B-Crystallin interacts with intermediate filaments in …...αB-Crystallin interacts with...

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INTRODUCTION Intermediate filaments (IFs) constitute major cytoskeletal com- ponents of the eukaryotic cytoplasm and the nuclear lamina. Depending on their cell type and developmental state, IFs can be assembled from single intermediate filament proteins (IFPs) or from a combination thereof. IFPs constitute a multigene family whose members can be grouped into six categories (I- VI) (Stewart, 1990; Fuchs and Weber, 1994). In a cellular context, cytoplasmic IFs are radially distributed from the nuclear membrane towards the cell surface (Goldman et al., 1985). This implies site-specific recognition between IF subunits, and binding to specific proteins of the different cellular structures. During development and differentiation, extensive rearrangement of the IF network is correlated with the expression of completely new subsets of IFPs which are tissue- specific, and developmentally regulated. These events imply a dynamic state of the cytoskeleton, where de novo synthesised IF subunits are incorporated into or next to pre-existing IFs. Remarkably, the rearrangement is observed during the differ- entiation of neurons, in which specific IFPs are expressed in a sequential, and overlapping pattern in correlation with the state of neuronal differentiation, and maturation (Cochard and Paulin, 1984; Escurat et al., 1990; Djabali et al., 1993; Liem, 1993). Furthermore, fully differentiated neurons show a highly dynamic IF system. In these neurons newly synthesised IFPs are added by the mechanism of lateral, and segmental incor- poration into the backbone of neurofilament arrays in the axon (Okabe et al., 1993; Takeda et al., 1994). Extensive remodelling of the cytoplasmic IF distribution has been observed in cells submitted to drug treatment. After such treatment, the IFs collapse towards the nucleus, and form a large juxta nuclear cap. After removal of the drug, the IFs reassemble into a normal network (Klymkowsky, 1988; Yang et al., 1992). Rearrangement of the IFs is also observed when cells are subjected to stress. Synthesis of heat shock proteins is induced following sublethal exposure to heat shock and various chemicals. The small heat-shock proteins (shsps) are one of the four most common groups of heat shock proteins (Hendrick and Hartl, 1993). These low molecular mass proteins are evolutionarily related to the vertebrate eye lens protein α-crystallin (Ingolia and Craig, 1982). α-Crystallin, usually found as large aggregates, consists of two types of subunits, αA and αB (Groenen et al., 1994). It occurs in various tissues; relatively high levels are found in heart, striated muscle, kidney and brain (Bhat and Nagineni, 1989; Kato et al., 1991; Klemenz et al., 1993). Functionally, the shsps, and α-crystallins are molecular chaperones as they can prevent stress-induced aggregation of proteins (Horwitz, 1992), and as they are able to convey thermotolerance (Klemenz et al., 1991b). Several reports suggest that αB-crystallin interacts with the cytoskeleton. In vitro, αB-crystallin interacts directly with actin and desmin filaments. In cardiomyocytes, αB-crys- tallin is distributed in the central region of the I bands (Z lines) together with desmin filaments (Bennardini et al., 1992). αB- Crystallin binds in vitro to GFAP, vimentin, and filensin/phakinin. Furthermore, αB-crystallin inhibits the in vitro assembly of vimentin and GFAP, and increases the soluble pool of these IFPs when added to preformed filaments (Nicholl and Quinlan, 1994; Carter et al., 1995). Here, we report that αB-crystallin binds to neuronal type III IFP peripherin, and type III IFP vimentin. The binding was found to be temperature dependent, and ATP independent. Fur- 2759 Journal of Cell Science 110, 2759-2769 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JCS9634 The small heat shock protein αB-crystallin interacts with intermediate filament proteins. Using a co-sedimentation assay, we showed that in vitro binding of αB-crystallin to peripherin and vimentin was temperature-dependent. Specifically, a synthetic peptide representing the first ten residues of αB-crystallin was involved in this interaction. When cells were submitted to different stress conditions such as serum starvation, hypertonic stress, or heat shock, we observed a dynamic reorganisation of the intermediate filament network, and concomitant recruitment of αB- crystallins on intermediate filament proteins. Under normal conditions αB-crystallin was extracted from cells by detergent. In stressed cells, αB-crystallin colocalised with intermediate filament proteins, and became resistant to detergent extraction. The intracellular state of αB-crys- tallin seemed to correlate directly with the remodelling of the intermediate filament network in response to stress. This suggested that αB-crystallin functions as a molecular chaperone for intermediate filament proteins. Key words: αB-Crystallin, Chaperone, Peripherin, Vimentin, Intermediate filament protein SUMMARY αB-Crystallin interacts with intermediate filaments in response to stress Karima Djabali*, Béatrice de Néchaud, Françoise Landon and Marie-Madeleine Portier Faculté de Médecine, Pitié-Salpêtrière, CNRS-URA 2115, Cytosquelette et Développement, 105, Boulevard de l’Hôpital, 75634 Paris Cedex 13, France *Author for correspondence

Transcript of B-Crystallin interacts with intermediate filaments in …...αB-Crystallin interacts with...

2759Journal of Cell Science 110, 2759-2769 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS9634

αB-Crystallin interacts with intermediate filaments in response to stress

Karima Djabali*, Béatrice de Néchaud, Françoise Landon and Marie-Madeleine Portier

Faculté de Médecine, Pitié-Salpêtrière, CNRS-URA 2115, Cytosquelette et Développement, 105, Boulevard de l’Hôpital, 75634Paris Cedex 13, France*Author for correspondence

The small heat shock protein αB-crystallin interacts withintermediate filament proteins. Using a co-sedimentationassay, we showed that in vitro binding of αB-crystallin toperipherin and vimentin was temperature-dependent.Specifically, a synthetic peptide representing the first tenresidues of αB-crystallin was involved in this interaction.When cells were submitted to different stress conditionssuch as serum starvation, hypertonic stress, or heat shock,we observed a dynamic reorganisation of the intermediatefilament network, and concomitant recruitment of αB-crystallins on intermediate filament proteins. Under

normal conditions αB-crystallin was extracted from cellsby detergent. In stressed cells, αB-crystallin colocalisedwith intermediate filament proteins, and became resistantto detergent extraction. The intracellular state of αB-crys-tallin seemed to correlate directly with the remodelling ofthe intermediate filament network in response to stress.This suggested that αB-crystallin functions as a molecularchaperone for intermediate filament proteins.

Key words:αB-Crystallin, Chaperone, Peripherin, Vimentin,Intermediate filament protein

SUMMARY

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INTRODUCTION

Intermediate filaments (IFs) constitute major cytoskeletal coponents of the eukaryotic cytoplasm and the nuclear lamDepending on their cell type and developmental state, IFs be assembled from single intermediate filament proteins (IFor from a combination thereof. IFPs constitute a multigefamily whose members can be grouped into six categoriesVI) (Stewart, 1990; Fuchs and Weber, 1994). In a cellucontext, cytoplasmic IFs are radially distributed from thnuclear membrane towards the cell surface (Goldman et1985). This implies site-specific recognition between subunits, and binding to specific proteins of the differecellular structures.

During development and differentiation, extensivrearrangement of the IF network is correlated with texpression of completely new subsets of IFPs which are tissspecific, and developmentally regulated. These events impdynamic state of the cytoskeleton, where de novo synthesIF subunits are incorporated into or next to pre-existing IRemarkably, the rearrangement is observed during the difentiation of neurons, in which specific IFPs are expressed sequential, and overlapping pattern in correlation with the sof neuronal differentiation, and maturation (Cochard aPaulin, 1984; Escurat et al., 1990; Djabali et al., 1993; Lie1993). Furthermore, fully differentiated neurons show a highdynamic IF system. In these neurons newly synthesised Iare added by the mechanism of lateral, and segmental inporation into the backbone of neurofilament arrays in the a(Okabe et al., 1993; Takeda et al., 1994).

Extensive remodelling of the cytoplasmic IF distribution hbeen observed in cells submitted to drug treatment. After s

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treatment, the IFs collapse towards the nucleus, and formlarge juxta nuclear cap. After removal of the drug, the Ireassemble into a normal network (Klymkowsky, 1988; Yanet al., 1992). Rearrangement of the IFs is also observed wcells are subjected to stress. Synthesis of heat shock protis induced following sublethal exposure to heat shock avarious chemicals. The small heat-shock proteins (shsps)one of the four most common groups of heat shock prote(Hendrick and Hartl, 1993). These low molecular maproteins are evolutionarily related to the vertebrate eye leprotein α-crystallin (Ingolia and Craig, 1982). α-Crystallin,usually found as large aggregates, consists of two typessubunits, αA and αB (Groenen et al., 1994). It occurs invarious tissues; relatively high levels are found in heart, striamuscle, kidney and brain (Bhat and Nagineni, 1989; Katoal., 1991; Klemenz et al., 1993). Functionally, the shsps, aα-crystallins are molecular chaperones as they can prevstress-induced aggregation of proteins (Horwitz, 1992), andthey are able to convey thermotolerance (Klemenz et 1991b). Several reports suggest that αB-crystallin interactswith the cytoskeleton. In vitro, αB-crystallin interacts directlywith actin and desmin filaments. In cardiomyocytes, αB-crys-tallin is distributed in the central region of the I bands (Z linetogether with desmin filaments (Bennardini et al., 1992). αB-Crystallin binds in vitro to GFAP, vimentin, andfilensin/phakinin. Furthermore, αB-crystallin inhibits the invitro assembly of vimentin and GFAP, and increases tsoluble pool of these IFPs when added to preformed filame(Nicholl and Quinlan, 1994; Carter et al., 1995).

Here, we report that αB-crystallin binds to neuronal type IIIIFP peripherin, and type III IFP vimentin. The binding wafound to be temperature dependent, and ATP independent.

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thermore, a synthetic peptide representing the first ten residof human αB-crystallin interacted in vitro with both peripherin, and vimentin. Finally, we explored the in vivo interactiobetween αB-crystallin and these IFPs. By exposing cells heat shock, hypertonic stress, and serum starvation, we fothat αB-crystallin was recruited on the rearranged type III IFThis suggested that the in vitro reaction between αB-crystallinand IFPs reflected a genuine physiological mechaniTherefore, we suggest that αB-crystallin functions as amolecular chaperone for IFPs.

MATERIALS AND METHODS

Cell cultureNIH 3T3 were plated on glass coverslips and grown overnightstandard medium: DMEM supplemented with 10% fetal calf ser(FCS), 2 mM glutamine and antibiotics (100 i.u./ml penicillin; 10mg/ml streptomycin). When cells were submitted to serum starvatreatment, the medium was the same medium as above supplemwith 0.5% FCS, as cells were allowed to grow for 5 days beffixation. For cells submitted to heat shock, the medium was replawith DMEM supplemented with 10% FCS and 10 mM Hepes, pH 7to maintain a constant pH. Cells were placed in a 43°C water bath1 hour, and were returned to 37°C. For recovered cells, the medwas replaced with fresh standard medium, and cells were fixetimed intervals as indicated in the text. Control cells were held at 3in standard medium. For treatment of the cells with a high conctration of potassium, the medium was prepared by adding 1.0 M stock solution to standard medium and brought to 100 mM KCl in final concentration. Cells grown on coverslips were incubated forhours and 48 hours in high potassium medium; some coverslips wimmediately processed for immunohistochemistry; others wreturned to standard medium and allowed to recover for a perio24 hours.

Plasmid construction and purification of recombinantperipherin and αB-crystallinFull-length cDNA for mouse peripherin or mouse αB-crystallin(Aoyama et al., 1993) were subcloned into NdeI and HindIII of the T7RNA polymerase promoter-based PT7.7 vector (Tabor and Richson, 1985). Expression of full-length peripherin or αB-crystallin inBL21 (DE3) pLysS bacteria was induced by the addition of isopropβ-D-thiogalactopyranoside. Peripherin was purified from bactelysates prepared as follows. Bacteria were harvested and resuspein lysis buffer (25 mM MES, 150 mM NaCl, 2 mM MgCl2, 1 mMPMSF, 1 mM DTT, pH 6.0), containing 1/1,000 volume of proteainhibitor stock solution (2 mg/ml of pepstatin, leupeptin, aprotinin),for all other solutions used in this procedure, and lysed by freethawing. By centrifugation (Sorvall, 30 minutes 15,000 g), the pelletwas extracted 2 times in high salt and Triton X-100 buffer (600 mKCl, 50 mM Tris-HCl, pH 7.6, 2 mM MgCl2, 1 mM PMSF, 1 mMDTT). The remaining pellet was washed once in 150 mM NaCl,mM Tris-HCl, pH 7.6, and the pellet was extracted for 2 hours at rotemperature with 10 volumes of 8 M urea, 20 mM Tris-HCl, pH 71 mM EDTA, 1 mM PMSF, 1 mM DTT. After centrifugation at 12,00g for 40 minutes (18°C), the supernatant was collected and mixed diaminoethyl cellulose (DE52; Whatman, Maidston/Kent, UK) equibrated in the above buffer. The resin was pelleted, washed withvolumes of urea buffer and loaded into a column. Bound proteins weluted with a linear gradient of 0-150 mM NaCl (in the same ubuffer). Fractions enriched for peripherin were pooled and further chmatographed on a hydroxylapatite column (Bio-Rad, Richmond, CThe column was eluted with a gradient of 10-100 mM Na3PO4 in 8 Murea, 10 mM Na3PO4, pH 7.5, 1 mM DTT, 1 mM PMSF.

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αB-Crystallin was purified from bacteria resuspended in 50 mMTris-HCl, pH 7.5, 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1 mMPMSF and other protease inhibitors as described above. Cells wlysed by freeze-thawing. After centrifugation the pellet was resupended in 6 M urea, 10 mM Tris-HCl, pH 9.0, 1 mM DTT and PMSFThe urea-soluble fraction was obtained by centrifugation (Sorvall, minutes at 15,000 g). The clarified supernatant was batch-incubatewith DEAE 52-cellulose and unbound material was dialysed again8 M urea, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT andPMSF overnight at room temperature. Dialysed material was loadonto a DEAE 53-cellulose column equilibrated in the same buffeBound material was eluted with a gradient of 0-100 mM NaCl in M urea buffer. Fractions containing purified αB-crystallin were iden-tified on 12.5% polyacrylamide gels.

Protein chemical proceduresαB-crystallin from bovine lens was purified as described by Bennadini et al. (1992) and bovine vimentin as described by Merdes et (1991). Fractions containing purified αB-crystallin were identified on12.5% polyacrylamide gels. Purified αB-crystallin was biotinylatedusing an ImmunoProbeTM biotinylation kit as specified by the manu-facturer (Sigma Chemical Co., St Louis, MO). Total extracts fromNIH 3T3 cells were prepared as follows: cells from treated ountreated conditions were rinsed twice in PBS supplemented wprotease inhibitors as described above, and the total cell numbereach condition of growth were determined. The final cell pellets weresuspended in 100 µl of LB buffer (9.5 M urea, 2% NP40, 5% beta-mercaptoethanol) per ten million cells and 100 µl of each sample wasseparated by two-dimensional gel electrophoresis (O’Farrell et a1977).

AssaysLigand-blotting assays were performed as described previou(Djabali et al., 1991). Immunoblotting assays were performed as pviously described (Georgatos and Blobel, 1987). In brief, proteinwere resolved in 10%-12% SDS-acrylamide gels and transferred onitrocellulose filters. Filters were first incubated with buffer A containing 0.9% NaCl, 20 mM Tris-HCl, 0.1% Tween-20, and 0.1%gelatin, pH 7.3, for 15 hours at room temperature and then probwith primary antibodies followed by a horseradish peroxidase-conjgated secondary antibody. Blots were rinsed in PBS and developwith enhanced chemiluminescence (Amersham). For ligand-blottiassays the nitrocellulose filters were probed with buffer A containin0.2 µg/ml of biotinylated protein at room temperature for 2 hoursAfter several washes, blots were incubated with horseradish perodase-coupled ExtraAvidin (Sigma Chemical). Blots were washed adeveloped as described above.

Sedimentation assays were done as follows: peripherin, vimentand αB-crystallin were dialysed for 24 hours at 4°C against a depolmerisation buffer (2 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 1 mMDTT, 1 mM PMSF). Samples were pre-centrifuged for 30 minutes 100,000 g in TL-100 (Beckman Instruments). The supernatant fromeach sample was concentrated in a Centriprep 30 or Centriprepconcentrator (Amicon, Beverly, MA, USA) and the protein concentration was measured using a Bio-Rad kit (Bio-Rad, Richmond, CAFor binding experiments, protein samples were mixed in a bindibuffer: 10 mM Tris-HCl, pH 7.3, 150 mM NaCl, 1 mM MgCl2, 0.1%Tween-20. Samples were incubated for 45 minutes at different teperatures: RT°C (room temperature between 22°C and 25°C), 3744°C. Aggregated IFPs were then removed by centrifugation at 5,0g for 15 minutes. The supernatants were collected and the pellets wresuspended at the same initial volume; both fractions were analyby 12.5% SDS-PAGE.

Immunological and immunochemical proceduresThe amino-terminal peptide Pcrys (MDIAIHHPWI), corresponding toresidues 1-10 of human αB-crystallin and identical to the mouse

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sequence (Aoyama et al., 1993) was purchased from NeosysteA., France. The following antibodies were employed in this studanti-Pcrys rabbit developed against Pcrys was obtained from Nocostra Laboratories Ltd, UK (NCL-ABcrys, batch 1670). The monclonal antibody 7A3 recognising vimentin was used as previoudescribed (Papamarcaki et al., 1991) and was kindly provided bD. Georgatos.

Affinity matrices were made by coupling Pcrys peptide to derivtised agarose HiTrapTM affinity columns as specified by the manufacturer (Pharmacia Biotech). The affinity chromatography wperformed as follows to purify the anti-Pcrys IgG population of tserum aPcrys: (1) 200 µl of aPcrys serum was diluted with 1 ml oPBS and loaded onto a Protein A affinity column (Pharmacia Biotenology); (2) the IgG fraction recovered was applied to a HiTrapTM-Pcrys column and incubated 15 hours at 4°C, the flowthrough collected, and the column was washed with 20 ml of PBS buffer.release the bound antibodies, the column was eluted with 10 m200 mM glycine-HCl, pH 2.3, 500 mM NaCl. Eluted antibodies werecovered in 1 ml aliquots and immediately neutralised with 2 M Tbase solution. The fraction containing anti-Pcrys IgG was dialyagainst PBS at 4°C and was concentrated in an Amicon concent(Amicon, Beverly, MA, USA).

For conventional indirect immunofluorescence microscopy, N3T3 were grown on glass coverslips. The cells were washed with Pfixed with 3% paraformaldehyde in PBS for 15 minutes at room teperature. After quenching with 50 mM NH4Cl in PBS, cells were per-meabilised for 30 minutes in blocking buffer (PBS containing 1BSA, 0.2% gelatin, 0.2% Triton X-100). For some samples, cells wlysed before fixing by washing first with PBS containing 5 mM MgC2and 3 mM EGTA, and then incubated for two minutes in PBS ctaining 0.5 mM MgCl2, 3 mM EGTA, 0.5 mM PMSF, 1 mM DTT,0.5% NP-40. Cells were fixed and processed as described above. were incubated with anti-Pcrys IgG (diluted 10 µg/ml) and anti-vimentin 7A3 antibodies (diluted at 10 µg/ml) for 30 minutes at roomtemperature. After appropriate washes, cells were further incubwith secondary antibodies: FITC-coupled goat anti-mouse antibodor rhodamine-conjugated goat anti-rabbit (both from Silenus labo

Fig. 1. In vitro interaction between αB-crystallin and IFP. (A) Cosedimentation ofperipherin with αB-crystallin. Control paneldenotes sedimentation assays conducted at43°C; peripherin (tracks 1and 1′), αB-crystallin (tracks 2 and 2′) and peripherin inthe presence of trypsin inhibitor (tracks 3 and3′) at a molar ratio 1:2. The supernatants (1 to3) and the pellets (1′ to 3′) were then analysedby SDS-PAGE. Note that αB-crystallinremains in the supernatant. RT°C denotesroom temperature (between 22°C and 25°C),37°C and 43°C denote assays performed at theindicated temperatures. Peripherin was mixedwith αB-crystallin at molar ratios of 1:4 (lanes4, 4′, 6, 6′, 8 and 8′) or 1:2 (lanes 5, 5′, 7, 7′, 9and 9′). Samples 4 to 9 correspond tosupernatants and 4′ to 9′ to pellets. Note thatαB-crystallin interacts with peripherin at 37°Cand 43°C. (B) Cosedimentation of vimentinwith αB-crystallin. Control panel representssedimentation assays performed at 43°C;vimentin (tracks 1and 1′), αB-crystallin (tracks2 and 2′) and vimentin mixed with trypsininhibitor at a molar ratio of 1:2 (tracks 3 and 3′). Supernatants correspco-sedimentation assays conducted at RT°C, 37°C and 43°C. Vim(tracks 4 to 6) and pellets (tracks 4′ to 6′) were then analysed by SDS37°C and remains nearly identical at 43°C.

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RESULTS

In vitro interaction of αB-crystallin with peripherinand vimentinWe used a co-sedimentation assay to investigate the bindbetween isolated αB-crystallin and the IFPs peripherin anvimentin. Incubation in the binding buffer (Materials anMethods) induced a very rapid polymerisation of peripherand vimentin. In the absence of IFP, αB-crystallin could notbe sedimented at any temperature, and behaved as in coconditions (Fig. 1A and B). Thus, the αB-crystallin sedi-menting with the recombinant peripherin, or bovine levimentin must have bound to the IF polymer. At 37°C thamount of αB-crystallin interacting with peripherin wasalready detectable; at 43°C the amount of αB-crystallin inter-acting was significantly higher, i.e. more αB-crystallincosedimented with peripherin (Fig. 1A). Thus, raising thtemperature from 37°C to 43°C (heat shock treatmeclearly promoted the interaction. The amount of αB-crys-tallin interacting with vimentin at room temperatur(between 22°C and 25°C) was comparable to that interacwith peripherin at 37°C (Fig. 1). Increasing the temperatuto physiological temperature (37°C) increased the interactbetween vimentin and αB-crystallin to a significantly higherlevel comparable to that between peripherin and αB-crys-tallin at 43°C (Fig. 1). A further increase in temperature 43°C did not increase the level of binding between vimenand αB-crystallin. These results revealed a different bindinaffinity of αB-crystallin for the two IFP subunits. No association with peripherin or vimentin could be detected at a

onded to lane 1 to 3 and pellets to 1′ to 3′. The next panel corresponds toentin was mixed with αB-crystallin at a molar ratio of 1:2. Supernatants-PAGE. Note that interaction of αB-crystallin with vimentin is detected at

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Fig. 2Binding of αB-crystallin to type III IFPdetected by ligand-blotting assay. (A) Binding ofbiotinylated αB-crystallin to peripherin andvimentin. Recombinant mouse peripherin (Pe),bovine lens vimentin (Vi) and bovine serum albumin(BSA) were subjected to electrophoresis through12% SDS polyacrylamide gels and either stainedwith Coomassie blue, or transferred to nitrocellulose.Blots were then incubated with either αB-crystallinor trypsin inhibitor, each biotinylated as described inMaterials and Methods. Note the reaction of αB-crystallin with peripherin and its degradationproducts (open arrowhead) and with vimentin.(B) Direct association of the decapeptide Pcrys withtype III IFP. The Coomassie stained panel shows the

proteins used in this study. Gels similarto the left panel were transferred tonitrocellulose filters and used for theligand-blot assays. Blots were incubatedfor 5 hours at room temperature withincreasing amounts of purified bovinelens αB-crystallin (none (−), 125 µg/ml(+) and 250 µg/ml (++)), washed andincubated with biotinylated Pcrys for 2hours and further processed as specifiedin Materials and Methods. Note thatPcrys interacts specifically withperipherin and vimentin and that bindingis inhibited by αB-crystallin.

temperature in the presence of trypsin inhibitor, a proteinsimilar size that is also used as a control for in vitro studby other groups (Bennardini et al., 1992; Nicholl anQuinlan, 1994). Trypsin inhibitor remained in the solubfraction at 43°C (control panel, Fig. 1). When recombinamouse αB-crystallin was used in the co-sedimentatioassays, we observed the same interaction with the IFPs (not shown). These results suggested that the interactionαB-crystallin with polymeric peripherin and vimentin watemperature dependent.

To further confirm that αB-crystallin interacted directly withtype III IFP, we performed a ligand blot assay (Djabali et a1991). We used biotinylated αB-crystallin, trypsin inhibitor,and a peptide Pcrys (corresponding to the first ten amiterminal residues of human αB-crystallin) to probe nitrocellu-lose blots containing recombinant peripherin, bovine vimentand BSA. While biotinylated trypsin inhibitor yielded nosignal, αB-crystallin bound to peripherin and vimentin, but noto BSA (Fig. 2A). We then examined the possibility of an assciation between the amino-terminal peptide of αB-crystallinand IFPs. Probing blots of recombinant peripherin, bovivimentin, and BSA with biotinylated Pcrys revealed that Pcrassociated with peripherin, and vimentin (Fig. 2B). No bindiof Pcrys to BSA was detected. To prove the specificity of association between Pcrys and the IFPs, we performed a cpetition experiment in which the nitrocellulose blot waincubated first with an increasing amount of αB-crystallin, andthen with biotinylated Pcrys. Under these conditions, when Isites were saturated by αB-crystallin, Pcrys could no longerbind to the IFPs (Fig. 2B). The data supported three concsions: (1) αB-crystallin interacted with peripherin andvimentin; (2) the binding was specific; (3) the amino-termin

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Biochemical characterisation of αB-crystallin in NIH3T3 cells under normal and stress conditionsWe first tested the specificity of aPcrys IgG fraction affinity-purified on the amino-terminal peptide of αB crystallin fordifferent protein preparations separated by non-equilibriumtwo-dimensional gels (Fig. 3A). The aPcrys IgG fractionrecognised the αΒ-crystallin from lens soluble extract, anddecorated equally well the phosphorylated, and unphosphorlated forms αB1 and αB2, respectively (Iwaki et al., 1990).Furthermore, the antibody showed a weak cross reactivity αA-crystallin (Fig. 3A, b), probably due to the high sequencehomology between the α-crystallins and also observed amongthe different species (Van Der Ouderraa et al., 1974; Kato al., 1992). In soluble fractions of cardiac muscle, aPcrys Igrecognised one single form of αB-crystallin that probably cor-responded to the non phosphorylated form αB2-crystallin(Iwaki et al., 1990). Recombinant mouse αB-crystallin gave astrong signal with aPcrys IgG and exhibited similar coordinates on NEPHGE gels to the αB-crystallin from tissue(Fig. 3A). Hence, the affinity-purified aPcrys antibody recognised equally well theαB-crystallin from different sources.

In order to fully characterise our cellular model NIH 3T3cells, we investigated whether αB-crystallin accumulated inresponse to stress as previously described (Klemenz et a1991a). We first analysed the level of αB-crystallin expressionin NIH 3T3 cells grown under standard conditions. Due to thlow level of expression, αB-crystallin cannot be detected innon-stressed cells (Klemenz et al., 1991b). We analysedhighly concentrated cellular extract of αB-crystallin to cir-

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Fig. 3. Identification andcharacterisation of αB-crystallinin NIH 3T3 cells.(A) Characterisation of the amino-terminal decapeptide antibody.Various preparations of αB-crystallin were subjected to non-equilibrium pH-gradient gelelectrophoresis (NEPHGE)followed by SDS-PAGE,transferred to nitrocellulose filters,and probed with affinity-purifiedaPcrys-IgG (diluted 0.10 ug/ml).(a,c,e) Coomassie blue stainedgels. (b,d,f) The correspondingwestern blots probed with aPcrysIgG. (a) The profile of an enrichedfraction of α-crystallin from lens.B denotes αB-crystallin. Adenotes αA-crystallin. B1 and A1indicate, respectively,phosphorylated αB and αAcrystallins and B2, A2, the nonphosphorylated forms. (c) Theprofile of a soluble fraction of ratcardiac muscle. (d) Thecorresponding western blot whereone spot corresponding to αB2-crystallin was detected by theantibody. (e) The profile of apurified fraction of recombinantmouse αB-crystallin.(f) Recombinant αB-crystallin wasdetected by aPcrys IgG. (B) Characterisation of the level of expression of αB-crystallin in NIH 3T3 grown under standard and stress conditions.Total cellular extracts of 10 million cells for each condition of growth were separated by two-dimensional gel (NEPHGE). (a,b) Cells weregrown under standard conditions. (c,d) Cells were submitted to serum starvation for a period of 5 days. (e,f) Cells were heat shocked for 1 hourat 43°C and collected 8 hours after recovery at 37°C. (a,c,e) The lower part of the gels stained by Coomassie blue. αB-Crystallin was barelydetectable on the Coomassie blue stained gels. The position of αB-crystallin was indicated by an arrow. (b,d,f) The corresponding western blotsprobed with aPcrys IgG (diluted 0.10 ug/ml). One spot of αB-crystallin was detected in b and d, and two spots were detected in f. Note theaccumulation of αB-crystallin after heat shock and the appearance of an acidic form of αB-crystallin in f.

cumvent the problem of detection. A total extract of ten milliocells was separated by non-equilibrium two-dimensional gand αB-crystallin was identified on a two-dimensional westeblot (Fig. 3B, a and b). We observed a detectable amounαB-crystallin using affinity-purified aPcrys IgG. This wastriking in that others reported that αB-crystallin was barelydetectable. Our ability to detect a significant level of expressmay have been for two reasons. Firstly, we analysed a very ccentrated NIH 3T3 extract. Secondly, we used an antiboraised against the amino-terminal peptide of αB-crystallinwhile others used antibodies raised against the entire αB-crys-tallin protein, or against the carboxy-terminal peptide. Henepitope masking or incomplete denaturation may account the differing levels of signal obtained with different antibodie(Klemenz et al., 1991b; Iwaki et al., 1994).

We then investigated the accumulation of αB-crystallinwhen cells were submitted to serum starvation, or to heat shtreatment by analysing an equal amount of extract to that ufor the control cells. The autoradiography of the western blwas performed in parallel such that the signals were saturated and that an approximate quantification was obtausing NIH image version 1.6. When cells were submittedserum starvation, one spot corresponding to αB2-crystallin was

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detected (Fig. 3B, c and d). By scanning the autoradiograwe observed that the amount of αB-crystallin in serum starvedcells remained equal to the signal obtained from the contcells. When cells were submitted to heat shock and colleceight hours after recovery, the aPcrys IgG recognised two spof similar coordinates to those of lens αB-crystallin (Fig. 3B,e and f). The results suggested that the major spot corresponto the non phosphorylated form, and the more acidic spotthe phosphorylated form of αB-crystallin. By comparing thesignal from the control and the heat shocked cells, we obtainafter eight hours recovery an approximately tenfold greataccumulation of αB-crystallin, as previously reported(Aoyama et al., 1993).

Intra-cellular localisation of αB-crystallin andvimentin in NIH 3T3 cells under normal growthconditions or serum starvation To investigate the intracellular state of αB-crystallin in NIH3T3 cells we used affinity-purified antibody aPcrys IgG. Oformaldehyde fixed NIH 3T3 cells, aPcrys-IgG decorated αB-crystallin distributed throughout the cell, with no specific subcellular localisation (Fig. 4b). The same cells probed with thanti-vimentin antibody 7A3 showed, however, a well organise

2764

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Fig. 4.Distribution of vimentin and αB-crystallin in NIH 3T3 after serumstarvation as revealed by indirectimmunoflorescence. Cells were grown oncoverslips in standard medium (a to d) orwere submitted for 5 days to serumstarvation (e to h). Cells were either fixedwith 3% paraformaldehyde (a,b,e,f) orlysed prior to fixation (c,d,g,h) asdescribed in Materials and Methods.Samples were simultaneously decoratedwith anti-vimentin 7A3 antibody (a,c,e,g)and aPcrys serum (b,d,f,h). Bar, 20 µm.

vimentin network extending from the nuclear surface to thplasma membrane (Fig. 4a). When the same cells were pmeabilised briefly prior to fixation (see Materials and Methodstaining of the vimentin filaments remained unchanged (F4c). Furthermore, αB-crystallin was no longer detected byaPcrys IgG (Fig. 4d). This result indicated that αB-crystallinwas completely extracted by mild detergent lysis, suggestthat αB-crystallin was not tightly bound to any cellular structures.

NIH 3T3 cells subjected to serum starvation for a period five days appeared more flattened and showed well expanvimentin filaments thicker than control cells (Fig. 4e). αB-Crystallin clearly accumulated in the nucleus. But the mostriking observation was the distribution of αB-crystallin incytoplasmic aggregates concentrated in perinuclear caps,

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organised along tracks which seemed to emanate from tnuclear periphery (Fig. 4f). When the distribution of αB-crys-tallin was compared to the vimentin network, the cluster of αB-crystallin aggregates appeared to be distributed along traccoinciding with vimentin filaments. When cells were permeabilised before fixation the vimentin network was well resolveand the vimentin filaments appeared to have a larger diameand were sometimes organised into thick cable structures (F4g). Surprisingly, the αB-crystallins which were in the solublepool under control growth conditions switched to the insolublfraction under serum starvation; permeabilisation prior tfixation permitted a strong staining of the cells with aPcrys IgG(Fig. 4h). The patterns of fluorescence observed with anvimentin and aPcrys antibodies were remarkably similar. αB-Crystallins appeared to be recruited into the vimentin netwo

2765αB-Crystallin acts as molecular chaperone for IFPs

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Fig. 5.Distribution of vimentin and αB-crystallin in cells submitted to high extracellular K+ concentration. NIH 3T3 were cultured for 24 hours(a to d) and 48 hours (e to h) in medium containing 100 mM KCl or treated for 24 hours with medium containing 100 mM KCl and thenallowed to recover for 24 hours in standard medium (i to l). Specimens were either fixed with 3% paraformaldehyde (a,b,e,f,i,j) or lysed beforefixation (c,d,g,h,k,l). Samples were then double stained with the anti-vimentin monoclonal antibody 7A3 (a,c,e,g,i,k) and aPcrys serum(b,d,f,h,j,l). Bar, 10 µm.

and were localised in a continuous fashion along the filameFurthermore, αB-crystallins appeared to be colocalised witthe nuclear membrane where aPcrys IgG gave a strong nucrim staining and at the cytoplasmic surface where staining walso observed along the edges.

Effect of elevated extracellular K + on the vimentinnetwork and on αB-crystallin localisationNIH 3T3 cells submitted to hypertonic stress showed copletely different dynamics. Cells appeared to be more flatteby an increase of K+ concentration to 100 mM for 24 hours inthe medium (Fig. 5a,d). αB-Crystallin accumulated in responseto high potassium, and a strong staining could be observethe nucleus as well as in the cytoplasm (Fig. 5b,d). A vimennetwork was redistributed around the nuclear membrane aperinuclear aggregate with some thick and short vimenfilaments extending toward the periphery without contactithe cytoplasmic membrane (Fig. 5a,c). αB-Crystallin appearedto colocalise with the collapsed vimentin network, and wivimentin bundles. In fixed cells, αB-crystallin not only con-centrated at the level of vimentin filaments, but a small amoof αB-crystallin remained diffused in the cytoplasm anshowed that the cellular volume was not affected by t

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rearrangement of the vimentin network (Fig. 5b). In cells lysprior to fixation, αB-crystallin localised as in the fixed cells:nucleus and vimentin filaments. We also noted a strong nuclrim staining with aPcrys IgG, which strongly resembled thnuclear lamin staining (Fig. 5b,d).

In cells submitted to high potassium for 48 hours, a mosevere cytoskeletal abnormality was detected. The ceappeared more damaged, in that they lost volume, and thflattened aspect (Fig. 5e to h). The vimentin IFs werearranged in a button-like pattern, and αB-crystallins wereconcentrated at the same location as vimentin. Cells lysed pto fixation showed that αB-crystallins remained attached to thecollapsed vimentin area (Fig. 5g,h). Longer exposure to hipotassium medium resulted in cell death (data not showInterestingly, cells treated with elevated K+ for 24 hours andthen allowed to recover for 12 hours in normal growth mediuwere able to recover their normal phenotype (Fig. 5i to l). Tvimentin network completely restored its normal cyto-archtectural shape, and the αB-crystallin became redistributedthroughout the cell. Furthermore, cells lysed prior to fixatiolost completely their αB-crystallin components (Fig. 5l),suggesting that αB-crystallins became soluble after the cellhad completely recovered from their stress.

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Fig. 6.Distribution of vimentin and αB-crystallin in heat-shockedand recovered NIH 3T3. Cells were heat-shocked at 43°C for 1 hoas described in Materials and Methods, and then were allowed torecover at 37°C for 1 hour (a,b), 2 hours (c,d), 8 hours (e,f) and 24hours (g,h). Cells were then fixed with 3% paraformaldehyde anddouble-stained with an anti-vimentin monoclonal antibody 7A3(a,c,e,g) and aPcrys antibody (b,d,f,h). Bar, 10 µm.

Effect of heat shock on the organisation of vimentinfilaments and αB-crystallinAfter a 43°C heat shock for one hour, cells were stained wanti-Pcrys and an anti-vimentin antibody at different timesrecovery at 37°C. One hour after heat shock, the vimen

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filament appeared to undergo an extensive rearrangemresembling a retraction of the IF network toward the nucleperiphery (Fig. 6a). The αB-crystallins were distributed incytoplasmic aggregates concentrated at the same positionthe vimentin filaments. Furthermore, αB-crystallin aggregateswere in close apposition with the nucleus (Fig. 6b). Two houafter heat shock, vimentin filaments were dramatically altereand appeared to have completely collapsed towards the nucperiphery; a clear empty space between the IF network and cytoplasmic surface could be seen. The αB-crystallin followedthe same cytoplasmic localisation as the vimentin cables (F6c,d). In most cells the clusters of αB-crystallin completelysurrounded the nucleus. When cells were allowed to recovfor eight hours at 37°C, the vimentin network re-extendetoward the cytoplasmic surface, while the αB-crystallinremained essentially around the nucleus and colocalised wthe still compact vimentin filaments (Fig. 6e,f). After a 24 hourecovery at 37°C, most cells had restored their vimentnetwork and appeared morphologically similar to the controcells (Fig. 6g). The αB-crystallin re-diffused throughout thecytoplasm (Fig. 6h).

DISCUSSION

Our results suggested the following conclusions. (1) In vitroperipherin, a type III neuronal IFP, interacted specifically witαB-crystallin in a temperature-dependent manner. (2) A costitutive level of αB-crystallin was expressed in non stresseNIH 3T3 cells. (3) Serum starvation did not affect the level oαB-crystallin expression, while heat shock treatment induceaccumulation of αB-crystallin with the apparition of an acidicform. (4) When cells were submitted to different stress coditions, αB-crystallins colocalised with the IFP network.

In vitro, αB-crystallin associated with intermediatefilament proteinsWe extended earlier in vitro studies reporting interactiobetween αB-crystallin and desmin (Bennardini et al., 1992)Firstly, we showed that αB-crystallin interacted also withperipherin. Secondly, we showed that the interaction betweαB-crystallin and both peripherin and vimentin was temperature-dependent (Fig. 1). The ability of αB-crystallin to bind toIFP at high temperatures strongly suggested a possible regutory role for αB-crystallin during the heat shock response. Inrecent work, the small heat shock protein Hsp25 is shown interact with citrate synthase (CS) at elevated temperatu(43°C) (Ehrnsperger et al., 1997). Using an in vitro aggregtion assay, the authors show that interaction of Hsp25 with Cprevents aggregation of this enzyme by immobilisinunfolding CS intermediates. Interestingly, Hsp25 did noprevent the inactivation of CS. Instead, reactivation of the Ccould be restored by addition of oxaloacetic acid (a substraand ligand of CS) or in the presence of another chaperoHsp70 in an ATP-dependent manner (Ehrnsperger et al., 199In analogy to the interaction between αB-crystallin and thetype III IFPs, αB-crystallin may also help to stabilise theseproteins from irreversible aggregation and denaturatioHowever, currently the analogy between the αB-crystallin-IFPand the Hsp25-CS interactions cannot be pushed further sinin vitro activity is not measurable for the IFPs. Furthermore

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2767αB-Crystallin acts as molecular chaperone for IFPs

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we showed, that the interaction between IFP-αB-crystallin wasATP-independent and did not imply any specific phosphorytion sites. These properties were consistent with the fact small heat shock proteins possess an ATP-independmolecular chaperone activity (Lee et al., 1995) Another smheat shock protein Hsp18.1 from Pisum sativum(pea) alsoprevents heat induced aggregation of CS and other model strates such as malate dehydrogenase and glyceraldehydphosphate dehydrogenase (Lee et al., 1997). Furthermore, of the substrates binds to Hsp18.1 at a specific and defined perature. In analogy, vimentin and peripherin also showedifferent temperature-dependence for their interaction wαB-crystallin. The fact that the vimentin-αB-crystallincomplex was highly abundant at 37°C may be predictedoccur in vivo. As reported for extracts of eye lens, vimenimmunoprecipitates with α-crystallin (Nicholl and Quinlan,1994). α-Crystallins can increase the soluble pool of GFAwhen added to preformed filaments (Nicholl and Quinla1994). Yet, when in vitro assembly of phakinin/filensin waperformed in the presence of α-crystallins there was no IF sol-ubilisation. Similarly, under our experimental conditions αB-crystallin did not induce solubilisation of polymerised peripherin, or vimentin. Thus, it seemed that αB-crystallin interactedwith soluble subunits (IFP polymers up to tetramers), as was with the fully polymerised 10 nm filament structures. addition, using a ligand-blotting assay we showed a specbinding of αB-crystallin with peripherin, and vimentin (Fig.2A). This assay provided a strong indication for a direct asciation between αB-crystallin and the IFPs. Furthermore, wshowed that the amino-terminal peptide (MDIAIHHPWI) oαB-crystallin interacted specifically with the IFPs (Fig. 2BThis result did not exclude the possibility that residues outsthe Pcrys region might also bind to the IFPs and/or play a rin the modulation of the interaction between αB-crystallin andIFPs. However, in vitro studies using recombinant αB-crys-tallin with mutations within the phenylalanine-rich regioindicate strongly that this region is involved in bindinunfolded proteins (Plater et al., 1996). When the N-terminaspartic acid (position 2) is point-mutated to a glycine,reduced efficiency in the chaperone activity is observed (Plaet al., 1996). Together with our data, this result seemedsuggest that the binding domain of αB-crystallin may beexpanding from the first N-terminal residue to, at least, tphenylalanine-rich region. However, further studies will brequired to characterise the binding region between αB-crys-tallin and IFPs more precisely.

Small heat-shock proteins, such as the α-crystallins(Klemenz et al., 1991b) act as molecular chaperones formcomplexes with a variety of cellular proteins (Schlesing1996). α-Crystallins are not the only chaperones associawith IFPs; several reports have shown interactions of hspwith IFP: (1) in vitro interaction of yeast hsp70 with vimentiand nuclear lamins is ATP-dependent (Georgatos et al., 19(2) the association of hsp70 with K8/K18 is ATP-dependeand promoted by heat stress; the addition of purified hsp 7keratin 8/18 during or after filament assembly does not affthe assembly process of IFPs (Liao et al., 1995). Interestinthe αB-crystallin-IFP interaction is ATP independent whereathe hsp70-IFP interaction is ATP dependent. This differenmay originate from a possible different mechanism in tchaperone activities of αB-crystallin and hsp70.

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αB-crystallin expressed at a constitutive level undernormal and stressed conditionsWe showed that αB-crystallin was expressed at a constitutivlevel in NIH 3T3 cells grown under standard condition (Fig3B) but the level of αB-crystallin remained very low comparedto that of the total proteins as previously reported (Klemenzal., 1991a). In cells submitted to serum starvation the levelexpression remained equal to that of the control cells. Cesubmitted to heat shock treatment showed an increase inaccumulation of αB-crystallin by a factor of ten. It is knownthat αB-crystallin could also accumulate in response to othstress conditions such as cadmium or arsenite expos(Klemenz et al., 1991b). Here, we reported for the first timthe accumulation of an acidic form of αB-crystallin after heatshock. Furthermore, on two-dimensional gels the coordinaof this acidic form were similar to the phosphorylated form olens αB-crystallin (Fig. 3). In the eye lens the phosphorylateresidues of α-crystallin are known. In αA-crystallin a singleserine at position 122 can be phosphorylated while in αB-crys-tallin two or three serines at positions 19, 45 and 59, resptively, can be phosphorylated (Voorter et al., 1986; Smith et a1992).

In vivo, αB-crystallin acted as a chaperone inresponse to stress on the vimentin IF networkIn vivo interaction between vimentin and αB-crystallin wasclearly detected when cells were submitted to different typof stress. Under standard conditions, αB-crystallin was dis-tributed throughout the cytoplasm and the majority remainsoluble (Fig. 4). Similar observations were previousdescribed for hsp24 in chicken embryo fibroblasts or hsp23Drosophilasalivary gland cells (Collier and Schlesinger, 1986Leicht et al., 1986). Serum starvation, however, inducedrearrangement of the vimentin network into thick, anstretched filaments, spreading from the nuclear periphery to cytoplasmic surface (Fig. 4). Interestingly, the αB-crystallinswere rearranged into small aggregates distributed radially aappearing to dock to the vimentin filaments. Furthermore, αB-crystallin seemed to be recruited from the soluble compartmto the insoluble fraction. Yet, the pattern of distribution of αB-crystallin followed the localisation of the vimentin filamentsPrevious in vitro studies have shown that α-crystallinsassociate with preformed GFAP filaments regularly every nm (Nicholl and Quinlan, 1994). Such results are in accordanwith the continuous pattern of decoration we observed in viv

When cells were submitted to high potassium, the vimennetwork collapsed toward the nuclear membrane and αB-crys-tallin relocalised at the same place (Fig. 5). We observed ta minor subpopulation of vimentin filaments remained linketo the nuclear envelope. These results indicated that, depenon the type of stress, αB-crystallin interacted with pre-existingIFs which appeared unaltered, but also followed aggregatedcollapsed IFs. This indicated a potential mechanism of intaction of αB-crystallin with well spread filaments and withaggregated IFs. It has been established that αB-crystallinincreased when C6 glioma cells were submitted to elevated+

concentration (Iwaki et al., 1995). It has been suggested tαB-crystallin accumulation supports the survival of the cellbut colocalisation of GFAP, and αB-crystallin has not beeninvestigated, previously.

When cells were subjected to heat shock treatment,

2768

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observed a complete rearrangement of the vimentin netwand a relocalisation of αB-crystallin at the same localisation avimentin (Fig. 6). Furthermore, the recovery of the stresscells led to the redistribution of the vimentin filaments whicslowly re-extended through the cytoplasmic space. The restoration of the vimentin network was concomitant with trelocalisation of the αB-crystallin throughout the cytoplasmSimilar observations during heat shock were reported bothinvertebrate, and vertebrate cell lines (Coss and Linnema1996). The induction of αB-crystallin upon heat shock has alsbeen related to a transient state of thermotolerance (Klemet al., 1991b; Head et al., 1994). Upon serum starvation, hytonic, or heat shock stress, we observed that αB-crystallin wastranslocated from the cytoplasm to the nucleus. Furthermothe labelling of αB-crystallin clearly showed a diffuse stainingin the nucleus, and a ring-like labelling. Our observations msuggest the recruitment of the αB-crystallin into the laminnetwork, but this remains to be explored. From these in vlocalisation experiments, we concluded that both the rearranment of the IFs and the relocalisation of αB-crystallin werefully reversible events during hypertonic, and heat shock streThe direct association of αB-crystallin and IFP may be signif-icant for the mechanism by which αB-crystallin protects theintegrity of the IFs either by modulating the level of their alteation, or by mediating the state of IFs aggregation.

We thank Dr R. Klemenz (University of Zurich, Switzerland) foproviding mouse αB-crystallin cDNA, Dr A. Tardieu and Dr Fr.Bonnete (University of Pierre et Marie Curie, France) for sharibovine lens extracts. We are grateful to Drs S. D. Georgatos (Unisity of Crete), B. Rost (EMBL, Heidelberg) and D. Martin for criticareading of the manuscript and B. Eddé (Collège de France, Parisuseful discussion. This work was supported by Centre National dRecherche Scientifique (CNRS, URA 2115), Institut National deSanté et de la Recherche Médicale (INSERM, grant 910810), Asciation Française contre les Myopathies (AFM) and Direction dRecherches-Etudes et Techniques (DRET, grant 94-2541A).

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(Received 25 April 1997 - Accepted 4 September 1997)