Characterization of a heat shock-induced insoluble complex in the nuclei

of cells

T. D. LITTLEWOOD*, D. C. HANCOCK and G. I. EVAN

Liidwig Institute for Cancer Research, MRC Centre, Hills Road, Cambndge CB2 2QH, England

Summary

The formation of an insoluble complex in iso-lated nuclei incubated at physiological tempera-ture (37°C) is demonstrated. A similar complex isshown to form in the nuclei of intact cells sub-jected to temperatures that induce the classicalheat-shock response. The formation of this com-plex occurs rapidly in response to hyperthermiaand is induced by small increases in temperature

both in vitro and in vivo. We have characterizedthe formation of the complex in isolated nucleiand the nuclei of intact cells. A small number ofthe subset of nuclear proteins involved in thecomplex have been identified. The significance ofthe loss of solubility of these proteins in thenucleus following hyperthermia is discussed.

Key words: heat shock, insoluble complex, p62c"mvir.

Introduction

The nuclear matrix is thought to be a major structuralcomponent of nuclei (Berezney & Coffey, 1977; Capcoet al. 1982). Several nuclear functions such as DNAreplication and transcription have been reported to beassociated with the nuclear matrix (see review byHancock & Boulikas, 1982), which is isolated fromnuclei by exhaustive digestion with nuclease followedby extraction of the soluble chromatin fraction withhigh-salt solutions or polyanion-containing buffers(Lebkowski & Laemmli, 1982-6). The residual insol-uble structure is the nuclear matrix, although itsprecise composition varies according to the methodof preparation and source of nuclei. Lebkowski &Laemmli (1982a,b) have described the preparation oftwo distinct nuclear matrix structures. Type I struc-tures obtained by extraction of isolated nuclei withbuffers containing 2 M salt or the polyanions dextransulphate/heparin retain 10-15% of the total nuclearproteins, including the three nuclear lamins and severalhigh molecular weight components. Type II matricesprepared in the presence of reducing agents such as2-mercaptoethanol consist almost exclusively of thenuclear lamins. Type I matrix structures could bestabilized (i.e. resistant to the effect of 2-mercapto-ethanol) if metal-depleted nuclei were exposed to aslittle as 1 X 10~6M-CaCl2 at 37 °C (Lebkowski &Laemmli, 1982a). Mirkovitch et al. (1984), employing

Journal of Cell Science 88, 65-72 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

a low-salt extraction method, subsequently indicatedthat incubation of isolated nuclei at 37 °C alone issufficient to stabilize the nuclei and obtain type Istructures.

We have previously described a change, in the appar-ent nature of the nuclear matrix in isolated nucleiexposed to temperatures above 35 °C (Evan & Han-cock, 1985). A subset of nuclear proteins that aresoluble in isolated nuclei prepared at 4°C formed ahigh-salt-insoluble complex if the nuclei were sub-jected briefly to temperatures above 35 °C. Further-more, we showed that the products of certain trans-forming genes, notably p62c""-v<r, p58v-"'vf and p45v-'"-v6,were components of that insoluble complex. Thepresence of 2-mercaptoethanol in the extraction bufferhad no effect on the number of components detectablein the insoluble complex of heated nuclei. Thus, thecomplex that forms in response to heat shock does notinvolve cross-linking by disulphide bonds.

Preliminary data (Evan & Hancock, 1985) suggestedthat the temperature-dependent formation of the insol-uble complex observed in isolated nuclei also occurredin the nuclei of intact cells subjected to heat shock.Clearly, the formation of an insoluble complex in thecell nucleus may have a profound effect on thosenuclear processes, such as DNA replication and tran-scription, which have been attributed to the nuclearmatrix (Jackson & Cook, 1985, 1986). We report herean investigation of the solubility of nuclear proteins in

65

isolated nuclei and intact cells following transienthyperthermia.

Materials and methods

Cell cultureThe Ela-transformed human embryonic kidney cell line 293(Graham et at. 1977) was maintained in Dulbecco's modifiedEagle's medium supplemented with 10% foetal bovineserum, 2mM-L-glutamine, 50 units ml"1 penicillin G and50/xgml"1 streptomycin sulphate. Heat shock of nuclei andcells was carried out in calibrated water baths for the periodsand temperatures indicated in the figure legends. Cells werealso stressed by the addition of 5 mM-L-azetidine-2-carboxylicacid or SOmM-sodium arsenite to the culture medium.

Isolation of nuclei and nuclear matricesNuclei were prepared as described by Lebkowski & Laemmli(19826). Washed nuclei were digested with 50^gml~' deoxy-ribonuclease 1 for 40 min at 4°C, followed by incubation atvarious temperatures. The nuclei were recovered by centrifu-gation at 700£ for 5 min at 4°C and proteins extracted with avariety of buffers for 60 min on ice. Buffers used were asfollows: A, 2M-NaCl, 10mM-Tris- HC1, p H 9 0 , 10mM-EDTA, 1% thiodiglycol, 0 1 % digitonin, OlmM-PMSF(phenylmethylsulphonyl fluoride), 1 % aprotinin (Lebkowski& Laemmli, 19826) supplemented with 140mM-2-mer-captoethanol; B, 2mgml~' dextran sulphate, 02mgml~ 'heparin, lOmM-Tris • HC1 (pH90) , 10mM-EDTA, 0 1 %digitonin, 0-1 mM-PMSF, 1% aprotinin (Lebkowski &Laemmli, 19826); C, 5 mM-Hepes/NaOH, pH7-4, 0-25 mM-spermidine, 2mM-EDTA/KOH, pH7-2, 2mM-KCl, 0 1 %digitonin, 25 mM-lithium 3,5-di-iodo-sahcylic acid (Mirko-vitch et al. 1984); and D, 50mM-NaCl, 25 mM-Tns-HC1,pH8-2, 0-5% NP40, 0-5% sodium deoxycholate, 0 1 %sodium dodecyl sulphate, 0-1 % aprotinin.

The remaining insoluble material was recovered by centri-fugation at 13 000 £ for 5 min at 4CC and resuspended inSDS-PAGE sample buffer (Laemmli, 1970). The super-natant (chromatin fraction) was diluted with an equal volumeof the same buffer.

Gel electroplioresis and immunoblottingAll samples were boiled for 3 min before fractionation by one-dimensional electrophoresis on 10% polyacrylamide-SDSgels (Laemmli, 1970) or by two-dimensional electrophoresisaccording to the procedure described by O'Farrell (1975).Except where indicated each lane contained 107 cell equival-ents. Gels were stained in Coomassie Brilliant Blue.

Electroblotting of proteins onto nitrocellulose paper wascarried out as described (Evan & Hancock, 1985). Each lanecontained 5X106 cell equivalents. p62c"""'c and p53 weredetected by incubation with the appropriate antibody: re-spectively, Mycl-9E10 (Evan el al. 1985) or PAbl22. Themouse monoclonal antibodies were detected with affinity-purified l25l-labelled rabbit anti-mouse Ig (F(ab')2 frag-ment). Filters were exposed to pre-fogged Fuji X-ray film at-70°C.

Recovery of p62c""°'r and p53 was assessed by immuno-precipitation and comparison of trichloroacetic acid-precipi-table counts in various fractions. A total 5X 107 293 cells wereincubated for 60 min in the presence of 2mCi of [ 5S]methio-nine (1130Cimmol~', Amersham). Nuclei were isolated asdescribed above and extracted with buffer A. p62c"'"v'" andp53 were immunoprecipitated with the appropriate antibody,pan-wye (Evan et al. unpublished) or PAbl22 (Gurney et al.1980), as described (Evan et al. 1984), precipitated withtnchloroacetic acid and collected onto GF/C discs.

Results

Effect of thermal stress on isolated nucleiNuclei were isolated from 293 human embryonickidney cells by cell lysis and density gradient centrifu-gation in sucrose-Percoll as described in Materials andmethods. Such nuclear preparations are free of majorcontaminating cytoplasmic components (Lebkowski &Laemmli, 19826). Actin and intermediate filamentproteins such as vimentin and prekeratin are alsoabsent from these preparations (Fig. 1). Type I andtype II nuclear matrices were isolated from such nucleiby exhaustive digestion with deoxyribonuclease 1 at4°C followed by extraction of chromatin-associatedproteins with one of several buffers (see Materials andmethods for details). Fig. 1A shows the result ofextracting nuclease-digested nuclei prepared at either4°C or 37°C. If the extraction is carried out at 4°C with2M-NaCl in the presence of 2-mercaptoethanol, type IInuclear matrices are obtained (Lebkowski & Laemmli,19826). These preparations consist almost exlusively ofthe three nuclear lamins, whereas type I structurescontain additional high molecular weight species (datanot shown). The low-salt extraction method (Mirko-vitch et al. 1984) and extraction with buffer D generatesimilar type I preparations. Nuclei extracted with thepolyanion buffer dextran sulphate/heparin (Lebkowski& Laemmli, 19826) are devoid of most of the laminproteins. When isolated nuclei were incubated at 37°Cfor 15 min prior to extraction, a complex formed thatwas resistant to solubilization with any of the extractionbuffers described, and which comprised a subset ofapproximately 50 proteins (Fig. IB). We refer to thecomplex, which forms in isolated nuclei at tempera-tures above 35 °C, as the in vitro heat-shock complex.Two of these proteins have been identified in appropri-ate cells as p62c-"'-vf (Evan & Hancock, 1985) and p53,both products of cellular transforming genes. Fig. 1shows an immunoblot of the fractions prepared withdifferent extraction buffers probed with monoclonalantibodies directed against p62c"'"-vc or p53. It is clearthat both of these proteins are present in the solublechromatin fraction of nuclei isolated at 4°C. However,both are immobilized in the insoluble complex thatforms when isolated nuclei are subjected to 37°C, and

66 T. D. Littleivood et al.

1 2 3 4 5 6 7 8 B 1 2 3 4 5 6 7 8

p53- —̂ - - - 1 • t 1 •Fig. 1. Extraction of proteins from nuclei by various buffers. Nuclei were prepared from 293 cells, digested with nucleaseat either 4°C (A) or 37°C (B), and extracted with a variety of buffers as described in Materials and methods. Lanes 1, 3, 5,7 are the extractable 'chromatin' fractions (5X106 cell equivalents) and lanes 2, 4, 6, 8 the residual insoluble nuclear matrix.Buffers were as follows: buffer A, lanes 1, 2; buffer B (dextran sulphate/heparin), lanes 3, 4; buffer C (low salt), lanes 5,6; and buffer D (RIPA), lanes 7, 8. Molecular weight markers are myosin (200XlO3^) , /3-galactosidase (116x 103jUr),phosphorylase B (92-5X 103A/r), bovine serum albumin (66x 103A/r) and ovalbumin (45X 103jl/r). The same fractionselectroblotted onto nitrocellulose and probed with monoclonal antibodies directed against p53 and p62c""'-vr are shownbelow.

cannot be extracted from this complex by any of theextraction buffers described above. Indeed, the pres-ence of these two proteins in the insoluble fraction hasproved to be diagnostic of the formation of the in vitroheat-shock complex in the nucleus. Comparison oftrichloroacetic acid-precipitable counts in nuclear frac-tions with the original cell lysate revealed that morethan 97% of p62c""-vc and p53 was recovered in thenuclear fractions (data not shown).

We sought to characterize some of the parametersgoverning the formation of the in vitro heat-shockcomplex. To examine the temperature dependence ofcomplex formation we treated isolated nuclei withnuclease and then incubated them at various tempera-tures prior to extraction with high-salt buffer. Theresults are shown in Fig. 2A. The in vitro heat-shockcomplex only forms at temperatures above 35 °C.Indeed, nuclei can be incubated for extended periods(18 h) at temperatures as high as 30°C without anyinsolubilization occurring (data not shown). The invitro heat-shock complex forms extremely rapidly attemperatures of 35 °C or greater, as shown in Fig. 2B.Within 10 min of incubation at 37°C, p62c""°'f, p53 andseveral other undefined proteins are found in theinsoluble fraction. To date we have been unable todetect any difference in the rates of insolubilization ofthe different components of the complex. Finally, wehave shown that the formation of the in vitro heat-shock complex is independent of the presence of DNA

or RNA. Exhaustive digestion of nuclei with DNase Iprior to extraction with 2 M salt, a procedure thatremoves more than 98% of DNA, or with RNase, hasno effect on formation of the nuclear complex or itscomposition (data not shown).

Characterization of the effect of heat stress on thenuclei of intact cells

The in vitw heat-shock complex described above isinduced by incubation of isolated nuclei at tempera-tures above 35 °C. Clearly, this is below the tempera-ture at which mammalian cells grow in culture and so itis difficult to see how this in vitro complex might berelated to any in vivo phenomenon. However, we notedthe possibility that isolated nuclei might have a some-what different thermosensitivity from nuclei in intactcells. Accordingly, we sought to identify some physio-logical process that was also known to be induced by asmall increase in temperature, albeit a slightly differenttemperature to the one inducing in vitro heat-shockcomplexes in isolated nuclei. When cells of mostspecies are exposed to temperatures a few degreesabove their normal physiological temperature theyundergo gross morphological changes, which are es-pecially profound within the cell nucleus (Pelham,1984). Cells actively respond to such hyperthermicstress by expressing a specific set of genes. This is theclassical 'heat-shock response' and the genes thereby

The nuclear matrix following heat shock 67

induced are the 'heat-shock genes' (for reviews, seeVoellmy, 1984; Schlesinger et al. 1982).

Despite the differing temperature dependence ofclassical heat shock (e.g. 41-43°C for 293 cells) on theone hand and the formation of the in vitro heat-shockComplex on the other (35-36°C), we felt that therewere substantial similarities between the two processes.We therefore investigated the possibility that an insol-uble nuclear complex forms in intact cells after heatshock. Intact 293 cells were subjected to heat shock at43°C for 60min, conditions shown to induce the heat-shock genes in mammalian cells. Nuclei were thenisolated from these heat-shocked cells and digestedwith DNase I at either 4°C or 37°C. The ability of 2M-NaCl buffer (buffer A) to extract proteins from thesenuclei is shown in Fig. 3. A high-salt-resistant com-plex, similar in composition to the in vitro heat-shockcomplex, was found in the nuclei of heat-shocked cells,even if the nuclei were prepared at 4°C, i.e. below thetemperature required to induce in vitro heat-shockcomplex formation in isolated nuclei. Close examin-ation of the proteins in these complexes (Fig. 3, lanes 6and 9; and two-dimensional analysis, not shown)suggests that most, if not all, of those present in the invitro heat-shock complex are involved in the complexfollowing heat shock of intact cells (referred to as the invivo heat-shock complex). The presence of p62c"'"-v<:

A 1 2 3 4 5 6 7 8 9 10

and p53 in an insoluble complex is therefore a consist-ent feature both of isolated nuclei incubated at 37 °Cand of nuclei derived from heat-shocked cells. We havesince found that several other nuclear oncoproteins,such as P750-"1**, p66i;j-"'-vc

) p58v-'"vf, P45V-"W* andSV40 large T antigen, are also sequestered in both invivo and in vitro heat-shock complexes.

In order to compare the in vitro and in vivo heat-shock complexes we examined the temperature depen-dence of in vivo complex formation in 293 cells. Thenuclear complex did not form when these cells weremaintained at 40°C for 4-5 h (data not shown).However, a salt-insoluble complex, containing p53and p62c'"'-vc, formed within 20 min when the cellswere incubated at temperatures higher than 40-5°C(Fig. 4A). Clearly not all of the p53 and p62c-"'-Vf issequestered into the insoluble complex in vivo. Pre-sumably, the extractable component of these proteinsmay bet nascent uncomplexed protein, since synthesisof p53 and p62c"'"-vc continues throughout the earlyperiod of hyperthermia (unpublished observations).

Cells recover from transient hyperthermia if cul-tured at their normal growth temperature. We weretherefore interested in determining whether this recov-ery required, or was correlated with, removal of theinsoluble nuclear complex. Accordingly, we investi-gated the fate of the in vivo heat-shock complex in 293cells during recovery from a 1-h heat shock at 43 °C.

B 1 2 3 4 5 6 7 8 9 10 11 12

p62iC-

Fig. 2. Characterization of the formation of heat-shock complex in isolated nuclei. A. The effect of different incubationtemperatures. Nuclei isolated from 293 cells were incubated at various temperatures for 15 min prior to extraction withbuffer A. The extractable (lanes 1-5) and insoluble (lanes 6—10) fractions are shown: 4°C, lanes 1, 6; 15°C, lanes 2, 7;30°C, lanes 3, 8; 35°C, lanes 4, 9; 37°C, lanes 5, 10. B. The speed of formation of the complex in isolated nuclei at 37°C.Again extractable (lanes, 1-6) and insoluble (lanes 7-12) fractions are shown. Time points are Omin, lanes 1, 7; 5min,lanes 2, 8; 7-5 min, lanes 3, 9; 10 min, lanes 4, 10; 12-5 min, lanes 5, 11; and 15 min, lanes 6, 12. Data for p53 and p62c""'v<r

and p53 correspond to lane 10. Molecular weight markers are the same as in Fig. 1.

68 T. D. Littlevmod et al.

1 2 3 4 5 6 7 8 9

Fig. 3. The effect of heat shock of intact cells on thesolubility of nuclear proteins. Nuclei from 293 cellscultured at 37°C (lanes 1-6) or heat-shocked at 43°C for60min (lanes 7-9) were digested with nuclease at 4°C(lanes 1-3, 7-9) or 37°C (lanes 4-6) followed by proteinextraction with buffer A. Fractions shown are total nuclei(lanes 1, 4, 7), soluble chromatin (lanes 2, 5, 8) andinsoluble matrix (lanes 3, 6, 9). Molecular weight markersare myosin (200X 103jV/r), /3-galactosidase (116x itfM,),phosphorylase B (92-5xl03iV/r), bovine serum albumin(66xlO3Mr) and ovalbumin (45xlO3Mr) on the left andphosphorylase B, bovine serum albumin, ovalbumin andcarbonic anhydrase (i\X\03MT) on the right.

Appreciable levels of p53 and p62c""'-vc were detected inthe soluble fraction (chromatin) within 2-5 h of recov-ery, correlating with the reappearance of matricescharacteristic of nuclei of non-heat-shocked cells (datanot shown). Furthermore, the loss of this insolublecomplex was accompanied by the reappearance ofnormal nuclear morphology (data not shown). Thusrecovery from heat shock correlates well with thedisappearance of the insoluble nuclear complex.

Heat shock is not the only insult capable of inducingheat-shock genes. Treatment of cells with amino acidanalogues, arsenite or hypoxia are also capable ofinducing heat-shock genes (Welch & Feramisco, 1982;Johnston et al. 1980). One possibility we thereforewished to examine was that the formation of a high-salt-insoluble nuclear complex might also be a featureof these insults. We examined the nuclei of 293 cellsexposed to agents such as L-azetidine-2-carboxylic acidand sodium arsenite. Both of these agents inducedinsoluble nuclear complexes in 293 cells (Fig. 5).However, these complexes contained only 10-20 pro-teins (Fig. 5, lanes 2 and 3), not all of which werecomponents of the in vitro and in vivo heat-shockcomplexes described above. Moreover, although p53

was always present in such complexes, p62c*'"-vf wasnot. Thus, formation of an insoluble nuclear complexdoes appear to be a common feature of insults thatinduce heat-shock genes. However, the precise compo-sition of the complex formed differs, depending uponthe agent inducing it.

Discussion

We have characterized the formation of salt-insolublepfotein complexes in isolated nuclei subjected to tem-peratures above 35 °C and in intact cells subjected toclassical heat shock. The heat-inducible nuclear com-plex is resistant to buffers containing a variety of saltconcentrations up to 2 M, polyanion buffers such asdextran sulphate/heparin, non-denaturing detergents,reducing and chelating agents. It is, however, com-pletely solubilized by SDS or urea. Furthermore, the!complex forms at the same rate in isolated nucleiirrespective of the presence of chelating agents, thiolreagents and a variety of divalent cations includingCa2+, Cu2+, Zn2+ and Mg2+ (Evan & Hancock, 1985,and data not shown), implying that disulphide bondsor metal ions are not involved in its formation. Itseems, therefore, that heat-shock complex formation isnot related to the Cu-dependent stabilization of type Imatrix structures described by Lebkowski & Laemmli(19826).

We have identified proteins common to both in vitroand in vivo heat-shock complexes, for example p53 andp62c""°'c. We have observed that, in appropriate celllines, some other nuclear oncoproteins, such as SV40large T antigen, p45v-"'-v6, p58v-"'-vr, p75c-"'-v6, p66N-'"vf

and Ela protein, are also associated (to varying de-grees) with the insoluble complexes formed in intactcells or in isolated nuclei when subjected to hyperther-mia (unpublished data). Both in vitro and in vivocomplexes form extremely rapidly, are resistant tosolubilization by a variety of buffers, and contain a verysimilar subset of nuclear proteins. Minor differences inprotein composition between the in vivo and in vitrocomplexes can be resolved by two-dimensional gelanalysis. The presence of additional components onlyin the in vivo heat-shock complex could be due to theirtransloeation to the nucleus during heat shock and theirsubsequent sequestration into the insoluble complex.Apparent transloeation of some proteins to the nucleusunder conditions of stress has been described (Kim etal. 1984; Tanguay, 1985). Alternatively, additionalproteins seen in in vivo heat-shock complexes may benuclear proteins that are lost during preparation ofisolated nuclei and are hence unavailable during invitro complex formation. Clearly, however, these ad-ditional proteins cannot be essential for the complex toform. In summary, therefore, the formation of thecomplex in isolated nuclei appears to be a process

The nuclear matrix following heat shock 69

1 2 3 4 5 6 7 8 9 10 B 1 2 3 4 5 6 7

p62c - myc »>

Fig. 4. The formation of insoluble nuclear complexes in intact cells subjected to heat shock. A. Temperature dependence.Formation of the insoluble complex in response to different incubation temperatures of intact cells was determined. 293cells were incubated at 40°C (lanes 1, 6), 41°C (lanes 2, 7), 42°C (lanes 3, 8), 43°C (lanes 4, 9) and 44°C (lanes 5, 10) for60min, the nuclei were isolated, digested with nuclease at 4CC and extracted with buffer A. Chromatin (lanes 1-5) andinsoluble fractions (lanes 6—10) are shown. B. Time dependence. The speed of nuclear complex formation in intact cellswas determined. Insoluble fractions from 293 cells were prepared at different times after the onset of heat shock at 43°C.Time points are 0 (lane 1), lOmin (lane 2); 20min (lane 3); 30min (lane 4); 40min (lane 5); 50min (lane 6); and 60mm(lane 7). The same fractions were also electroblotted and probed with antibodies to pS3 and p62c""'v'\ Molecular weightmarkers are as described in the legend to Fig. 3.

similar to that occurring in intact cells subjected to heatshock. Analysis of the formation and nature of the invitro heat-shock complex may thus provide clues as tothe early cellular events following heat shock.

Heat shock of intact cells causes the rapid formationof the nuclear complex (within 20min). During recov-ery from heat shock the complex is degraded quiteslowly, over a period of several hours. The disappear-ance of the complex correlates with the gradual re-appearance of p53 and p62c'"'-vc in the soluble nuclearfraction. Conceivably, this reappearance of soluble p53and p62c"'"vf may be due to resolubilization of com-ponents of the complex. Indeed, Lewis & Pelham(1985) have suggested that a function of the major heat-shock protein, hsp70, is to solubilize protein aggregatesthat form during heat shock by an ATP-dependentprocess. Accordingly, we have attempted to determinewhether the addition of various levels of ATP toisolated nuclei prevents or slows the formation of the invitro heat-shock process. However, we have not ob-served any such effects (T.D.L. and G.I.E., unpub-lished observations). It is also of note that 293 cellsconstitutively express high levels of a member of the

mammalian hsp70 family (Wu et al. 1986), closelyrelated in sequence and antigenicity to hsp70, andoccupying the same location in the cell (Pelham, 1986).This is, however, clearly not sufficient to preventformation of the in vivo heat-shock complex in thesecells. An alternative explanation for the reappearanceof soluble p53 and p62c""-vr in recovered cells is thatthey arise from de novo synthesis. This latter seems tous to be the most likely explanation.

Sequestration of certain nuclear proteins into aninsoluble complex following thermal stress may ac-count for the observed stabilization of certain otherwiseshort-lived proteins following heat shock (Munro &Pelham, 1984). We have, ourselves, noted that the half-life of p62c""'-vf is greatly extended in heat-shocked cells(G.I.E., unpublished observations).

There is some evidence that the method of prep-aration of nuclei and nuclear matrices may generateartefacts (Hancock, 1982). Specifically, high salt con-centrations (2M-NaCl) appear to result in the precipi-tation of transcription complexes onto the nuclearmatrix (Mirkovitch et al. 1984). To try and avoid suchartefactual precipitation, we have employed a number

70 T. D. Littlewood et al.

p53:

Fig. 5. Induction of insoluble nuclear complexes bychemical inducers of heat-shock proteins. 293 cells wereincubated at 37°C in medium alone (lanes 1, 4) or mediumsupplemented with 5 niM-L-azetidine-2-carboxylic acid (lane2) or 50fiM-sodium arsenite (lane 3) for 16 h. Nuclei weredigested with nuclease at 4°C (lanes 1, 2, 3) or 37°C (lane4) and insoluble fractions prepared by extraction withbuffer A and fractionated by SDS-polyacrylamide gelelectrophoresis. Molecular weight markers are as describedin the legend to Fig. 3. The same fractions wereelectroblotted and probed with monoclonal antibodiesdirected to p53 and p62c'"°r; these data are also shown.

of different methods for isolating nuclear matrices.However, neither low-salt (Mirkovitch et al. 1984) norpolyanion-containing (Lebkowski & Laemmli, 19826)buffers are able to extract any of the proteins thatbecome insolubilized in the heat-induced complex inisolated nuclei or in the nuclei of intact cells. It hasbeen suggested further that isolation of nuclei in low-salt buffers may lead to disruption of nuclear structure.Conceivably, the in vitro heat-shock complex could bea result of such disruption. Our own studies, however,indicate that isolation of nuclei under isotonic con-ditions has no effect on the temperature-dependent

formation of the insoluble nuclear complex (unpub-lished data). Furthermore, there are substantial simi-larities between the complex that forms in isolatednuclei and that which forms within intact cells sub-jected to heat shock: both complexes have a similarconstitution and temperature dependence. Thisimplies that the in vitro complex is related to aphenomenon that occurs in intact cells and is thusunlikely to be some irrelevant artefact resulting simplyfrom the method of isolation and/or extraction ofnuclei.

The mechanism of formation of the heat-shockcomplex is unclear. Our data demonstrate thathyperthermia is not the only insult capable of causinginsolubilization of nuclear components. Treatment ofcells with either sodium arsenite or the amino acidanalogue L-azetidine-2-carboxylic acid also inducesheat-shock genes and also results in formation ofnuclear complexes, albeit of different constitution. It istherefore difficult to see how these complexes mightrepresent fixation of an existing dynamic structure, ashas been suggested (Evan & Hancock, 1985). It isperhaps more likely that hyperthermia, arsenite andamino acid analogues all cause a conformational changein one or more 'key' proteins. If these key proteins existin close physical proximity to other nuclear proteins,perhaps within a small nuclear compartment, then theresult could be the cooperative destabilization of aspecific set of proteins. Such a 'catastrophic' cascadeevent is supported by the sharp temperature transitionseen during the formation of both the in vitro and /;;vivo nuclear heat-shock complex. The proteins presentin heat-shock complexes may thus exist within discretecompartments of the nucleus and may perhaps haveinterrelated functions. Further investigation of thenature of such proteins may have implications for ourunderstanding of the architecture of the interphasenucleus.

The formation of the nuclear heat-shock complex isan early result of hyperthermia and does not appear torequire synthesis of novel proteins (very similar com-plexes form in vitro and in vivo). Insults capable ofeliciting the classic heat-shock response share thecommon property of inducing the formation of aninsoluble nuclear complex. Thus the formation of thecomplex may initiate, or contribute to, induction ofheat-shock genes. Clearly, further detailed investi-gation of the components within the heat-shock com-plex is required in order to evaluate the full relevance ofthis phenomenon.

The authors thank Drs H. R. B. Pelham and J. P. Moorefor their valuable comments and Mary-Ann Starkey forsecretarial assistance.

The nuclear matrix following heat shock 71

References

BEREZNEY, R. & COFFEY, D. S. (1977). Nuclear matrix.Isolation and characterization of a framework structurefrom rat liver nuclei, J. Cell Biol. 73, 616-637.

CAPCO, D. G., WAN, K. M. & PENMAN, S. (1982). The

nuclear matrix: three-dimensional architecture andprotein composition. Cell 29, 847-858.

EVAN, G. I. & HANCOCK. D. C. (1985). Studies on the

interaction of the human c-myc protein with cell nuclei:p62c"mv': as a member of a discrete subset of nuclearproteins. Cell 43, 253-261.

EVAN, G. I., LEWIS, G. K. & BISHOP, J. M. (1984).

Isolation of monoclonal antibodies specific for productsof the avian oncogene myb. Molec. Cell Biol. 4,2843-2850.

EVAN, G. I., LEWIS, G. K., RAMSAY, G. & BISHOP, J. M.

(1985). Isolation of monoclonal antibodies specific forhuman c-myc proto-oncogene product. Molec. Cell Biol.5, 3610-3616.

GRAHAM, F. L., SMILEY, J., RUSSELL, W. C. & NAIRN, R.

(1977). Characteristics of a human cell line transformedby DNA from human adenovirus type 5..7. gen. Virol.36, 59-72.

GURNEY, E. G., HARRISON, R. O. & FENNO, J. (1980).

Monoclonal antibodies against Simian virus 40 Tantigens: evidence for distinct subclasses of large T andfor similarities among nonviral T antigens. J. Virol. 34,752-763.

HANCOCK, R. (1982). Topographical organisation ofinterphase DNA: the nuclear matrix and other skeletalstructures. Biol. Cell 46, 105-122.

HANCOCK, R. & BOULIKAS, T. (1982). Functional

organisation in the nucleus. Int. Rev. Cytol. 79, 165-214.JACKSON, D. A. & COOK, P. R. (1985). Transcription

occurs at a nucleoskeleton. EMBOJ. 4, 919-925.JACKSON, D. A. & COOK, P. R. (1986). Replication occurs

at a nucleoskeleton. EMBOJ. 6, 1403-1410.JOHNSTON, D., OPPERMAN, H., JACKSON, J. & LEVINSON,

W. (1980). Induction of four proteins in chick embryocells by sodium arsenite. J. biol. Chem. 255, 6975-6980.

KIM, Y.-J., SHUMAN, J., SETTE, M. & PRZYBYLA, A.

(1984). Nuclear localization and phosphorylation of three25-kilodalton rat stress proteins. Molec. Cell Biol. 4,468-474.

LAEMMLI, U. K. (1970). Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature, Land. 227, 680-685.

LEBKOWSKI, J. S. & LAEMMLI, U. K. (1982«). Evidence

for two levels of DNA folding in histone-depleted HeLainterphase nuclei. J. molec. Biol. 156, 309-324.

LEBKOWSKI, J. S. & LAEMMLI, U. K. (19826). Non-histone

proteins and long-range organization of HeLa interphasenuclei. J. molec. Biol. 156, 325-344.

LEWIS, M. J. & PELHAM, H. R. B. (1985). Involvement of

ATP in the nucleolar functions of the 70 kd heat shockprotein. EMBOJ. 4, 3137-3143.

MIRKOVITCH, J., MIRAULT, M.-E. & LAEMMLI, U. K.

(1984). Organization of the higher-order chromatin loop:specific DNA attachment sites on nuclear scaffold. Cell39, 223-232.

MUNRO, S. & PELHAM, H. R. B. (1984). Use of peptide

tagging to detect proteins expressed from cloned genes:deletion mapping functional domains of Drosophilahsp70. EMBOJ. 3, 3087-3093.

O'FARRELL, P. H. (1975). High resolution two-dimensionalelectrophoresis of proteins. J . biol. Chem. 250,4007-4021.

PELHAM, H. R. B. (1984). Hsp70 accelerates the recoveryof nucleolar morphology after heat shock. EMBOJ. 3,3095-3100.

PELHAM, H. R. B. (1986). Speculations on the functions ofthe major heat shock and glucose-regulated proteins. Cell46, 959-961.

SCHLESINGER, M. J., ASHBURNER, M. & TlSSIERES, T.(eds) (1982). Heat Shock from Bacteria to Man. NewYork: Cold Spring Harbor Laboratory Press.

TANGUAY, R. M. (1985). Intracellular localization andpossible functions of heat shock proteins. In Changes inEukaryotic Gene Expression in Response toEnvironmental Stress (ed. G. Atkinson & D. B.Walden), pp. 91-113. New York, London: AcademicPress.

VOELLMY, R. (1984). The heat shock genes: a family ofhighly conserved genes with a superbly complexexpression pattern. BioEssay 1, 213-217.

WELCH, W. J. & FERAMISCO, J. R. (1982). Purification ofthe major mammalian heat shock proteins. J. biol. Chem.257, 14949-14959.

Wu, B. J., HURST, H. C , JONES, N. C. & MORIMOTO, R.

J. (1986). The E1A 13S product of adenovirus 5activates transcription of the cellular human HSP70gene. Molec. Cell. Biol. 6, 2994-2999.

(Received 25 March 1987 -Accepted 18 May 1987)

72 T. D. Littlewood et al.