HSP25, a Small Heat Shock Protein Associated with Dense Bodies and M-lines of Body Wall Muscle in Caenorhabditis elegans

Abstract

HSP25, a previously uncharacterized member of the α-crystallin family of small heat shock proteins inCaenorhabditis elegans, has been examined using biochemical and immunological techniques. HSP25 is the second largest of 16 identifiable small heat shock proteins in the nematode and is expressed at all developmental stages under normal growth conditions. Recombinant HSP25 produced in Escherichia coli exists predominantly as small oligomers (dimers to tetramers) and possesses chaperone activity against citrate synthase in vitro. InC. elegans, HSP25 is localized to dense bodies and M-lines in body wall muscle, to the lining of the pharynx, and to the junctions between cells of the spermathecal wall. Affinity chromatography of nematode extracts on a column of immobilized HSP25 resulted in specific binding of vinculin and α-actinin but not actin, as revealed by Western blotting. These results suggest a role for HSP25 in the organization or maintenance of the myofilament lattice and adherens junctions in C. elegans. HSP25, a previously uncharacterized member of the α-crystallin family of small heat shock proteins inCaenorhabditis elegans, has been examined using biochemical and immunological techniques. HSP25 is the second largest of 16 identifiable small heat shock proteins in the nematode and is expressed at all developmental stages under normal growth conditions. Recombinant HSP25 produced in Escherichia coli exists predominantly as small oligomers (dimers to tetramers) and possesses chaperone activity against citrate synthase in vitro. InC. elegans, HSP25 is localized to dense bodies and M-lines in body wall muscle, to the lining of the pharynx, and to the junctions between cells of the spermathecal wall. Affinity chromatography of nematode extracts on a column of immobilized HSP25 resulted in specific binding of vinculin and α-actinin but not actin, as revealed by Western blotting. These results suggest a role for HSP25 in the organization or maintenance of the myofilament lattice and adherens junctions in C. elegans. small heat shock protein polyacrylamide gel electrophoresis RNA interference assays The small heat shock proteins (smHSPs),1 whose relationship to the α-crystallin family was first noted in Drosophila(1.Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 855-859Crossref PubMed Scopus (106) Google Scholar) and Caenorhabditis elegans (2.Russnak R.H Jones D. Candido E.P.M. Nucleic Acids Res. 1983; 11: 3187-3205Crossref PubMed Scopus (43) Google Scholar), have been found in all kingdoms of life and, thus, have an evolutionary history dating back to the common ancestor of all present day living cells. The subunit molecular weights of smHSPs range from approximately 12,000 to 43,000, although most fall within 17,000–30,000, and generally exist as large multimeric assemblies in solution (3.$$$$$$ ref data missingGoogle Scholar, 4.Ehrnsperger M. Buchner J. Gaestel M. Fink A.L. Goto Y. Molecular Chaperones in the Life Cycle of Proteins. Marcel Dekker, Inc., New York1997: 533-575Google Scholar, 5.de Jong W.W. Caspers G.-J. Leunissen J.A.M. Int. J. Biol. Macromol. 1998; 22: 151-162Crossref PubMed Scopus (439) Google Scholar). The first three-dimensional structure determined for a smHSP, Hsp16.5, from the thermophileMethanococcus jannaschii, revealed a spherical array of 24 subunits of a polypeptide consisting largely of β-sheet (6.Kim K.K Kim R. Kim S.-H. Nature. 1998; 394: 595-599Crossref PubMed Scopus (789) Google Scholar). An ATP-independent chaperone activity has been demonstrated in most smHSPs studied based upon their ability to prevent the aggregation and precipitation of denatured substrate proteins (7.Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1745) Google Scholar). In multicellular organisms, smHSPs are among the most highly induced heat shock proteins under stress conditions, and some smHSPs may also be subject to developmentally regulated expression in the absence of stress, as first noted in Drosophila studies (8.Arrigo A.-P. Landry J. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). Studies on the protective effect of mammalian hsp27 revealed that cells overexpressing this protein were mainly resistant to the heat-induced disruptions seen in the microfilament lattice (9.Laszlo A. Davidson T. Hu A. Landry J. Bedford D. Int. J. Radiat. Biol. 1993; 63: 569-581Crossref PubMed Scopus (22) Google Scholar, 10.Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar), and it was suggested that actin might be a major target of the protective effect of hsp27 (10.Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar). Subsequent work suggested that this protective effect was dependent on the ability of hsp27 to be phosphorylated in vivo (11.Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar). Recently, hsp27 was found to interfere with apoptosis induced by tumor necrosis factor α, probably by decreasing the level of reactive oxygen species and increasing the level of glutathione (12.Mehlen P. Préville X. Kretz-Remy C. Arrigo A.-P. EMBO J. 1996; 15: 2695-2706Crossref PubMed Scopus (518) Google Scholar). This protective function was found to be dependent on the formation of large hsp27 aggregates, and in contrast to the earlier work, mutants of hsp27 in which key phosphorylation sites were eliminated also formed large aggregates and conferred protection against tumor necrosis factor α (13.Mehlen P. Hickey E. Weber L.A. Arrigo A.-P. Biochem. Biophys. Res. Commun. 1997; 241: 187-192Crossref PubMed Scopus (198) Google Scholar). The ability to form large aggregates is a property of most smHSPs, however, and is not necessarily dependent upon phosphorylation, since some members of this family are not phosphorylated (14.Suzuki T.C. Krawitz D.C. Vierling E. Plant Physiol. 1998; 116: 1151-1161Crossref PubMed Scopus (42) Google Scholar). Our laboratory has extensively analyzed the expression and chaperone activity of four 16-kDa smHSPs in the nematode C. elegans(2.Russnak R.H Jones D. Candido E.P.M. Nucleic Acids Res. 1983; 11: 3187-3205Crossref PubMed Scopus (43) Google Scholar, 15.Jones D. Russnak R.H. Kay R.J. Candido E.P.M. J. Biol. Chem. 1986; 261: 12006-12015Abstract Full Text PDF PubMed Google Scholar, 16.Russnak R.H. Candido E.P.M. Mol. Cell. Biol. 1985; 5: 1268-1278Crossref PubMed Scopus (83) Google Scholar, 17.Jones D. Dixon D.K. Graham R.W. Candido E.P.M. DNA (N. Y.). 1989; 8: 481-490Crossref PubMed Scopus (38) Google Scholar, 18.Leroux M.R. Melki R. Gordon B. Batelier G. Candido E.P.M. J. Biol. Chem. 1997; 272: 24646-24656Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). These smHSPs, which are strictly stress-inducible and act as molecular chaperones, likely play important roles in enhancing survival of the animal under conditions of chemical and physical stress. The completion of the C. elegans genome sequence (19.The C. elegans Sequencing ConsortiumScience. 1998; 282: 2012-2018Crossref PubMed Scopus (3613) Google Scholar) provides a unique opportunity to investigate the range of functions that members of the smHSP family carry out under normal and stress conditions in a multicellular animal. Here we describe HSP25, a novel member of the C. elegans smHSP family that possesses chaperone activity and is associated with specific structures in the body wall muscle, pharynx, and spermatheca. hsp25 was amplified from first-strand cDNA with primers LD3 (5′-gat cat ATGCCC ACG TAC ACT CGA ACT-3′, forward primer, containing first ATG) or LD15 (5′-gat cat ATG TCG GAA CGC CGT ATC GAC-3′, forward primer, containing second ATG) and LD4 (5′-acg aag ctt TCA TTG CTG GAT TGC CAA-3′, reverse primer). In the primer sequences, restriction sites are underlined, and nucleotides in uppercase correspond to the genomic sequence. The amplifiedhsp25 gene (nucleotides 43–660 of thehsp25-coding region, Fig. 1 B, derived from GenBank™ sequence C09B8.6) was cloned into theNdeI-HindIII sites of pRSET (Invitrogen) following digestion with these enzymes. hsp25 was subcloned from pRSET23 into the NcoI-HindIII sites of pET28a(+) (Novagen) with primers LD22 (5′-tat cca tgg gc ATG TCG GAA CGC CGT ATC-3′, forward primer) and LD23 (5′-acc aag cttCTG GAT TGC CAA TTG TGG-3′, reverse primer). Thehsp25 gene was digested from pET23H6 and subcloned into XbaI and XhoI sites of pBluescript-SK(+) (Stratagene). pRSET23 was transformed into Escherichia coliBL21(DE3) cells, and soluble protein was expressed following induction with 1 mmisopropyl-1-thio-β-d-galactopyranoside (20.Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) 185, 60–89Google Scholar). Bacterial cell pellets were suspended in TEND buffer (50 mm Tris, 1 mm EDTA, 50 mm NaCl, 1 mmdithiothreitol, pH 7.5) with protease inhibitor mixture (2 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 5 mm EDTA). After sonication, the cell lysate was centrifuged at 12,000 ×g for 10 min, and the supernatant was passed through a DEAE column (1.5 × 5.4 cm, pre-equilibrated with TEND buffer). The unbound fraction was loaded on a Sephacryl S-300HR column (1.5 × 100 cm, 177-ml bed volume, 0.15 ml/min, 5 ml/fraction, pre-equilibrated and eluted with 50 mm Tris, 1 mm EDTA, 50 mm NaCl, 1 mm dithiothreitol, pH 8.0). Elution was monitored at 254 nm since HSP25 lacks tryptophan residues. Fractions were analyzed by 15% SDS-PAGE (21.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar). The purified protein was at pET23H6 was transformed into E. coliBL21(DE3) cells and in with μg/ml and the protein was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside (20.Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) 185, 60–89Google Scholar). Bacterial cell pellets were suspended in buffer (50 mm Tris, pH mm NaCl, and with mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml pepstatin A. After sonication, the cell lysate was centrifuged at 12,000 × for 10 The supernatant was loaded on a column 1 × cm, bed After with 50 of the column was with 10 of buffer (50 mm pH and eluted with in and was purified by chromatography on a Sephacryl S-300HR column as of purified HSP25 was with and to were four with of HSP25 with were purified from by precipitation with by against mm NaCl, mm pH were on S. PubMed Google Scholar) at were on a at for to at for nematode were suspended in SDS-PAGE buffer (21.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) and at 100 for was by Proteins were on 15% SDS-PAGE and to an by were with with E. coli E. D. Cold Spring Harbor Laboratory, Cold Spring Harbor, or with actin by or were by of developmental were as to approximately with the aggregation assays were out with nm citrate synthase with or HSP25 in 50 mm pH mm NaCl, mm were monitored at nm in a with a cell to The of citrate synthase at of was as an approximately of was loaded on a column pre-equilibrated with buffer and extensively with the nematode previously at were by min, in of buffer containing mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml pepstatin A. After at 12,000 × for 10 min, the supernatant was to the After unbound the column was first with 10 bed of with bed of buffer and eluted with proteins were against The proteins from the column were to a column and eluted with in and unbound proteins from the were analyzed by and the were with or to an by and with of were at or at as previously R.H. Candido E.P.M. Mol. Cell. Biol. 1985; 5: 1268-1278Crossref PubMed Scopus (83) Google Scholar, 17.Jones D. Dixon D.K. Graham R.W. Candido E.P.M. DNA (N. Y.). 1989; 8: 481-490Crossref PubMed Scopus (38) Google Scholar, 18.Leroux M.R. Melki R. Gordon B. Batelier G. Candido E.P.M. J. Biol. Chem. 1997; 272: 24646-24656Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). was based on the of and J. 1993; PubMed Google Scholar). were at in in at for and digested with in buffer mm Tris, pH for at After in at for 1 were with at with E. coli E. D. Cold Spring Harbor Laboratory, Cold Spring Harbor, or at for and with or at for In was with 1 HSP25 After were with mm pH containing 1 μg/ml and by RNA was from (nucleotides 43–660 of the hsp25 to A. S. Nature. 1998; PubMed Scopus Google Scholar). RNA was into the of of an Mol. Cell. Biol. PubMed Scopus Google Scholar) and a of the body wall with the protein gene a from A. This was by Western were to on with for approximately to and to for to and to for an The of the was monitored by HSP25 was as of 16 smHSP in a of the C. elegans genome using the for with the sequence of the stress-inducible protein as These are in Fig. 1 as a using Nucleic Acids Res. 22: PubMed Scopus Google Scholar) and 1996; Google Scholar). The largest of smHSPs are the with and are the stress-inducible members that have been previously D. Russnak R.H. Kay R.J. Candido E.P.M. J. Biol. Chem. 1986; 261: 12006-12015Abstract Full Text PDF PubMed Google Scholar, 16.Russnak R.H. Candido E.P.M. Mol. Cell. Biol. 1985; 5: 1268-1278Crossref PubMed Scopus (83) Google Scholar). be noted that and which are are also R.H. Candido E.P.M. Mol. Cell. Biol. 1985; 5: 1268-1278Crossref PubMed Scopus (83) Google Scholar), and these are as and and are 16-kDa proteins that and is not proteins are The second largest of four smHSPs, which are the members of this in or The form structures to in large and to be of chaperone activity in the assays M.R. Batelier G. Melki R. Candido E.P.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, M.R. Candido E.P.M. Jong W.W. 1998; PubMed Scopus Google Scholar). is a previously studied smHSP with chaperone activity that is expressed and for elegans B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). HSP25, the are and The have not been studied to HSP25 a of sequence with a protein from the nematode The of is between the of the proteins is in the with the C. it to HSP25 1 A, that these proteins may be of cDNA are for hsp25 and that this gene is since of these to the it was to and the cDNA by reverse as under and to cDNA using primer LD3 the in the GenBank™ by were primer LD15 the second Fig. 1 was in of a was A, and the cDNA was cloned into the of the is to or the protein in at to be the HSP25 cDNA was expressed in E. a soluble protein with an molecular of approximately was B, In the of we to to this protein as HSP25, although it is by that by the chromatography of HSP25 a with most of the protein between and and These data also with the results of using in which the species were and not HSP25 to form most smHSPs, the C. elegans M.R. Melki R. Gordon B. Batelier G. Candido E.P.M. J. Biol. Chem. 1997; 272: 24646-24656Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). HSP25 was in a chaperone M.R. Melki R. Gordon B. Batelier G. Candido E.P.M. J. Biol. Chem. 1997; 272: 24646-24656Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, Buchner J. R. U. Nature. 1992; PubMed Scopus Google Scholar, S. T. Buchner J. Scopus Google Scholar), which the heat-induced aggregation of citrate a of synthase of citrate synthase aggregation was and a of resulted in of aggregation HSP25 is an chaperone in this in specific activity to smHSPs that have been investigate the developmental of HSP25, nematode extracts were from all stages and with against Western that HSP25 is produced at all developmental stages in at normal 5 HSP25 is not in of following a heat shock 5 In these actin level of which is through was as an for In these the of and HSP25 were 5 B, and The developmental expression of HSP25 was examined by In and was in body wall B, and and in the lining of the and an of HSP25 was in the as was that the seen is specific to The is localized to a of cells the that these may be the cells J.N. R. Sci. PubMed Scopus Google Scholar). These cells at the of the and to the lining the HSP25 was also at the junctions between cells the spermathecal wall The is of cells that are by an of J.N. R. Sci. PubMed Scopus Google Scholar). HSP25 to be associated with in the spermatheca. In body wall muscle, HSP25 was localized to a of to the of the with consisting of B, and This is with the of HSP25 to the dense bodies and M-lines of the The dense bodies in nematode are the sites of of the actin or and correspond to in M-lines are to of and from the of the of the or R.H. The Caenorhabditis Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar). investigate the of the HSP25 in body muscle, we it to the in with an to the R.H. The Caenorhabditis Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar, R.H. J. Biol. 1985; PubMed Scopus (198) Google Scholar). is localized to dense bodies and M-lines in the nematode R.H. J. Biol. 1985; PubMed Scopus (198) Google Scholar). that the seen within the with and are the of HSP25 to the dense bodies and The however, also the junctions between cells C. J. Biol. PubMed Scopus Google Scholar), which was not the with Fig. and of HSP25 a that the M-lines of nematode R.H. J. Biol. 1985; PubMed Scopus (198) Google Scholar) was found to to the seen in the HSP25 cells investigate the that HSP25 might with specific in dense HSP25 a was to a and as an from were to the and proteins were eluted with Proteins that to the column were analyzed by and the were and with to dense body of the proteins and revealed a with an molecular and a of at molecular the and unbound were by Western with to actin and HSP25 A, and the was as The HSP25 eluted from the column likely resulted from the formation of oligomers between the immobilized HSP25 and HSP25 in the was in the unbound fraction but not in the In contrast to actin, Western of HSP25 column with to vinculin or α-actinin revealed that a large fraction of these proteins to the column A, molecular in the using these likely of vinculin and the proteins from the HSP25 column were to a HSP25, vinculin and α-actinin to that the is specific for the HSP25 B, RNA act as a for of expression in C. elegans A. S. Nature. 1998; PubMed Scopus Google Scholar, A. 15: Full Text Full Text PDF PubMed Scopus Google Scholar). of RNA to the of a gene results in and specific interference with the expression of that The effect is seen in the animal and and the of the gene in the of an HSP25 we out using RNA from the HSP25 gene (nucleotides conditions that produced of a gene in approximately of the of effect of HSP25 on was and the of the not The α-crystallin family of smHSPs are molecular in and cells (3.$$$$$$ ref data missingGoogle Scholar, 4.Ehrnsperger M. Buchner J. Gaestel M. Fink A.L. Goto Y. Molecular Chaperones in the Life Cycle of Proteins. Marcel Dekker, Inc., New York1997: 533-575Google Scholar, 5.de Jong W.W. Caspers G.-J. Leunissen J.A.M. Int. J. Biol. Macromol. 1998; 22: 151-162Crossref PubMed Scopus (439) Google Scholar). In these proteins act as of protein aggregation and precipitation (7.Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1745) Google Scholar). are of the of polypeptide that smHSPs are to denatured proteins and in a for with which may M. S. Gaestel M. Buchner J. EMBO J. 1997; Scopus Google Scholar, Vierling E. EMBO J. 1997; PubMed Scopus Google Scholar, S. Buchner J. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the genome sequence for C. elegans (19.The C. elegans Sequencing ConsortiumScience. 1998; 282: 2012-2018Crossref PubMed Scopus (3613) Google Scholar) provides a unique opportunity to the of small gene structure and function in an animal of the nematode genome for members of this family revealed 16 1 studies have that the 16-kDa smHSPs are strictly stress-inducible D. Russnak R.H. Kay R.J. Candido E.P.M. J. Biol. Chem. 1986; 261: 12006-12015Abstract Full Text PDF PubMed Google Scholar, 16.Russnak R.H. Candido E.P.M. Mol. Cell. Biol. 1985; 5: 1268-1278Crossref PubMed Scopus (83) Google Scholar, Jones D. Candido E.P.M. Mol. Biol. Cell. 1992; PubMed Scopus Google Scholar) and likely function to prevent protein under conditions. smHSPs, as B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and the four smHSPs are produced under normal conditions at specific stages C. elegans M.R. Batelier G. Melki R. Candido E.P.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, M.R. Candido E.P.M. Jong W.W. 1998; PubMed Scopus Google Scholar). This that may functions within or may with specific of protein The studies that HSP25 is localized to the dense bodies and M-lines of the within body wall This is the first of specific of elegans small heat shock The dense bodies in nematode cells, which are to the of cells and the dense of R.H. The Caenorhabditis Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar), are structures containing the proteins R.H. The Caenorhabditis Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar, R.H. J. Biol. 1985; PubMed Scopus (198) Google Scholar), vinculin R.J. R.H. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar), α-actinin R.H. The Caenorhabditis Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar, R.J. R.H. PubMed Scopus Google Scholar), R.H. R.J. Mol. Biol. Cell. 1996; PubMed Scopus Google Scholar), and actin J. J. Biol. PubMed Scopus Google Scholar). In to the of HSP25 to dense bodies and vinculin and α-actinin were found to to an HSP25 a between these The binding of vinculin and proteins associated in vivo with dense body to and the of binding of a actin, suggest that the a The of a the of actin, and proteins to specific sites in R.H. R.J. Mol. Biol. Cell. 1996; PubMed Scopus Google Scholar), and the of a smHSP to these sites in nematode body wall that HSP25 may be in the or of roles for HSP25 might be as a chaperone associated with protein or in the maintenance of structures within cells. has been that of a major fraction of proteins the A.L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and the of proteins cell in the the A.L. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in of heat shock factor in mammalian cells A. Morimoto R.J. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar), and of all members of the heat shock factor family in cells Y. A. M. J. Biochem. 1998; PubMed Scopus Google Scholar), in the induction of all of the In this a of HSP25 might not to a in the absence of or of large protein with this is the that member of the smHSP and the protein A. Y. Y. M. S. S. J. Biol. 1998; PubMed Scopus Google Scholar). this chaperone has been in to from heat-induced the smHSP is not heat-induced in but is This is with the that cells are and to heat and and that the of smHSP might be In this it is that the sites of HSP25 may sites from which the chaperone be stress. at present we out the that the vinculin and α-actinin by HSP25 may have been and the of with actin might that actin is under the conditions may be for the of effect seen in hsp25 it is that of member of the smHSP family might have for a in the of an HSP25 might be under specific conditions. the present of the of we out the that gene may be resistant to The of an hsp25 among these smHSP, from the mammalian nematode D. is to C. elegans HSP25 This protein was also found to be and that to to the to the on the of and W. 1996; PubMed Scopus Google Scholar). The dense bodies of body wall cells through to the and, to the R.H. The Caenorhabditis Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar). the of D. is with that of HSP25 in C. elegans at the level of that these proteins may functions in nematode noted smHSPs containing substrate proteins have been to with chaperones, in the of protein these and results for the of molecular and for smHSPs in the maintenance of of C. elegans body wall of and molecular in C. elegans, be to the roles of HSP25 in these are to for of and for and to for