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• W2051139405 abstract "Synemin is a cytoskeletal protein originally identified as an intermediate filament (IF)-associated protein because of its colocalization and copurification with the IF proteins desmin and vimentin in muscle cells. Our sequencing studies have shown that synemin is an unusually large member (1,604 residues, 182,187 Da) of the IF protein superfamily, with the majority of the molecule consisting of a long C-terminal tail domain. Molecular interaction studies demonstrate that purified synemin interacts with desmin, the major IF protein in mature muscle cells, and with α-actinin, an integral myofibrillar Z-line protein. Furthermore, expressed synemin rod and tail domains interact, respectively, with desmin and α-actinin. Analysis of endogenous protein expression in SW13 clonal lines reveals that synemin is coexpressed and colocalized with vimentin IFs in SW13.C1 vim+ cells but is absent in SW13.C2 vim− cells. Transfection studies indicate that synemin requires the presence of another IF protein, such as vimentin, in order to assemble into IFs. Taken in toto, our results suggest synemin functions as a component of heteropolymeric IFs and plays an important cytoskeletal cross-linking role by linking these IFs to other components of the cytoskeleton. Synemin in striated muscle cells may enable these heterofilaments to help link Z-lines of adjacent myofibrils and, thereby, play an important role in cytoskeletal integrity. Synemin is a cytoskeletal protein originally identified as an intermediate filament (IF)-associated protein because of its colocalization and copurification with the IF proteins desmin and vimentin in muscle cells. Our sequencing studies have shown that synemin is an unusually large member (1,604 residues, 182,187 Da) of the IF protein superfamily, with the majority of the molecule consisting of a long C-terminal tail domain. Molecular interaction studies demonstrate that purified synemin interacts with desmin, the major IF protein in mature muscle cells, and with α-actinin, an integral myofibrillar Z-line protein. Furthermore, expressed synemin rod and tail domains interact, respectively, with desmin and α-actinin. Analysis of endogenous protein expression in SW13 clonal lines reveals that synemin is coexpressed and colocalized with vimentin IFs in SW13.C1 vim+ cells but is absent in SW13.C2 vim− cells. Transfection studies indicate that synemin requires the presence of another IF protein, such as vimentin, in order to assemble into IFs. Taken in toto, our results suggest synemin functions as a component of heteropolymeric IFs and plays an important cytoskeletal cross-linking role by linking these IFs to other components of the cytoskeleton. Synemin in striated muscle cells may enable these heterofilaments to help link Z-lines of adjacent myofibrils and, thereby, play an important role in cytoskeletal integrity. Intermediate filaments (IFs), 1The abbreviations used are:IFintermediate filamentBSAbovine serum albuminECLenhanced chemiluminescencekbkilobase(s)mAbmonoclonal antibodypAbpolyclonal antibodyPAGEpolyacrylamide gel electrophoresisUTRuntranslated region1The abbreviations used are:IFintermediate filamentBSAbovine serum albuminECLenhanced chemiluminescencekbkilobase(s)mAbmonoclonal antibodypAbpolyclonal antibodyPAGEpolyacrylamide gel electrophoresisUTRuntranslated region along with actin-containing microfilaments and tubulin-containing microtubules, are one of the three major classes of cytoskeletal filaments in multicellular animals (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 2Robson R.M. Curr. Opin. Cell Biol. 1989; 1: 36-43Crossref PubMed Scopus (58) Google Scholar, 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar, 4Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (830) Google Scholar). The IFs, which are considered to play an important role in structure and mechanical integration of cellular space (5Lazarides E. Nature. 1980; 283: 249-256Crossref PubMed Scopus (1443) Google Scholar, 6Goldman R.D. Khuon S. Chou Y.H. Opal P. Steinert P.M. J. Cell Biol. 1996; 134: 971-983Crossref PubMed Scopus (310) Google Scholar), are composed of cell type-specific proteins that have been divided into classes based upon sequence comparisons (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar, 7Klymkowsky M.W. Curr. Opin. Cell Biol. 1995; 7: 46-54Crossref PubMed Scopus (102) Google Scholar). The members of this protein superfamily have within their sequence a conserved rod domain, which promotes coiled-coil interactions between two individual IF proteins and formation of an IF protein dimer, the first step in assembly of the ∼10 nm diameter IFs (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar, 8Ip W. Heuser J.E. Pang Y.Y. Hartzer M.K. Robson R.M. Ann. N. Y. Acad. Sci. 1985; 455: 185-199Crossref PubMed Scopus (21) Google Scholar, 9Meng J. Khan S. Ip W. J. Biol. Chem. 1996; 271: 1599-1604Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Herrmann H. Aebi U. Curr. Opin. Struct. Biol. 1998; 8: 177-185Crossref PubMed Scopus (141) Google Scholar, 11Herrmann H. Haner M. Brettel M. Ku N.O. Aebi U. J. Mol. Biol. 1999; 286: 1403-1420Crossref PubMed Scopus (213) Google Scholar). Flanking the rod domain are N-terminal head and C-terminal tail domains that vary considerably in size and sequence among the IF protein classes (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar, 4Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (830) Google Scholar). Most IF proteins are grouped into five major classes or types (I–V) based upon sequence analysis (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 2Robson R.M. Curr. Opin. Cell Biol. 1989; 1: 36-43Crossref PubMed Scopus (58) Google Scholar, 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar, 4Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (830) Google Scholar). Some classes of IF proteins, such as the type I and II keratins (3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar) and the type IV neurofilament proteins (12Lee M.K. Xu Z. Wong P.C. Cleveland D.W. J. Cell Biol. 1993; 122: 1337-1350Crossref PubMed Scopus (315) Google Scholar, 13Ching G.Y. Liem R.K. J. Cell Biol. 1993; 122: 1323-1335Crossref PubMed Scopus (231) Google Scholar), are known to form obligate heteropolymers in vivo, resulting in IFs that consist of at least two different IF proteins. In contrast, IFs containing type III proteins, such as desmin or vimentin, often are considered homopolymeric IFs (3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar, 14Abumuhor I.A. Spencer P.H. Cohlberg J.A. J. Struct. Biol. 1998; 123: 187-198Crossref PubMed Scopus (13) Google Scholar) because each of these individually purified proteins readily assemble into synthetic IFs in vitro (8Ip W. Heuser J.E. Pang Y.Y. Hartzer M.K. Robson R.M. Ann. N. Y. Acad. Sci. 1985; 455: 185-199Crossref PubMed Scopus (21) Google Scholar,15Huiatt T.W. Robson R.M. Arakawa N. Stromer M.H. J. Biol. Chem. 1980; 255: 6981-6989Abstract Full Text PDF PubMed Google Scholar, 16Geisler N. Weber K. Eur. J. Biochem. 1980; 111: 425-433Crossref PubMed Scopus (84) Google Scholar, 17Ip W. Hartzer M.K. Pang Y.Y. Robson R.M. J. Mol. Biol. 1985; 183: 365-375Crossref PubMed Scopus (111) Google Scholar). intermediate filament bovine serum albumin enhanced chemiluminescence kilobase(s) monoclonal antibody polyclonal antibody polyacrylamide gel electrophoresis untranslated region intermediate filament bovine serum albumin enhanced chemiluminescence kilobase(s) monoclonal antibody polyclonal antibody polyacrylamide gel electrophoresis untranslated region The type III IF proteins vimentin and desmin are the major IF proteins of developing and mature striated muscle cells, respectively (18Bennett G.S. Fellini S.A. Toyama Y. Holtzer H. J. Cell Biol. 1979; 82: 577-584Crossref PubMed Scopus (168) Google Scholar, 19Granger B.L. Lazarides E. Cell. 1979; 18: 1053-1063Abstract Full Text PDF PubMed Scopus (218) Google Scholar). Synemin and paranemin, a pair of relatively high molecular weight proteins identified in the early 1980s, were initially described as IF-associated proteins because they copurified in the initial purification steps with desmin and vimentin and colocalized with them in muscle cells (2Robson R.M. Curr. Opin. Cell Biol. 1989; 1: 36-43Crossref PubMed Scopus (58) Google Scholar, 20Granger B.L. Lazarides E. Cell. 1980; 22: 727-738Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 21Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar, 22Price M.G. Lazarides E. J. Cell Biol. 1983; 97: 1860-1874Crossref PubMed Scopus (77) Google Scholar, 23Foisner R. Wiche G. Curr. Opin. Cell Biol. 1991; 3: 75-81Crossref PubMed Scopus (94) Google Scholar, 24Bellin R.M. Sernett S.W. Robson R.M. Kreis T. Vale R. Guidebook to the Cytoskeleton and Motor Proteins. 2nd Ed. Oxford University Press, UK1999: 322-324Google Scholar). Recent cloning and sequencing studies in our laboratory, however, demonstrate that both synemin and paranemin contain the ∼310-amino acid rod domain characteristic of IF proteins and, therefore, are members of the IF protein superfamily (25Becker B. Bellin R.M. Sernett S.W. Huiatt T.W. Robson R.M. Biochem. Biophys. Res. Commun. 1995; 213: 796-802Crossref PubMed Scopus (40) Google Scholar, 26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Those results, along with their colocalization (20Granger B.L. Lazarides E. Cell. 1980; 22: 727-738Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 22Price M.G. Lazarides E. J. Cell Biol. 1983; 97: 1860-1874Crossref PubMed Scopus (77) Google Scholar, 26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 27Bilak S.R. Sernett S.W. Bilak M.M. Bellin R.M. Stromer M.H. Huiatt T.W. Robson R.M. Arch. Biochem. Biophys. 1998; 355: 63-76Crossref PubMed Scopus (59) Google Scholar), suggest synemin and paranemin may form heteropolymeric IFs with the type III proteins desmin and/or vimentin in vivo (26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar,27Bilak S.R. Sernett S.W. Bilak M.M. Bellin R.M. Stromer M.H. Huiatt T.W. Robson R.M. Arch. Biochem. Biophys. 1998; 355: 63-76Crossref PubMed Scopus (59) Google Scholar). Our hypothesis is that synemin acts as a component of heteropolymeric IFs with vimentin and/or desmin and helps attach these IFs to other cytoskeletal structures. Based upon localization of IFs at the periphery of, and between, Z-lines of adjacent myofibrils (19Granger B.L. Lazarides E. Cell. 1979; 18: 1053-1063Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 28Richardson F.L. Stromer M.H. Huiatt T.W. Robson R.M. Eur. J. Cell Biol. 1981; 26: 91-101PubMed Google Scholar, 29Tokuyasu K.T. Dutton A.H. Singer S.J. J. Cell Biol. 1983; 96: 1727-1735Crossref PubMed Scopus (120) Google Scholar), synemin-containing heteropolymeric IFs may help link adjacent myofibrils in striated muscle cells. In this paper, we describe the complete sequence of synemin, which establishes it as a unique IF protein with a long C-terminal extension, which is not readily grouped with any of the well established IF protein types. Transfection of full-length synemin into SW13 clonal lines demonstrates that synemin requires another IF protein for assembly into IFs. We demonstrate specific molecular interactions between synemin and desmin, the major IF protein present in most mature muscle cells (18Bennett G.S. Fellini S.A. Toyama Y. Holtzer H. J. Cell Biol. 1979; 82: 577-584Crossref PubMed Scopus (168) Google Scholar, 19Granger B.L. Lazarides E. Cell. 1979; 18: 1053-1063Abstract Full Text PDF PubMed Scopus (218) Google Scholar), and between the large tail domain of synemin and α-actinin, an integral protein of myofibrillar Z-lines (30Yamaguchi M. Izumimoto M. Robson R.M. Stromer M.H. J. Mol. Biol. 1985; 184: 621-644Crossref PubMed Scopus (85) Google Scholar) and costameres (31Imanaka-Yoshida K. Danowski B.A. Sanger J.M. Sanger J.W. Cell Motil. Cytoskeleton. 1996; 33: 263-275Crossref PubMed Scopus (20) Google Scholar) of striated muscle cells, and of adhesion plaques of many other cell types (32Yamada K. Geiger B. Curr. Opin. Cell Biol. 1997; 9: 76-85Crossref PubMed Scopus (517) Google Scholar). In toto, the studies herein help establish synemin as an important member of the IF protein superfamily and one that likely functions as a component of heteropolymeric IFs that can interact with α-actinin and, thereby, enable IFs to link other components of the cytoskeleton. Initial cloning studies on synemin from our laboratory (25Becker B. Bellin R.M. Sernett S.W. Huiatt T.W. Robson R.M. Biochem. Biophys. Res. Commun. 1995; 213: 796-802Crossref PubMed Scopus (40) Google Scholar) described only sequence of the rod domain portion of synemin. Additional clones encoding parts of the full-length synemin cDNA were retrieved from the same λgt11 library, prepared from adult chicken gizzard, by hybridization screening. Sequencing of the entire length of both strands of clones 47, 108, 135 and 244, and multiple internal sites of all other clones shown in Fig. 1, was done on Applied Biosystems 373 and 377 sequencers at the Iowa State University Sequencing and Synthesis Facility. Confirmation of the 5′ end of the sequence was done by 5′-rapid amplification of cDNA ends (33Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4329) Google Scholar) with a kit from Life Technologies, Inc., by using a primer (nucleotides 144–162) from the 5′ end of clone 108. The longest rapid amplification of cDNA ends clones produced start at the same nucleotide as the 244 clone. Computer analysis of the synemin cDNA sequence was carried out by using version 10 of the Wisconsin Package, Genetics Computer Group (GCG), Madison, WI, and the NCBI BLAST server (34Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69731) Google Scholar). Synemin polyclonal antibodies (pAb) 2856 were produced in rabbits injected with native purified protein essentially as described (35Knudson K.A. Anal. Biochem. 1985; 147: 285-288Crossref PubMed Scopus (136) Google Scholar). The pAbs were characterized by Western blotting, and they labeled only the 230-kDa synemin band present in fresh, avian whole muscle homogenates. Additionally, these antibodies labeled purified samples of both the expressed rod and C-terminal tail domains of synemin. Aliquots of these antibodies also were affinity purified by utilizing a column of purified, intact synemin coupled to CNBr-activated Sepharose 4B (Sigma). Vimentin monoclonal antibody (mAb) AMF-17b (developed by Dr. A. B. Fulton) was obtained from the Developmental Studies Hybridoma Bank. Immunocytochemistry studies with SW13.C1 vim+ and SW13.C2 vim− cells were done similarly to those described in Hemkenet al. (26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) but utilized synemin pAb 2856. For Western blotting, cell lysates of SW13.C1 vim+ and SW13.C2 vim− cells were separated into supernatant and pellet fractions to concentrate the cytoskeletal proteins in the pellets by the method of Athlan et al. (36Athlan E.S. Sacher M.G. Mushynski W.E. J. Neurosci. Res. 1997; 47: 300-310Crossref PubMed Scopus (26) Google Scholar). The resulting samples were analyzed by standard procedures (37Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44846) Google Scholar), using enhanced chemiluminescence (ECL) and blot stripping according to the manufacturer's procedure described in the ECL Western blotting protocols guidebook (Amersham Pharmacia Biotech). Total RNA was prepared from SW13.C1 vim+ and SW13.C2 vim− cells by the standard guanidine isothiocyanate method (38Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons Inc., New York1997Google Scholar), and blots were probed with avian cDNA probes corresponding to either the synemin rod or tail domain by using the GeneImages kit (Amersham Pharmacia Biotech). The kit instructions were followed, except that the gel transfer was carried out by using 50 mmNaOH as the transfer fluid, and the hybridization and final stringency washes were done at 47 °C. Studies were carried out essentially as described (26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), with the following changes. Full-length synemin cDNA was assembled from overlapping clones and inserted into the pcDNA3 eukaryotic expression vector (Invitrogen). The cDNA construct was transfected into SW13.C2 vim− cells with FuGENE 6 reagent (Roche Molecular Biochemicals), utilizing an empirically determined ratio of 3 μl of transfection reagent to 1 μg of DNA for a 60-mm diameter dish. Proteins were visualized approximately 40 h after transfection by immunocytochemistry. All proteins purified from tissue were prepared from adult turkey gizzards quick frozen immediately postmortem to minimize proteolysis. Synemin, in particular, is highly susceptible to proteolytic degradation (27Bilak S.R. Sernett S.W. Bilak M.M. Bellin R.M. Stromer M.H. Huiatt T.W. Robson R.M. Arch. Biochem. Biophys. 1998; 355: 63-76Crossref PubMed Scopus (59) Google Scholar). Intact synemin (27Bilak S.R. Sernett S.W. Bilak M.M. Bellin R.M. Stromer M.H. Huiatt T.W. Robson R.M. Arch. Biochem. Biophys. 1998; 355: 63-76Crossref PubMed Scopus (59) Google Scholar), desmin (15Huiatt T.W. Robson R.M. Arakawa N. Stromer M.H. J. Biol. Chem. 1980; 255: 6981-6989Abstract Full Text PDF PubMed Google Scholar), and α-actinin (39Craig S.W. Lancashire C.L. Cooper J.A. Methods Enzymol. 1982; 85: 316-321Crossref PubMed Scopus (39) Google Scholar) were purified by standard methods. The rod (nucleotides 138–1047) and tail (nucleotides 1048–4917) domains of synemin were produced by bacterial expression using pProEX HT vectors (Life Technologies, Inc.). The rod domain was expressed inEscherichia coli XL-I Blue (Stratagene) and purified by using nickel-nitrilotriacetic acid resin (Qiagen) in non-denaturing conditions. The tail domain was expressed in the protease-deficientE. coli strain BL21 (DE3) (Stratagene) and purified from inclusion bodies by dissolving the pellet fraction resulting from centrifugation (40,000 × g for 30 min) in 6m urea, 10 mm Tris-HCl, pH 8.5, and dialyzing into 1 mm EGTA, 10 mm Tris-HCl, pH 8.5, before nickel-nitrilotriacetic acid (Qiagen) chromatography. The interaction of purified intact synemin with purified desmin was tested by using three different sets of conditions as follows: 1) for soluble desmin, desmin and synemin were mixed in 10 mm Tris-HCl, pH 8.5; 2) for filament forming conditions, desmin and synemin were first mixed in 10 mm Tris-HCl, pH 8.5, and then the mixture was adjusted to IF-forming conditions by titrating the pH to 7.0 with addition of 2m imidazole HCl, pH 6.0, and by addition of MgCl2 and NaCl to 1 and 100 mm, respectively; 3) for pre-formed filaments, desmin by itself was first assembled into filaments in 100 mm NaCl, 1 mmMgCl2, 10 mm imidizole HCl, pH 7.0, and then the desmin filaments and synemin were mixed. For each of the three sets of conditions, an equal amount by weight (25 μg) of purified intact synemin and purified intact desmin were used in each sample. Bovine serum albumin (BSA) (Sigma) was added (10 μg) to each sample as an internal control. High speed centrifugation conditions (100,000 ×g for 20 min), chosen to sediment desmin filaments but not synemin alone, were used to sediment desmin IFs and any associated protein(s). The resulting supernatants and pellets were analyzed by SDS-PAGE. Individual samples of desmin and of synemin, in each of the three sets of conditions, also were subjected to the same high speed centrifugation in order to test their sedimentation behavior in the absence of the other protein. The interaction of the synemin rod domain with desmin, under filament-forming conditions, also was tested as described for intact synemin. Highly purified samples of desmin, α-actinin, and synemin prepared from turkey gizzard, together with a sample of whole gizzard homogenate, were subjected to SDS-PAGE. The proteins were transferred electrophoretically to a nitrocellulose membrane (37Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44846) Google Scholar, 40Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5889) Google Scholar), which was then blocked by incubation in IF buffer (100 mm NaCl, 1 mm MgCl2, 10 mm imidazole HCl, pH 7.0) containing 0.1% (v/v) Tween 20 and 5% (w/v) non-fat milk powder. Blots were incubated with purified synemin or with bacterially expressed synemin domains, at 10 μg/ml in IF buffer containing 0.1% (v/v) Tween 20 and 1% (w/v) non-fat milk powder, and then washed thoroughly with several fresh changes of the latter buffer. A control blot was treated identically but incubated with buffer containing no synemin. Protein interactions were detected with affinity purified synemin pAb 2856, diluted 1:10,000 in phosphate-buffered saline containing 0.1% Tween 20 and 5% non-fat milk powder, and visualized by ECL. Several overlapping cDNA clones that encompass the complete cDNA sequence for avian muscle synemin were obtained (Fig. 1). The full-length sequence is 8,615 base pairs (GenBankTM accession numberU28143), which corresponds well with the 8.4-kb transcript size we have shown previously by Northern blot analysis of avian smooth muscle RNA with a synemin cDNA probe (25Becker B. Bellin R.M. Sernett S.W. Huiatt T.W. Robson R.M. Biochem. Biophys. Res. Commun. 1995; 213: 796-802Crossref PubMed Scopus (40) Google Scholar). Analysis of the sequence reveals a single open reading frame followed by a long (3,330 base pairs) 3′-untranslated region (UTR) (Fig. 1). The open reading frame (4,812 base pairs) of the complete sequence codes for a protein with a predicted molecular mass of 182,187 Da. The sequence mass is smaller than that estimated (230 kDa) for synemin by SDS-PAGE (20Granger B.L. Lazarides E. Cell. 1980; 22: 727-738Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 27Bilak S.R. Sernett S.W. Bilak M.M. Bellin R.M. Stromer M.H. Huiatt T.W. Robson R.M. Arch. Biochem. Biophys. 1998; 355: 63-76Crossref PubMed Scopus (59) Google Scholar). This size difference may be explained, as it was for the IF protein paranemin (26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), by the acidic nature (pI = 4.85) of synemin, which has been shown for other proteins to repel SDS and result in a slower relative migration by SDS-PAGE (41Bryan J. J. Muscle Res. Cell Motil. 1989; 10: 95-96Crossref PubMed Scopus (28) Google Scholar). As we reported in a preliminary study (25Becker B. Bellin R.M. Sernett S.W. Huiatt T.W. Robson R.M. Biochem. Biophys. Res. Commun. 1995; 213: 796-802Crossref PubMed Scopus (40) Google Scholar), and as shown in Fig. 2, the synemin sequence contains the conserved, ∼310 amino acid rod domain characteristic of IF proteins. Of the sequences in GenBankTM, this region has the highest sequence identity (∼31 to 33%) with the type III IF proteins desmin, vimentin, peripherin, and glial fibrillary acidic protein, which is much lower than the >70% identity generally observed for IF proteins within the same “type” (3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1275) Google Scholar). Thus, synemin can not be classified as a specific type IF protein solely by cDNA sequence comparisons. The rod domain (304 amino acids) of synemin is flanked by a short N-terminal head domain of 10 amino acid residues, and a C-terminal tail domain of 1,290 amino acid residues (Fig. 2). This tail domain is extremely long for an IF protein and lacks significant homology with all other proteins. Other than the homology with the rod domains of other IF proteins, the sequence of synemin shows no significant homology/identity to other known proteins in GenBankTM. There are, however, notable regions of identity with a human EST sequence (gb_est24:AB077476) and with an unidentified human cDNA clone (GenBankTMaccession number AB002351). In order to determine the ability of synemin to assemble into IFs in vivo, the SW13 cell line was utilized. This cell line has been separated into specific clonal lines, including the SW13.C1 vim+, which has an endogenous vimentin IF network, and the SW13.C2 vim−, which lacks any cytoplasmic IFs (42Sarria A.J. Lieber J.G. Nordeen S.K. Evans R.M. J. Cell Sci. 1994; 107: 1593-1607PubMed Google Scholar). These clonal lines have been used in several studies (e.g. Refs. 26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar and 43Chen W.J. Liem R.K. J. Cell Sci. 1994; 107: 2299-2311PubMed Google Scholar) to characterize assembly of transfected IF proteins into IFs in the presence and absence of the cytoplasmic IF protein vimentin. We first characterized the cell line before we used it for the transfection studies. Western blot analysis of lysates of these clonal lines (Fig. 3) showed, as expected, that synemin was absent in the SW13.C2 vim− cells (Fig. 3, panel A, lanes 4 and 5) but, surprisingly, that synemin was already present in the cytoskeletal protein-containing pellet fraction of SW13.C1 vim+ cells (Fig. 3,panel B, lane 3). Northern blot analysis showed that the mRNA for synemin (∼9 kb) was present in SW13.C1 vim+ cells but absent in the SW13.C2 vim− cells. Thus, synemin has the same pattern of transcription as previously shown for the mRNA of vimentin in these clonal lines (42Sarria A.J. Lieber J.G. Nordeen S.K. Evans R.M. J. Cell Sci. 1994; 107: 1593-1607PubMed Google Scholar). Double label immunofluorescence of SW13.C1 vim+ cells, utilizing the same antibodies as for the Western blotting experiments, also show that they express synemin as a component of their vimentin-containing IF network (Fig.4, panels A and B). And, consistent with the Western and Northern blot analyses, immunofluorescence labeling of the SW13.C2 vim− cells, known to lack an endogenous vimentin IF network, with synemin pAbs showed no labeling of synemin (Fig. 4, panels C and D).Figure 4Immunofluorescence localization of synemin and vimentin in SW13 cells. Panels on the left(A, C, and E) depict immunofluorescence labeling with synemin pAb 2856. Panels on theright (B, D, and F) depict fluorescence labeling with vimentin mAb AMF-17b. Panels Aand B show endogenous synemin/vimentin expression in a filamentous pattern typical of IFs in the SW13.C1 vim+ cells.Panels C and D show the lack of endogenous synemin/vimentin expression in the SW13.C2 vim− cells. Note that synemin colocalizes with vimentin in SW13.C1 vim+ cells (panels A and B) but is absent from SW13.C2 vim− cells as is vimentin (panels C and D). Panels Eand F show immunofluorescent labeling of SW13.C2 vim− cells after transfection with full-length synemin cDNA. Note that the synemin expressed in the SW13.C2 vim− cells appears in a non-filamentous, punctate pattern when vimentin is absent (panel E). Bar, 10 μm for A–F.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Transfection of SW13.C2 vim− cells with full-length synemin cDNA resulted in cells that contain punctate aggregates when observed by immunofluorescence (Fig. 4, panels E and F). The punctate aggregates were similar to those seen for paranemin expressed in the same vimentin-negative cell line (26Hemken P.M. Bellin R.M. Sernett S.W. Becker B. Huiatt T.W. Robson R.M. J. Biol. Chem. 1997; 272: 32489-32499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and for assembly-deficient mutants of desmin in other cells lacking IFs (44Raats J.M. Pieper F.R. Vree Egberts W.T. Verrijp K.N. Ramaekers F.C. Bloemendal H. J. Cell Biol. 1990; 111: 1971-1985Crossref PubMed Scopus (66) Google Scholar). These results suggest that synemin cannot form an IF network without another IF protein, such as vimentin, present. Experiments were conducted to characterize interactions between synemin and desmin. Analysis of the int" @default.
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• W2051139405 title "Molecular Characteristics and Interactions of the Intermediate Filament Protein Synemin" @default.
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