis (Bravo & Celis, 198On). The peptide map studies also revealed that m3 and m4 are related to IEF 31 (not shown), and that vd, (IEF 3P), vdJ (IEF 41).
24
ml
36 37 m6 m7 from mitotic cells. [?3] methionine-labelled were digested with S. aweus 1’8 protease as described in Materials and Methods. 26
m2
vdi
vd3
vds vd5
FIG. 9. One-dimensional peptide maps of polypeptides polypeptides
138
R. BRAVO
BT
AI,
WI, (IEF 44i) and vd, (IEF 47m) correspond to degradation products of vimentin (f(‘ig. 9). Since all the mitotic specific polypeptides migrat,ed at t’he acidic side of the corresponding spot. we labelled the mitotic cells for 15 minutes with [32P]orthophosphate t,o determine if they corresponded to phosphorylations. An enlargement of a selected area of the IEF gel is shown in Figure 10. These studies indicated that m2. m3, m4, m5. m6, m7 and m8 are phosphoproteins. I RF‘ 12. 24. m 1, vimentin. 31,35 and 36 are not det)ect,ably phosphorylated. Similar labellings of G, and asynchronous cells (Fig. 10) indicated that IEF 36 is phosphorylated in interphase to yield IEF 37. in agreement with the /35S]methionine labellings. which indicated t,hat there is a subst,antial amount of this prot.ein in int,erphase (Fig. 8). Only small amounts of phosphorylated mti and 1117can be detected in G,. while m”. rr13. m-C and m5 arc barely detected in the background (Fig. 10). In mit’osis. IE:F 37 is further phosphorylated to yield m6. m7 and m8. A few major phosphorylated polypeptides whose intensity increases in mitosis are indicated with small arrows in Figure 10. Many of these are also found in asynchronous ~11s but are barely dektable in C:, Also. one can detect phosphorylated polypeptides in asynchronous cells (arrowheads) that do not seem to be present in mitosis: these may correspond to modificat,ions occurring in the other phases of t,he cycble. A detailed study of t’he phosphorylated polypeptides of HeLa cells is now being made. The nature of the IEF 24 modification is unknown. Mitotic Triton cytoskeletons cont.ained, in addit,ion to the 1,-H-L cytoplast polypeptides, the polypeptides ml. m2. m3. m4. m5, mfi and m7 (Fig. 1 l(a)). These cytoskeletons contained about 90($, of vimentin, as compared with 99?,, normally found in cgtoskeletons from asynchronous cells (Table 1 : see also Fig. I l(b) and (c) for a comparison of the Triton supernat,ants). The relative proportion of the other L-H-L cytoplast polypept,ides in the mitotic Triton cytoskeleton was very similar to that observed in asynchronous cells, although small but significant, differences were observed in the cases of IEF 31 and 36 (Table 1).
4. Discussion The high sensitivity of the two-dimensional gel electrophoresis technique when used with ] 3sR]methionine-labelled polypeptides has enabled us to screen relatively small numbers of substrate-attached cultured cells without the need to resort to cell concentration via scraping procedures. Apart from being considerably more convenient, this approach has allowed a direct correlation between features of the cytoskeleton as seen in the electron microscope and their polypeptide composition. In this study we have focused our attention on the most insoluble polypeptides of the cytoskeleton. some of which show interesting changes in the cell cycle (Bravo & Celis, 198Ou). In tracing the polypeptides associated with the intermediat’e filament enriched cytoskeletons, the analysis of enucleated a,nd artin depleted preparations proved particularly useful. Seven abundant polypeptides (termed L-H-I, cytoplast polypeptides) emerged as t,he main components of these cyt,oskeletons (Table 1). The larger number of protein components found in these cytoskeletons as compared
E P
kl H
R. BRAVO
IEF
ET Al,
-
FIG. 11. Two-dimensional gel electrophoresis (IEF) of ~J58Jmethionine-labelled polypptides from (a) HeLa mitotic Triton cytoskeletons; (b) HeLa mitotic Triton supernatants; and (c) Triton supernatants from asynchronous HeLa cells.
MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
141
to previous studies reported by Franke et al. (1979), is most likely due to the different extraction procedures used and to the higher resolving power of the twodimensional gel electrophoretic method. 80 far. only vimentin (IEF 26) has been characterized (see also Franke et al.. 1979), while the identity of the other polypeptides remains unknown. Preliminary indicate that IEF 36 and 46 may correspond to experiments. however, cytokeratins, as these proteins migrate very close to PTK 2 cytokeratins (Franke ef al., 19786) and exhibit similar one-dimensional peptide maps (not shown). Further experiments, however, are needed to assess the identity of these proteins. We cannot exclude the possibility that IEF 31 may also correspond to a cytokerat’in but this polypeptide is different to skeletin (see also Lazarides, 1980). Init’ial experiments using antibodies raised against IEF31 showed that this antibody stains a filament, network similar to that of the intermediate filaments. It is not known whether IEF 12, 24 and 35 are an integral part of the intermediate filament network or whether they represent accessory proteins that may function in interfilament or filament-organelle interactions. The possibility that these proteins may not be associated with the intermediate filaments must also be considered. Experiments are now under way to raise antibodies against these polypeptides in an effort to determine their location in the cell. These polypeptides are clearly different to vimentin, as judged by one-dimensional peptide analysis (Fig. 6). IEF 12 and 24 are far easier to extract than the rest of the L-H-L cytoplast polypeptides (Table l), a fact that may explain why these polypeptides have not been described before in HeLa cells. A polypeptide with similar properties to IEF 12 has been described by Lehto et al. (1978) in human embryonic skin fibroblasts and by Wang et al. (1980) in non-neuronal cells and skeletal myofibrils. IEF 12. 24 and 35 are abundant polypept’ides in HeLa cells and together with vimentin (IEF 26) make up a polypeptide “quartet” that is widespread in many cell lines and primary cultures from hamster, human, mouse and rat origin as well as mouse tissues (Fey et ccl., 1981: Bravo et al.. 1981c,d). Analysis of the polypeptide pattern of a single Sarcoma 180 cell showed that these polypeptides coexist in a single cell (Bravo et al., 1981c). Studies of mitotic as well as of interphase cells showed that IEF 24, 26 (vimentin), 31 and 36 (via IEF 37) are modified preferentially during mitosis (Table 1). Vimentin is phosphorylated transiently during mitosis to yield polypeptides m2 and m3 (Table 1). Polypeptide m2 can also be detected in small amounts in interphase cells but its intensity is strongest in short-term labelled mitotic cells. Since most of the mitotic cells collected by shaking are in metaphase (66’+,). it is likely that these modifications take place in or very near to this phase of the mitotic cycle. We have observed a similar increase in the relative proportion of IEF m2 and m3 in mitotic 3T3B cells (Bravo & Celis, 19806). We do not know whether preferential phosphorylation during mitosis is a general feature of other cells : Cabral & Gottesmann (1979) have reported a minor phosphorylated form of vimentin in asynchronous Chinese hamster ovary cells that does not seem to increase in mitosis. Also, O’Connor et al. (1979) have reported phosphorylation of vimentin in chicken muscle and in non-muscle cells, but they have not determined the phase of the cycle in which the modification occurs. Furthermore, Cabbiani et
142
It.
BRAVO
ET AL
al. (1981) have recently described two isoelectric variants of vimentin in vascular smooth muscle cells. the less acidic variant of whirh comigrates with purified vimentin from different cell lines. Of interest is the appearance of the vimentin degradation products at a certain t,ime during mitosis. In interphase, the intensity of these polypeptides is strongest in (:, (Bravo & Crelis, 1980a), and can barely be detect,ed in G, or S. Based on the analysis of many hundreds of gels from HeLa and other cells, we believe that these polypeptides are not’ the product of non-specific degradation but rather correspond to some important event, in the process of cell division. A similar set of polypept,ides. called diagonal proteins, have been described by OYlonnor ef ~1. (1979) in myogenic cell cultures. The modification of IEF 36 is most interest.ing as IEF 37 is the major phosphorylated form of this protein observed in interphase. In mitosis. IEF 37 is furt)her phosphorylated to yield m6. m7 and m8. Small amounts of these phosphorylated polypeptides can be observed in interphase cells but their amount increases dramatically in mitosis. We have observed other phosphorylated polypeptides that occur mainly in mitosis (Fig. 10) but their identity is unknown. The nature of the modificat,ion of IEF 24 has not been established. Even though it is yet) t,oo early to envisage a mechanism by means of which these modificat,ions might regulate t,he stage of assembly of these cyt’oskeletal polypeptides, it seems relevant’ to not,e the small but significant increase of viment,in in the Triton supernatant of mit,otic cells (loo/A). Careful examination of t,he fluorograms of IEF gels from T&on supernatants indicates t,hat it is viment.in and not phosphovimentin (m2) that is extractable. Since most of the vimentin remains in the cytoskeleton during mitosis (Fig. 1 l(a) ; Table 1 ), however, it is likely that the extracted fraction may reflect changes in a second component that interacts with vimentin and that disassembles during mitosis. Since rearrangements in this 10 nm filament net (Blose, 1979: Hynes & Dest’ree, 1978; Aubin et al.. 1980) clearly accompany the other major changes in mit,osis, it is possible that the polypeptide modifications observed are instrumental in assuring at least part of the process of cell division. We would like to thank A. Celis, G. Langanger and P. Jertschin for expert assistance, and 0. Jensen and B. Thornsen for photography. One author (R.B.) is a recipient of a fellowship from the Medical and Natural Science Research Councils, and another (S.J.F.) is a recipient of a European Molecular Biology Organization long-term fellowship. This work was supported by grants from Euratom, the Danish Medical and Natural Science Research Council
(to J.E.C.)
and the Muscular
Dystrophy
Association
(to J.V.S.).
REFERENCES Aubin, J. E., Osborn, M., Franke, W. W. & Weber, K. (1980). Eq~pt. CeZZ Res. Bennett, G. S., Fellini, S. A. & Holzer, H. (1978). Diffeerentiation, 12, 71-82. Blase, S. H. (1979). Proc. Nat. Acad. Sci., U.S.A 76, 3372-3376. Bravo. R. & Celis, .J. E. (198Oa). J. Cell BioZ. 84, 795-802. Bravo, IX. & Celis, J. E. (1980b). Expt. Cell Res. 127, 249-260. Bravo, R. & Knowland, J. (1979). Differentiation, 13. 101-108.
Bravo, R., Bellatin, Bravo.
129, 149-165.
J. & Celis, J. E. (1981a). Cell BioZ. Int. Rep. 5, 93-96.
FL. Celis. A., Mosses, D. & Celis, .J. E. (1981b). Cetb BioZ. fnt. Rep. 5, 479-489.
MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
133
Bravo. K.. Fry, S. J., Small, ,J. V., Mose Larsen, P. & Celis, ?J. E. (1981c). CeZZ, 25, 195-207. Bravo, It., Fey, S. J. & Celis, J. E. (1981d). Carcivwgenesis, 2, 769-782. Brown, S., Levinson, W. & Spudich, J. A. (1976). J. Supramol. Struct. 5, 119-130. Cabral, F. & Gottesmann, M. M. (1979). J. Biol. Chem. 254, 6203-6206. Celis, ,J. E. & Bravo, R. (1981). Trends Biochem. Sci. 6, 197-201. Celis, J. E., Kaltoft, K. & Bravo, R. (1980). In Transfer of CeZZ Constituents into Eukayotic Cells (Celis, J. E., Graessmann, A. & Loyter, A., eds), pp. l-28, Plenum, New York. Celis, .J. E., Small, J. V., Andersen, P. & Celis, A. (1978u). Expt. Cell Res. 114, 335-348. Delis, J. E., Small, J. V., Kaltoft, K. & Celis, A. (1978b). Expt. Cell Res. 120, 79-86. Cleveland, I)., Fischer, S., Kirschner, M. & Laemmli, U. (1977). J. BioZ. Chem. 252, 110% 1106. Cooke, P. (1976). J. Cell BioZ. 68, 539-556. Edds, K. T. (1977). J. Cell BioZ. 73, 479-491. Fey, S. J., Bravo, R., Mose Larsen, P., Bellatin, J. & Celis, J. E. (1981). Cell Biol. Znt. Rep. 5. 491-500. Franke, \V. M’.. S(>hmid, E., Osborn, M. & kl’eber. K. (1978n). Prof. Xat. /lrad. A‘?&.. Ir.Q.rl. 7.5. .X)34-5038. Franke, \f’. LV., Svhmid, E., &born, M. & &‘eber, K. (1978b). Cytobiologie, 17, 394-411. Frankr. MT. W., Schmid, E., Weber, K. & Osborn, M. (1979). Expt. Cell Res. 118, 95-109. (:abbiani. (i.. Scahmid, E., ITinter. S., Chaponnier. C., De Chastonay, C., Vandekerckhove, .J.. LVrber, K. Sr Franke, W. W. (1981). Proc. Xat. ilcad. Sri., lJ.S.A. 78, X98-302. (iard. 1). I,.. Bell. P. B. $ Lazarides, E. (1979). Proc. Nat. ilcad. Sri., I!.S.A. 73, 4344-4348. Garrels. .I. I. (1979). Develop. Biol. 73, 1344152. H inssrrl. H (1979). Tn Cell Motility: Molecules and Organization (Hatano, S., Ishikawa. H & Sate. H _. eds). pp. 59-85. University of Tokyo Press, Tokyo. Hynes, fi. 0. B Destree, A. T. (1978). Cell, 13, 151-163. Krystal. (i.. Ratner, J. B. & Hamkalo, B. A. (1978). t’roc. I%!. Acad. Sri., cI.S..4. 75, 49774981. Laemmli. I’. (1970). Nuture (London), 227. 68&685. Laskev. K. A. & Mills, A. D. (1975). Eur. J. Biochem. 56, 335-341. Lazar&s. E. (1980). ~Vuture (London), 283, 249-856. Lazarides. E. Xr Hubbard, B. D. (1976). Proc. ~Vat. Acad. Sci., I7.S.A. 73, 434444348. Lrhto, Y.-P.. Virtanen. I. & Kurki, P. (1978). ‘Vatwe (London), 272, 175-177. Jlilrarrk. (‘. & Zahn. K. (1978). J. Cc/l BioZ. 79, 833-838. O’Connor. C. M.. Balzer, D. R. & Lazarides, E. (1979). Proc. ,Vat. Acad. Sri., C.S.rl. 76. 819823. O’Farrell. I’. H. (1975). J. BioZ. Chem. 250, 4(K)7-4021. O’Farrrll. P. %.. Goodman, H. M. & O’Farrell. P. H. (1977). Cell, 12, 1133-1142. Peterson. 1,. J. Kr McConkey, E. H. (1976). J. BioZ. Chem. 251, 548-554. Roberts, K. (1974). Progr. Biophys. ;ZloZ. BioZ. 28, 371-420. Small. .I. V. 8: (‘elis. J. E. (1978a). Cytobiologie, 16, 308-325. Small, .I. \-. 6 C’elis. *J. E. (19786). J. Cell Sci. 31. 393-409. Small. ,J. V. & Robieszek, A. (1977). J. Cell Sci. 23, 234-268. Small, ?J. V.. Isenberg, G. & Celis, J. E. (1978). Nature (London), 272, 638-639, Small, .J. V., Celis. J. E. & Isenberg, G. (1980). In Transfer ofCell Constituents into Eukuyotic CrlZs (Celis, J. E., Graessman, A. & Loyter, A., eds), pp. 75-111, Plenum, New York. Starger. .J. Bl. 8: (Goldman, R. D. (1977). P rot. Xat. rlcud. Sri., U.S..4 74, 2422-2426. St’orti. f<. V.. Horovitch, S. ,J.. Scott, M. P.. Rich. A. & Pardue, M. L. (1978). Cell, 13, 589598. Strand. 31. & August, J. T. (1978). Cell, 13, 399-408. Trrasima. T. B. & Tolmach, L. J. (1963). Expt. Cell Res. 30, 344-362. \Vang. (‘.. Asai. D. .J. & Lazarides, E. (1980). P rot. Nat. Acad. Sci., IJ.S.A. 77, 1541-1545. M:rhtcr. K. E.. Osborn, MI. & Weber. K. (1978). Expt. Cell Res. 117, 47-61. Edited
by IV. Franke
Biol. (1982) 154, 121-143
Architecture Modification
and Polypeptide Composition HeLa Cytoskeletons
of Cytoarchitectural
Polypeptides
of
during Mitosis
RODRIGO BRAVO'. ,J. VICTOR SMALL'. STEPHEN
LDitGion of Hio&uctrrro~l Chetnistr.y . l)epartment of Chemistry .+Inrhus 1‘tt&ersity . DK-X000 A arhus (‘. Denmark ‘Jnstit,ute oj’ Molecular Biology of the -4 ustrian Academy of Sciences, Wzburg, Austria (Received
3 =1pril 1981, and in revised form
I4 July
1981)
Substrate-attached asynchronous HeLa eells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton rytoskeletons showed actin filament bundles, microtubules. intermediate filaments, and actin networks in the substrate-associated lamellae, and cont,ained around 90 pol,y.peptides (48 basic, 42 acidic; 52“~ of total artin. 99”” of viment,in. 41°, of T-aetmm and 30?<;,of p-tubulin). Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a t,otal of 19 polypeptides (13 acidic!, 6 basic). Of these, 1%corresponded to abundant acidic proteins in the 47,000 to 70,000 ill, region as determined by staining with Coomassie blue and labelling with a mixture of 14C-labelled amino acids. ITsing 1,-H-L extracted cytoplasts, and employing an a&in depolymerising protein from slime moulds, seven abundant acidic IEFf polypeptides were shown to be present in these intermediate filament-enriched, substrat,e-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 111,)and six polypeptides (IEF 12, 68,000 112~:IEF 24. 56.000 M,: IEF 31. 50,000 Nr; IEF 35, 49,000 Mr: IEF 36. 48,509 *)I, and IEF 46, 43.500 W,) not previously reported as present in cytoskeletons. I’eptide analysis showed that these were not related as products of modification or proteolysis. JAabelling of mitotie and interphase cells with [3SS]methionine followed by onedimensional peptide map analysis showed that IEF 24,26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37. and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-I, cytoplast polypeptides may be important in the reorganization of @oskeletal elements that takes place during cell division.
1. Introduction \l’hile the exist,enee of different’ types of cytoplasmic filaments, i.e. actin filaments. intermediat,e-sized filaments and mirrotubules. has long been established (for t Author to whom all correspondence 1 IEP. isoelectric focusiug.
should be addressed 121
(M)22-4836/82/010121-23
$02.00/0
0 1982 Academic Press Inc. (London) Ltd.
reviews, see Roberts. 1971: Lazarides, 1980: Small et ~1.. 1!380), their general and mutual abundance in a wide variety of tissues and cultured cells has only recentI> recscived genera.1 attention. In our laboratories we are interested in determining the role of cytoskeletal proteins in regulating cell growth and proliferation (Celis et uZ.. 1978a,b ; Bravo & Celis, 1980a,b) as well as in cell motility (Small & Celis, 1978a,b; Small et al.. 1980). In earlier studies we developed methods, similar to those adopted independently by other investigators (Brown et al., 1976; Edds. 1977: Webster et al., 1978), for revealing the different components of cultured cell cytoskeletons directly in the electron microscope (Small & Celis, 1978a,b; Small et ul., 1980). Utilizing relatively mild extraction procedures with the non-ionic detergent Triton X-100: all three filameut systems could he demonstrated. together wit.h the delicate meshwork of actin filaments that occurs at, the spreading of locomoting regions of the cell edge. Additional extractiotls of these cytoskeletons with solutions of high and low salt were further shown to remove the microt’ubules and t’he bulk of act’in, leaving cytoskeletons composed primarily of the cell nucleus and the intermediate filaments (Small &, Celis. 19785). Such high salt extraction procedures, adapted from those used in previous studies of smooth muscle (Cooke, 1976: Small & Sobieszek, 1977; Lazarides $ Hubbard. 1976), are now commonly used to achieve an enrichment of this latter filament species (Starger 8r Goldman. 1977 : Bennett et al., 1978; Franke et al., 1978a,b; Gard et al.. 1979). In this paper we present’ a parallel study of the ultrastruct’ure and polypeptide composition of HeLa cells extracted using various procedures, which retain different
e1ernent.s
of t,he csytoskeletcrn.
on the most insoluble thr cell q&.
polypeptides.
Spec:ifically.
wt
have
focused
some of which show interesting
2. Materials
our
attent,ion
changes during
and Methods
(a) (‘rll and cdl .sync~hrorr?J HeLa cells free of mycoplasmas Culture Collection, catalogue number Dulberco’s modified Eagle’s medium (penicillin 100 units/ml. streptomycin detachment essentially as described
(GIBCO Rio-(Jult Ltd, st)ock source, ,imerican Type H3002) were grown routinely as monolayer cultures in containing I 0°, (v/v) fet,al calf serum and antibiotics 50 pg/ml). Mitotic cells were obtained by mechanical by Terasima & Tolmach (1963). Normally. 2 flasks
were used. The distribut.ion of phases (250 ml) containing about lo6 t,o 2 x 106 cells/flask among mit.otic population was determined by phase contrast microscopy. About. 6S”/, of the
cell population correspond to metaphases (Bravo & Celis, 19X&). Cells for electron mirroscopy were grown on silver hexagonal electron microscope grids (150 mesh : Teepe Brandsma. Hilversum, Holland) coated with a plastic carbon support film (Small & Celis, 1978u). (b) Labrllirq
qf nsytlrhronous
cullx with [ 35Q jmethioninr
(‘ells to be treated with Triton alone or to be further extracted with buffers of low and high (Bellco, U.S.A.) placed in 0.25 ml ionic strength were grown attached to 9 mm’ coverslips flat-bottomed microtiter plates (NIJNC, Denmark) containing 02 ml of DMEMt. To label t Abbreviations used : I)MEM, Dulbecco’s isoelectric focusing: SFPHGE. non-equilibrium bis(p-aminoethyl ether)S.~~‘-tetraacetic wid:
modified Eagle’s minimal essential pH gradient electrophoresis; EGTA, SDS. sodium dodecyl sulphate.
medium : I EF. ethyleneglycol-
MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
123
the ~11s (logarithmically growing culture). the normal medium was removed with the aid of a drawn-out Pasteur pipette and was replaced by 0.1 ml of home-made DMEM lacking fetal calf serum (Flow). methionine (1 g NaHCO,/I) and containing 1O0b (v/v) dialysed 100 &i of 135S]methionine (Amersham S-1204, England) and a final concentration of 1 mg rold methionine/l (Celis et al., 1980; Bravo & Celis, 198Ou; Celis & Bravo, 1981). The cells were usually labelled for 20 h at 37”C, during which time the microtiter plates were wrapped in Saranwrap to avoid evaporation. At the end of the labelling period, the slides were washed twice by dipping in wells containing 0.25 ml of Ca’+, Mg * +-free Hank’s buffer. Under these conditions, 5000 to 6000 cts/min were incorporat,ed per cell. Excess liquid was drained from the coverslips and the samples were dissolved immediately in 20 ~1 of lysis buffer (O’Farrell. 1975) or treated furt,her, as described below. For analysis of the polypeptides synthesized by whole cells. the cells were grown and labelled directly in the microtiter wells. For the production of labelled cytoplasts, the cells t’o be enucleated were grown on 2.5 cm discs cut, from Petri dishes and were labelled as above in 3 cm Petri dishes containing 3 ml of labelling medium with 100 &i of [ 35S]methionine/ml (Bravo rf al.. 19816).
(r) Preparation
qf cytopla~vts
Asynchronous cells grown att.arhed to 2.5 cm 135S]methionine as indicated above were enucleated with & Celis, 19786: Bravo et aZ., 19816). Cells were t,reated Cyt,ochalasin B per ml of medium and then centrifuged 11,000 revs/min in an SS34 rotor of a Sorval centrifuge. was higher than 90”,. The enurleated rells (cytoplasts) before any further treatment.
(d) Labelling
of mitofic
plastic discs and labelled with Cytochalasin B as desrribed (Small for 45 min at 37°C with 10~~ of in the same medium for 30 min at The proportion of enucleated cells were allowed to recover for 1 to 2 h
cells with f35S]methioninr
Mitotic cells (lo4 t,o 3 x 104) harvested at 4°C and washed with Hank’s buffer were labelled for various t,imes at 37°C in 0.1 ml of DMEM minus methionine containing 1 g NaHCO,/l. 10YO (v/v) dialysed fetal calf serum, 100 &i of [ %]methionine (Amersham SJ204, England) and 1 mg cold methionine/l (Celis et a/.. 1980: Bravo & Celis, 198Ou; Celis & Bravo, 1981). At the end of the labelling period. the cells were washed in Hank’s buffer and resuspended immediately in lysis buffer or extracted with 0.1’?& (v/v) Triton as described below.
(e) Labelling
of mitotic
cells with 13’P jorthophosphute
Mitotic cells isolated as described above were labelled for 15 min at 37°C in 1 ml of phosphate-free medium containing 2 mCi of [ 32P]orthophosphate. After labelling, the cells were processed for electrophoresis as described for [ 35S]methionine-labelled samples.
(r) T&ton
cytoskeletons
After appropriate times in culture, the small coverslips (9 mm’) or centrifuge discs (2.5 cm diam. for enucleat)ion) with the [ 35S]methionine-labelled cells, or electron microscope grids carrying unlabelled cells were removed, rinsed in Hank’s buffer and transferred to O.lsO (v/v) T&on X-100 in PIPES cytoskeleton buffer (a Ca2+ -free Hank’s solution containing 2 mMMgCI,, 2 mrv-EGTA, 5 m&r-PIPES, pH 6.1). Treatment with Triton X-100 was for 60 to 90 s at room temperature. After washing in Hank’s buffer the roverslips were placed in microtest wells rontaining 20~1 of lysis buffer, and the electron microscope grids were fixed in 250/,, glutaraldehyde in PIPES cytoskeleton buffer for 10 to 30 min before processing for electron microscopy. Mitotic cells in suspension were extracted with Triton as described above but were washed by means of centrifugation in conical tubes (5 min at 4000g).
144 (g) Intcrmediale
Jilament
R. HRAVO
fC’7’ itl,
enriched
cytoskelctons:
L-H-L
cytoskeletons
After treatment with Triton, the coverslips, centrifuge discs or electron microscope grids were washed twice in PIPES buffer and dipped for 90s in each of the following ice-cold solutions (Small 8r Sobieszek, 1977; Small & Celis, 197%): (1) 60 mM-KCI. 1 m&EDTA, 2 mM-EGTA. 1 mM-cysteine, 10 rnM-ATP, 40 mM-imidazole, pH 7.1 (L); (2) 0% M-KU, 1 rnhfEDTA, 2 mM-ATP, 1 mM-cysteine, 40 mM-imidazole, pH 7.1 (H); (3) as for step (1). The coverslips and centrifuge discs were then rinsed in the PIPES buffer and transferred to lysis buffer (O’Farrell, 1975). The electron microscope grids were fixed in 25O, glut,araldehyde in PIPES buffer for 10 to 30 min before processing for electron microscopy. These skeletons, extracted in high and low salt will be referred to as L-H-L cyt,oskeletons. For the more complete extraction of actin from these cytoskeletons. an additional treatment was employed using the actin depolymrrizing protein purified from slime mould (Hinssen, 1979). The purified protein, kindly provided by Dr H. Hinssen. was diluted to between @3 and 0.4 mg/ml in PIPES csytoskeleton buffer plus 2 rnM-CaCl, and then incubated with either the Triton or L-H-L cyt,oskeletons for 30 min at room temperature. The (*ells attached to the coverslip were then rinsed in buffer and processed either for elecatron microscopy or gel electrophoresis as desc,ribrd above. (h) Tuwdimunsiowl
qrl c1rctrophorr.G
The procedures followed have been described elsewhere (C)‘Farrell, 1975; O’Farrell et a/.. 1977; Fey rt al., 1981). Special note should be made. however, that currently available ampholytes can give very variable and sometimes unsatisfactory results. It was therefore necessary t)o check ampholyt,es very carefully and to make optimal mixtures to ensure satisfac%ory and reproducible separation over the whole pH range. The first dimension separations were performed either as (1) acidic isoelectrofocusing gels cont,aining 2”,, (IEF. 18 h at 400V) in 130mmx 12 mm 4”,, (w/v) polyacrylamide ampholines (IW, pH 5-T (LKB. Sweden): 0.4”,, pH 3.5-10 (LKB)) or (2) basic noneyuilibrium pH gradient electrophoresis (NEPHGE. 4.5 h at 400V) in 130 mm x I.2 mm 4!,, (w/v) polyacrylamide gels containing 1”, ampholines (l’?(~ pH 7-9 (LKB): 19, pH 8-9.5 (LKB)). Bot.h IEF and NEPHGE gels were immersed in solution 0 (@06 M-Tris (pH 68), 4O,, and incubated at room (w/v) SDS, loo,, (v/v) mercapt,oet,hanol, dW, (v/v) glycerol) temperature for 20 min before applying to the second SDS/polyacrylamide dimension. Gels to be stored frozen were incubated for I5 min only and the incubation completed while drfrost,ing. The second dimension SDS/pol?;acr,vlamide gcxl electrophoretic separations were’ ac*complished by laying the equilibriated gels ont,o a stacking gel (4,75?, (w/r) acrylamide. O~24~~,,(w/v) bisacrylamide, OIC)b (w/r) SDS. 0-375 MmTris. HCI, pH 6%). A 14,,, (w/v) agarose blue secured the gel in gel made up in solution 0 and containing 0.01 O(, (w/v) bromophenol place. Electrophoresis was performed at, room temperat,ure for 14 h at 11 to 13 mA or until the tracking dye reached 1 cm from the bottom. The slab measured I7 rrn x I4 cm. Polypeptides (IEF, NEPHGE) were numbered from the top of the first dimension and from the highest molecular weight. To define the position of a given polypeptjide, we designate each spot, by its molecular weight followed by its relative mobility with respect) to /3-a&n (IEF) or the NEPHGE spot 9 (NEPHGE). IVe found the latter measurement easier and more reproducible than t,he actual measurement of pH. as there is great discrepancy among the reported pl values of several cnellular polypeptides (Petersen &. McConkey. 1976: Strand Hr August, 1978: Storti ef al., 1978: Krpstal rt al., 1978; Milcarek h Zahn, 1978: Bravo & Knowland, 1979: Garrels, 1979). The gels were processed for fluorograqh? as described by Laskey & Mills (1975). Approximate]\, 200,000 trichloroacetica-prec~p~table cts/min were routinely applied per gel. for 12 h in lOl$, (v/v) Bio-solv For quantitation. the spots were cut out. resuspended solubilizer BBS-3 (Beckman, USA) in toluene-based scintillator and counted for 10 min. The maximum variability for the spots quantitated in this study was determined by counting gel spots from at least 4 independent samples.
P(Tptide maps of the cytoskeletal proteins were obtained by srtbjec,ting individual spots. vut from the 2-dimensional gels. to a second SDS/polyac~rylamide gel electrophoresis in the prewnw of the Stnphylo~occ~s awws V8 prot,ease (Cleveland P/ nl.. 1977 : Fey pf al.. 1981 ). l’\~o-dinrellsional gels were run as described above using loadings of up to 40 x IO’ c.ts/min: this permitted peptide analysis of proteins romprising down to WOI”,, of tht> total wllular proteins. The 2--dim&onal gels were fixed, dried and left for autol,adioyraph\- at room temperature overnight. Appropriate spots were cut from the drietl pt4. swollen in &tilled water and mashed between 2 piwes of clear plastic foil. The samplrs wcrtb thrn taken up in load buffer (012.5 M-Tris. HCI (pH 6%). 03’?‘,, (a/v) SDS. %?P(),,(w/v) I)tlxtran T .iOOand O.Ol(!, (w/v) bromophenol blue) and boiled for !i min before appliration to a I.‘,“,, (rv,‘r) polyaerylamide gel containing SDS (1.2 mm thick, acrylamide to hisacvylamidr~ ratio. 30 : 0.8), and overlayed with a further .5~1 of load buffer containing IO pp S. C(~WPW~S VX ~)rotraw/ml. Ele&rophoresis was c*arrietl out at 15 mA for I2 h using the Laemmli buffi~r system (Lat~mmli, 1970). The gels were processed for fluorography as described abovch (Laskey k Mills. 197;5), except that the last water wash was replaced by a wash in so,, (v/r) gl!~cvrol.
(‘tills grown on silver hexagonal electron microscope grids (Small 8: Delis. 1978a.h) ww ~~ro~vss~d for elec,tron microscopy either after the above extraction procedures or just fi)llo\ving thcb initial t.reatment with Triton .KlOO. Following the fixation step. in each casv thcb gricls bvcrc’ rrmored individually from the coverslips and negatively stained in thr c~)l(l w,ith aqueous I ‘),I (\v/v) uranyl acetate or at room temperature with 3”,, (w/v) sodium qilicotutlpstatt,. Wectron rniwowop>, was carried out in a Zeiss IO.4 operating at 80 kY.
3. Results
For whole mount preparations of’ HeI,a (*ells prepared for elrvtron rniwosvop~~ (SW Jlatwi& and AMethods). the distribution of filamentous cwmponents wuld by d-rrsolvecl only in cells that had sprea.d reasondAy well on the support film. In sucah WI/S. af’tw extraction with Triton both miwotubules and actin filaments c~)ukl Iw watlily visualized. the latter mainly owurring in hundIes or sheets of’ parallel filwtnents (Triton cytoskeletons). The actin filament huntlles. or sheds. c.hilr.Rc.trristic.~ll~ formed small paralld arrays tangential to the cdl nwlew (Fig, I (a)). As in other cultured cells ((‘elis et nl.. 1HiHrr : Small Rr (‘elis. lW80: Small vt t/l.. I!)xo). the concave (~11 edges \\x’re delimited l)y bundles of actin filaments. while t hr spreacling parts of the carIt perimeter \vtw wmpos~l of’ a tarninar cliaponat meshwork of actin filaments containing variabk numbers of radiating mic*rorilli. or microspikes (Fig. 1(a) anti (b)). The microspikes \vere sometimes notic*eabty long. extending up to many mirrometres and often being associated at their tips with nvighbouring cells. ~licwdnbules ivere numerous a.nd occurred at random. curved or’ straight t~lvmrnts or in parallel arra,ys. Ihe latter sometimes ext’ending in width from close to the. cell Iwripher?~ to the perinuclear region (not shown). The microtubules showed IIO pr&~rwcl orientation to the adin filament. bundles. alt,hough the? cwuld bc*
iwoDIFrc.~TIo~
0~ (~YT~SKEI,ETA~.
PoI,YPEPTII)bX
127
found t.ogether with actin. running in parallel groups. The third component of the cytoskeleton. t’he intermediate-sized filaments, could also be recognized t’ogether with microtubules and actin filaments (not shown). However, in cytoskelet’ons extracted only with Triton, the number of these filament,s seen in different regions of the cytoplasm was extremely variable. (ii) Intermediate jilament enriched cytoskeletons: L-H-L cyto,skeletons \Z’e showed earlier (Small & Celis, 19783) that the further extraction of Triton cytoskeletons with solut)ions of high and low salt served to remove both the microtubules and the bulk of actin and, as a result, revealed the extent of the int,ermediate filament net (L-H-L cytoskelet,on). The results of such extractions with HeLa cells are shown in Figure 2(a) and (b). As in other cultured cells (for a review. see Lazarides, 1980), the intermediate-sized filaments were seen to be mainly concentrated around the cell nucleus and became progressively depleted in number towards the cell periphery. The filaments were generally coiled and frecl ends could not be detected. A residual number of act)in filaments could also be recognized in the 1,-H-l, cytoskeleton mainly occurring at or close to the cell periphery (Fig. 2(a) and (b)). These appeared to correspond in the main to remnants of the actin bundles and meshwork found in normal cytoskeletons t,o delimit the cell perimeter. The caoexistence of the two types of filament in extracted cgtoskeletons correlated with the elrctrophoretic analyses described in the following sections. Essentially the same result was obtained with 1,-H-I, evtoskeletons of enucleated cells (rytoplasts : rcbsults not, shown: Small & Celis, 19786). By the further t’reatment of the 1,-H-L cytoskeletons with t,he actin modulatw protein of slime mould (Hinssen. 1979), all visible traces of F-a&in filaments were removed, as judged by electron microscopy (Fig. 2(c) and (d)). Triton cytoskelet,ons treated directly with the actin depolymerising prot’ein were also visibly deplet,ed of’ both actin and microtubules (the latter removed by the necessary inclusion of C”a2+ in the buffer) but retained a large complement, of their ribosomes attached to the LO nm filament net,.
Figure 3(a) shows a two-dimensional gel electrophoretic pattern (IEF and XEPHGE) of total [35S]methionine-labe11ed polgpept’ides from about IO0 asynchronous HeLa cells: 855 acidic (IEF) and 314 basics (NEPHGE) polypeptides
h:. 1. (a) IClectron micrograph of Triton cytoskeleton of a well-spread HeLa cell showing actin filament bundles and peripheral lamella regions. Microtubules and intermediate filaments are also present in such cytoskeletons but are not resolvable at this magnification. Negatively stained with wanyl acetate. Magnification 2250x. (b) E:nlargement of peripheral region of HeLa cell Triton ytoskeleton showing a microspike bundle of F-actin and the associated meshwork of artin tilamrnt,s. NegativeI>- stained w-ith sodium siiic:otungstat.e. Magnification 65.OOox
118
R.
BRAVO
ET
.-II,
MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
129
can be detected in overexposed fluorograms (Bravo et al., 1981a) but of these, only a tot,al of 450 can be seen in the photograph. The films have been exposed so as to keep the intensity of the spots within the linear range of the film response. For reference purposes and to maintain continuity with our previous work, we have used the numbering system described by Bravo et al. (1981a). An important proportion of the major cellular polypeptides labelled with [ 358]methionine are clustered in a region of the gels between M, 35,000 and 70,000 (Bravo & Celis, 198Ou,6; Bravo et al., 1981a). Of these the major polypeptide is actin. which shows the normal @ (IEF 47) and y (IEF 47a) variants described by others. Spots 19 plus 19b and 25 correspond to in and j+tubulin respectively; 8 to aactinin and 26 to a major component of the intermediate filaments (vimentin, Franke et al., 1978a). The percentages and co-ordinates of some of these proteins have been determined (Bravo et al., 1981a).t Polypeptides whose numbers have been underlined (IEF 12, 24, 26 (vimentin), 31, 35, 36 and 46; termed L-H-L cytoplast polypeptides) will be followed throughout this study as they correspond to the most abundant and insoluble polypeptides present in t,he cytoskeleton (see also Table 1). (ii) Triton
cytoskeletons Figure 3(b) illustrates the composition of the cytoskeleton-associated polypeptides from asynchronous HeLa cells treated with 010, (v/v) Trit’on X-100. The Triton cptoskeleton corresponding to that illustrated in Figure 1 contained about 90 polypeptides (48 basic and 42 acidic) and about 307; of the total counts present in whole cells. Spots indicated with large arrows in Figure 3(b) correspond to polypeptides that are mainly present in cytoskeletons (600/, or more), while t’hose indicated with small arrows are present in about equal amounts in the Triton supernatant and in the cytoskeleton. Even though 300/b of IY and /3-tubulin remain in t’he cytoskeleton, they are indicated with large arrows as they are part of the known cytoarchitectural structure. About 52% of total actin and 41% of a-actinin remain in the cytoskeletons. The percentages of the L-H-L cytoplast polypeptides present in these cytoskeletons are given in Table 1. (iii) Irrtermediate $Zument enriched cytoskeletons: L-H-L cytoskezeton We have already shown (Fig. 2; Small & Celis, 1978b) that the further extraction of Triton cytoskeletons with cold buffers of high and low ionic strength under conditions in which microtubules and most of the microfilaments are dissociated leaves a cytoskeleton (L-H-L cytoskeleton) that remains attached to the substrate and that is composed of the nucleus, a prominent web of intermediate-sized VI{:. 2. (a) and (b) Triton cytoskeletons after extraction in high salt: L-H-L cytoskeletons. The main part of the cytoplasm is composed of intermediate filaments (if). Residual actin filament bundles (mf) occur at the cell periphery. Uranyl acetate staining. Magnification: (a) 2340 x ; (b) 17,000 x (c) and (d) Triton 1,-H-I, cytoskeletons treated additionally with the actin modulator protein from slime mould. Actin filament, bundles and single actin filaments are removed. and only intermediate filaments remain. I!ranyI acetate staining. Magnification: (c) 3200 x : (d) 79.000 x t The percentages of the major HeLa cells proteins labelled with a mixture of 16 [“C].labelled aminoacids has now been submitted for publication to Clinical Chemistry (R. Bravo & J. E. Celis).
130
R. BRAVO
e---
NEPHGE
IEF
ET
AL.
w
(a)
92.5 69 55 43
30
PK. 3. (a) Two-dimensional gel electrophoresis of total [ s%]methionine-labelled polypeptides from 100 asynchronous HeLa cells (IEF and NEPHGE). The gel has been exposed so as to keep the intensit> ofthe’spots within the linear range of the film response. Spots were numbered starting from the top of the first dimension and from the highest molecular weight. Only the spots indicated with large numbers hare been quantitated in this study. at. m-tubulin : j3t, j%tubulin ; v, vimentin. fl and yactin are the major polypeptides species in HeLa cells. (b) Two-dimensional gel electrophoresis of ] 358]methionine-labelled from Triton cytoskeletons from asynchronous HeLa cells.
:
0
48.5/0.74 435/090
98
90 80 90 90
34
99 90 90 89 87 74
46 75 70 60 60
32
43 35
Cytoplasts
L’nknown Vimentin (_:nknown I’nknown 1Tnknown rnknown
41
cytoplnst
54 25 30 40 40 2
65
Constant Constant Constant
I)ecreases§ Constant
Constant Constant
Relative proportion during cell cycle$
polypeptides
Karyoplsstst
O/J Distribution
Vnknown
Identit?
%, in Triton cytoskeleton Asynchronous Mitotic cells cells
of L-H-L
1
ml (unknown) m2. m3 (phosphorylations) m4. m5 (phosphorylations) m6. m7 and m8 (phosphorylations)
Modified forms during mitosis (nature of modification)
t From Bravo el al. (19816). : From Bravo 8: Celis (1978). Only polypeptides whose relative proportion changed by g-fold or more were considered as variable in that study (based on 30 min labellings). § The decrease observed could be due in part to modification. The quantitations are based on the analysis of at least 4 independent samples.
49/1.18
36 46
56/097 54/1.19 50/O%
68/0.55
31 35
24 26
12
IF’P1
Polypeptide
Molecular weight ( x 10-q/ mobility relative to fi-actin
Some properties
TABLE
132
R. BRAVO +--
NEPHGE
ET
.-LL
IEF ---+
-69
-55 -43
-30
? 0 x $
*69 .55 -43
,30
FIG. 4. Acidic (IEF) and basic (NEPHGE) [35S]methionine-labelled polypeptides present in HeLa I,H-L cytoskeletons from (a) whole cells and (b) cytoplasts. Polypeptides indicated with small arrows and small numbers are present in variable amounts in these cytoskeletons. Spots indicated with an asterisk in (a) correspond to polypeptides that are mainly present in karyoplasts.
filaments and some residual, mainly peripheral actin. Electrophoretic analysis (Fig. 4(a)), shows that several of the major [ 35S]methionine-labelled polgpeptides remain in the L-H-L cytoskeletons. Beside the 1,-H-L cy%oplast polypept,ides. these cytoskeletons contain a sizeable amount’ of actin together with IEF polypept’ides 13. 13e. 33,38, as well as NEPHGE polypept,ides 3,6,8,1ls. 2lh and 23s. There are also other acidic and basic polypeptides that are present in variable amounts. examples being indicated with small arrows and numbers in Figure 4(a). Essentially the same patterns were obtained if the gels were stained with Coomassie blue (not, shown).
>MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
133
Enucleation of asynchronous cells prelabelled for 20 hours with [ 35SJmethioninc~ (see Materials and Methods) followed by exbraction with 0.194 (v/v) Triton S-ICH) and buffers of high and low salt concentration (L-H-L) showed that polypeptides IEF 13, 13e, 33, 38 and NEPHGE 3, 6 and 9 are mainly of karyoplastic origin (indicated with an asterisk in Fig. 4(a) and (b); Bravo et al., 1981b). Finally, analysis of the L-H-L cytoskeletons depleted of actin (Fig. 2(c) and (d)) using the actin depolymerizing protein from slime mould (Hinssen, 1979) showed the almost complete removal of actin, leaving the proportion of the L-H-L cytoplast polypeptides unchanged (Fig. 5(a); see also Fig. 4(a)). This has been further supported by quantitation of the spots (not given) and analysis of the supernatant. (Fig. 5(b)). IEF
---+-
IEF -
(0)
(b)
FIG. 5. (a) Acidic (IEF) polypeptides present in HeLa L-H-L cytoskeletons treated with the a&in depolymerising protein from slime mould (Hinssen, 1979). (b) Polypeptides present in the supernatants.
the substrate-attached. intermediat,e filament enriched To summarize. +oskeletons are composed of seven abundant polypeptides. which have been termed 1,-H-L cytoplast polypeptides. The ratio of these polypept,ides to total acids actin, as determined from labelling with a mixture of 16’ “C-labelled-amino are t.he following: IEF 12, @08; IEF 24, @14; IEF 26 (vimentin), 0.195; IEF 31, 0.02 : IEF 35.0.04 ; IEF 36,002 : and IEF 46,0.0.5. There are also basic polypeptides present in these cytoskeletons but these have not been investigated further. Similar studies by Franke et al. (1979) using one-dimensional gel electrophoresis have revealed four polypeptides besides t,otal actin in HeLa cells treated with 19,, (v/v) Triton X-100 and extracted with 1.5 M-KCI. The four polypeptide bands had a,pparent molecular weights of 57,000, 52,000, 48,000 and 46,000. (iv) I’eptidr analysis of L-H-L cytoplast polypeptides Having identified the major polypeptides that are present in the intermediate filament enriched. substrate-attached cytoskeletons, it was necessary to establish if they were all distinct or were related in peptide composition. Accordingly, onedimensional pept,ide analysis of the individual [3sS]methionine labelled spots recovered from two-dimensional gels was undertaken. the results of which are 5’
13-l
R. BRAVO
ET
AL
shown in Figure 6. As can be seen from this analysis, and given the molecular weight. and charge differences, t,here is no obvious homology between IEF spots 12. 24. 26 (vimentin), 31, 35, 36 and 46. (c) ModiJication
of L-H-L
cytoplast
polypeptides
during
mitosis
Recently. we showed that the relative proportion of a few polypeptides changes signiticantly- during mitosis (Bravo & Delis. 1980a). In Figure 7 we show enlargements of selected areas of IEF gels from mit.otic HeLa cells labelled for (a)
12
242631
35364647
PK. 6. One-dimensional peptide maps of I.-H-I, cytoplast polypeptides from HeLa cells. [35Slmethionine-labelled polypeptides were digested with S. r~z(teus V8 protease as described in Mabrials and Methods. The peptide map of actin (IEF 47) has been included as a reference.
MODIFICATION IEF :: 0-J
OF (:YTOSKELETAL IEF -
a
POLYPEPTIDES
)_
x
(a)
I
- 55 -43
FIG. i. Two-dimensional gel electrophoresis (IEF) of [35S]methionine-labelled polypeptides from mitotic- HeLa cells lahelled for (a) 10. (b) 22, (c) 30. (d) 35, (e) 40. and (f) 90min after collection. Polypeptides indicated by the letter m correspond to those whose relative proportion increases preferentially during mitosis. Only a fraction of the gel is shown. at and fit indicate a and fl-tubulin. respectiveI!
IO. (b) 22. (P) 30, (d) 35, (e) 40 and (f) 90 minutes after collection. The polypeptides that appear preferentially during mitosis are indicated by the letter m, followed by a number and. for convenience. t,hey have been termed mitosis spec*ific polypeptides. All these polypeptides occur transiently and none of t’hem can be detect,ed. at least at comparable intensities. in long-term labellings: that is. when the cells have already reached G,(Fig. 7(f)). The larger number of mitosis specific polypeptides detect’ed in this study as compared to our previous work (Bravo & (‘rlis. 198)cr) is due to the shorter labelling time of t)en minutes included in this study. With the exception of m2 (also called 26e: Bravo rt nl.. 1981c), which can be tlrtected in significant amounts in synchronized 6, cells (Fig. 8: Bravo & (Delis.
IEF
-
IEF
-
FK. 8. Two-dimensional gel electrophoresis (TEF) of [‘5S]methionine-labelled polypeptides from G ,. S and G, HeLa cells labelled for 80 min. m2 has also been called IEF 26e in the text. Only a fraction of the gel is shown. at and ,9t indicate a and fl-t,ubulin. respectively.
IEF -
-30
-43
-55
-69
:
0 T s-
MOIJIF’ICATIO~
OF (:YTOSKELETAL
POLYPEPTIDES
1x
198oU) and in lesser but detectable amounts in G, and S cells (Fig. 8). the other mitosis specific polypeptides can be det,ected only at vet-y low levels in I,& frotn interphase cells (Fig. 8). Similar results are obtained if the synchronized celis are labelled for ten minutes (not shown). The relative proportion of a few polypeptides has been shown to change in mitosis and G2? and these correspond to IEF 30, rd, (IEF 32). vd3 (IEF41), vd, (IEF44i) and vd, (IEF47m: Figs 7 and 8: Bravo Kr (‘elk. 198&L; Bravo et a.Z., 1981a). Since some of the mitosis specific polypeptides exhibit the same molecular weight and migrat.e closely to some of the L-H-L cytoplast polypept.ides, we determined their one-dimensional peptide maps in order t,o search for possible homologies. As shown in Figure !I, ml is related to IEF 24; m:! is related to vimentin: and IEF 37. m6 and m7 are related to IEF 36. We have previously reported that the relative proportion of IEF 37, which occurs in significant amounts in interphase, increases during mitosis (Bravo & Celis, 198On). The peptide map studies also revealed that m3 and m4 are related to IEF 31 (not shown), and that vd, (IEF 3P), vdJ (IEF 41).
24
ml
36 37 m6 m7 from mitotic cells. [?3] methionine-labelled were digested with S. aweus 1’8 protease as described in Materials and Methods. 26
m2
vdi
vd3
vds vd5
FIG. 9. One-dimensional peptide maps of polypeptides polypeptides
138
R. BRAVO
BT
AI,
WI, (IEF 44i) and vd, (IEF 47m) correspond to degradation products of vimentin (f(‘ig. 9). Since all the mitotic specific polypeptides migrat,ed at t’he acidic side of the corresponding spot. we labelled the mitotic cells for 15 minutes with [32P]orthophosphate t,o determine if they corresponded to phosphorylations. An enlargement of a selected area of the IEF gel is shown in Figure 10. These studies indicated that m2. m3, m4, m5. m6, m7 and m8 are phosphoproteins. I RF‘ 12. 24. m 1, vimentin. 31,35 and 36 are not det)ect,ably phosphorylated. Similar labellings of G, and asynchronous cells (Fig. 10) indicated that IEF 36 is phosphorylated in interphase to yield IEF 37. in agreement with the /35S]methionine labellings. which indicated t,hat there is a subst,antial amount of this prot.ein in int,erphase (Fig. 8). Only small amounts of phosphorylated mti and 1117can be detected in G,. while m”. rr13. m-C and m5 arc barely detected in the background (Fig. 10). In mit’osis. IE:F 37 is further phosphorylated to yield m6. m7 and m8. A few major phosphorylated polypeptides whose intensity increases in mitosis are indicated with small arrows in Figure 10. Many of these are also found in asynchronous ~11s but are barely dektable in C:, Also. one can detect phosphorylated polypeptides in asynchronous cells (arrowheads) that do not seem to be present in mitosis: these may correspond to modificat,ions occurring in the other phases of t,he cycble. A detailed study of t’he phosphorylated polypeptides of HeLa cells is now being made. The nature of the IEF 24 modification is unknown. Mitotic Triton cytoskeletons cont.ained, in addit,ion to the 1,-H-L cytoplast polypeptides, the polypeptides ml. m2. m3. m4. m5, mfi and m7 (Fig. 1 l(a)). These cytoskeletons contained about 90($, of vimentin, as compared with 99?,, normally found in cgtoskeletons from asynchronous cells (Table 1 : see also Fig. I l(b) and (c) for a comparison of the Triton supernat,ants). The relative proportion of the other L-H-L cytoplast polypept,ides in the mitotic Triton cytoskeleton was very similar to that observed in asynchronous cells, although small but significant, differences were observed in the cases of IEF 31 and 36 (Table 1).
4. Discussion The high sensitivity of the two-dimensional gel electrophoresis technique when used with ] 3sR]methionine-labelled polypeptides has enabled us to screen relatively small numbers of substrate-attached cultured cells without the need to resort to cell concentration via scraping procedures. Apart from being considerably more convenient, this approach has allowed a direct correlation between features of the cytoskeleton as seen in the electron microscope and their polypeptide composition. In this study we have focused our attention on the most insoluble polypeptides of the cytoskeleton. some of which show interesting changes in the cell cycle (Bravo & Celis, 198Ou). In tracing the polypeptides associated with the intermediat’e filament enriched cytoskeletons, the analysis of enucleated a,nd artin depleted preparations proved particularly useful. Seven abundant polypeptides (termed L-H-I, cytoplast polypeptides) emerged as t,he main components of these cyt,oskeletons (Table 1). The larger number of protein components found in these cytoskeletons as compared
E P
kl H
R. BRAVO
IEF
ET Al,
-
FIG. 11. Two-dimensional gel electrophoresis (IEF) of ~J58Jmethionine-labelled polypptides from (a) HeLa mitotic Triton cytoskeletons; (b) HeLa mitotic Triton supernatants; and (c) Triton supernatants from asynchronous HeLa cells.
MODIFICATION
OF CYTOSKELETAL
POLYPEPTIDES
141
to previous studies reported by Franke et al. (1979), is most likely due to the different extraction procedures used and to the higher resolving power of the twodimensional gel electrophoretic method. 80 far. only vimentin (IEF 26) has been characterized (see also Franke et al.. 1979), while the identity of the other polypeptides remains unknown. Preliminary indicate that IEF 36 and 46 may correspond to experiments. however, cytokeratins, as these proteins migrate very close to PTK 2 cytokeratins (Franke ef al., 19786) and exhibit similar one-dimensional peptide maps (not shown). Further experiments, however, are needed to assess the identity of these proteins. We cannot exclude the possibility that IEF 31 may also correspond to a cytokerat’in but this polypeptide is different to skeletin (see also Lazarides, 1980). Init’ial experiments using antibodies raised against IEF31 showed that this antibody stains a filament, network similar to that of the intermediate filaments. It is not known whether IEF 12, 24 and 35 are an integral part of the intermediate filament network or whether they represent accessory proteins that may function in interfilament or filament-organelle interactions. The possibility that these proteins may not be associated with the intermediate filaments must also be considered. Experiments are now under way to raise antibodies against these polypeptides in an effort to determine their location in the cell. These polypeptides are clearly different to vimentin, as judged by one-dimensional peptide analysis (Fig. 6). IEF 12 and 24 are far easier to extract than the rest of the L-H-L cytoplast polypeptides (Table l), a fact that may explain why these polypeptides have not been described before in HeLa cells. A polypeptide with similar properties to IEF 12 has been described by Lehto et al. (1978) in human embryonic skin fibroblasts and by Wang et al. (1980) in non-neuronal cells and skeletal myofibrils. IEF 12. 24 and 35 are abundant polypept’ides in HeLa cells and together with vimentin (IEF 26) make up a polypeptide “quartet” that is widespread in many cell lines and primary cultures from hamster, human, mouse and rat origin as well as mouse tissues (Fey et ccl., 1981: Bravo et al.. 1981c,d). Analysis of the polypeptide pattern of a single Sarcoma 180 cell showed that these polypeptides coexist in a single cell (Bravo et al., 1981c). Studies of mitotic as well as of interphase cells showed that IEF 24, 26 (vimentin), 31 and 36 (via IEF 37) are modified preferentially during mitosis (Table 1). Vimentin is phosphorylated transiently during mitosis to yield polypeptides m2 and m3 (Table 1). Polypeptide m2 can also be detected in small amounts in interphase cells but its intensity is strongest in short-term labelled mitotic cells. Since most of the mitotic cells collected by shaking are in metaphase (66’+,). it is likely that these modifications take place in or very near to this phase of the mitotic cycle. We have observed a similar increase in the relative proportion of IEF m2 and m3 in mitotic 3T3B cells (Bravo & Celis, 19806). We do not know whether preferential phosphorylation during mitosis is a general feature of other cells : Cabral & Gottesmann (1979) have reported a minor phosphorylated form of vimentin in asynchronous Chinese hamster ovary cells that does not seem to increase in mitosis. Also, O’Connor et al. (1979) have reported phosphorylation of vimentin in chicken muscle and in non-muscle cells, but they have not determined the phase of the cycle in which the modification occurs. Furthermore, Cabbiani et
142
It.
BRAVO
ET AL
al. (1981) have recently described two isoelectric variants of vimentin in vascular smooth muscle cells. the less acidic variant of whirh comigrates with purified vimentin from different cell lines. Of interest is the appearance of the vimentin degradation products at a certain t,ime during mitosis. In interphase, the intensity of these polypeptides is strongest in (:, (Bravo & Crelis, 1980a), and can barely be detect,ed in G, or S. Based on the analysis of many hundreds of gels from HeLa and other cells, we believe that these polypeptides are not’ the product of non-specific degradation but rather correspond to some important event, in the process of cell division. A similar set of polypept,ides. called diagonal proteins, have been described by OYlonnor ef ~1. (1979) in myogenic cell cultures. The modification of IEF 36 is most interest.ing as IEF 37 is the major phosphorylated form of this protein observed in interphase. In mitosis. IEF 37 is furt)her phosphorylated to yield m6. m7 and m8. Small amounts of these phosphorylated polypeptides can be observed in interphase cells but their amount increases dramatically in mitosis. We have observed other phosphorylated polypeptides that occur mainly in mitosis (Fig. 10) but their identity is unknown. The nature of the modificat,ion of IEF 24 has not been established. Even though it is yet) t,oo early to envisage a mechanism by means of which these modificat,ions might regulate t,he stage of assembly of these cyt’oskeletal polypeptides, it seems relevant’ to not,e the small but significant increase of viment,in in the Triton supernatant of mit,otic cells (loo/A). Careful examination of t,he fluorograms of IEF gels from T&on supernatants indicates t,hat it is viment.in and not phosphovimentin (m2) that is extractable. Since most of the vimentin remains in the cytoskeleton during mitosis (Fig. 1 l(a) ; Table 1 ), however, it is likely that the extracted fraction may reflect changes in a second component that interacts with vimentin and that disassembles during mitosis. Since rearrangements in this 10 nm filament net (Blose, 1979: Hynes & Dest’ree, 1978; Aubin et al.. 1980) clearly accompany the other major changes in mit,osis, it is possible that the polypeptide modifications observed are instrumental in assuring at least part of the process of cell division. We would like to thank A. Celis, G. Langanger and P. Jertschin for expert assistance, and 0. Jensen and B. Thornsen for photography. One author (R.B.) is a recipient of a fellowship from the Medical and Natural Science Research Councils, and another (S.J.F.) is a recipient of a European Molecular Biology Organization long-term fellowship. This work was supported by grants from Euratom, the Danish Medical and Natural Science Research Council
(to J.E.C.)
and the Muscular
Dystrophy
Association
(to J.V.S.).
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by IV. Franke