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• W2017408747 abstract "Three mutations (R120G, Q151X, and 464delCT) in the small heat shock protein αB-crystallin cause inherited myofibrillar myopathy. In an effort to elucidate the molecular basis for the associated myopathy, we have determined the following for these mutant αB-crystallin proteins: (i) the formation of aggregates in transfected cells; (ii) the partition into different subcellular fractions; (iii) the phosphorylation status; and (iv) the ability to interact with themselves, with wild-typeαB-crystallin, and with other small heat shock proteins that are abundant in muscles. We found that all three αB-crystallin mutants have an increased tendency to form cytoplasmic aggregates in transfected cells and significantly increased levels of phosphorylation when compared with the wild-type protein. Although wild-type αB-crystallin partitioned essentially into the cytosol and membranes/organelles fractions, mutant αB-crystallin proteins partitioned additionally into the nuclear and cytoskeletal fractions. By using various protein interaction assays, including quantitative fluorescence resonance energy transfer measurements in live cells, we found abnormal interactions of the various αB-crystallin mutants with wild-type αB-crystallin, with themselves, and with the other small heat shock proteins Hsp20, Hsp22, and possibly with Hsp27. The collected data suggest that eachαB-crystallin mutant has a unique pattern of abnormal interaction properties. These distinct properties of the αB-crystallin mutants identified are likely to contribute to a better understanding of the gradual manifestation and clinical heterogeneity of the associated myopathy in patients. Three mutations (R120G, Q151X, and 464delCT) in the small heat shock protein αB-crystallin cause inherited myofibrillar myopathy. In an effort to elucidate the molecular basis for the associated myopathy, we have determined the following for these mutant αB-crystallin proteins: (i) the formation of aggregates in transfected cells; (ii) the partition into different subcellular fractions; (iii) the phosphorylation status; and (iv) the ability to interact with themselves, with wild-typeαB-crystallin, and with other small heat shock proteins that are abundant in muscles. We found that all three αB-crystallin mutants have an increased tendency to form cytoplasmic aggregates in transfected cells and significantly increased levels of phosphorylation when compared with the wild-type protein. Although wild-type αB-crystallin partitioned essentially into the cytosol and membranes/organelles fractions, mutant αB-crystallin proteins partitioned additionally into the nuclear and cytoskeletal fractions. By using various protein interaction assays, including quantitative fluorescence resonance energy transfer measurements in live cells, we found abnormal interactions of the various αB-crystallin mutants with wild-type αB-crystallin, with themselves, and with the other small heat shock proteins Hsp20, Hsp22, and possibly with Hsp27. The collected data suggest that eachαB-crystallin mutant has a unique pattern of abnormal interaction properties. These distinct properties of the αB-crystallin mutants identified are likely to contribute to a better understanding of the gradual manifestation and clinical heterogeneity of the associated myopathy in patients. αB-crystallin (αBC) 2The abbreviations used are: αBC, αB-crystallin; sHsp, small heat shock protein; WTαBC, wild-type αB-crystallin; MTTαBC, mutant αB-crystallin; R120GαBC, αB-crystallin mutant R120G; Q151XαBC, αB-crystallin mutant Q151X; 464αBC, αB-crystallin mutant 464ΔCT; TH, yeast two-hybrid; CL, cross-linking; PD, pulldown; qFRET, quantitative fluorescence resonance energy transfer; AAFE, apparent average fluorescence resonance energy transfer efficiency; CFP, cyan fluorescent protein; Cit, citrine; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; DSS, disuccinimidyl suberate; ANOVA, analysis of variance. is a ubiquitously occurring small heat shock protein (sHsp) with particularly high abundance in skeletal and cardiac muscles (1Iwaki T. Kume-Iwaki A. Goldman J.E. J. Histochem. Cytochem. 1990; 38: 31-39Crossref PubMed Scopus (269) Google Scholar) in which it can be incorporated into the sarcomeric structure (2Lutsch G. Vetter R. Offhauss U. Wieske M. Grone H.J. Klemenz R. Schimke I. Stahl J. Benndorf R. Circulation. 1997; 96: 3466-3476Crossref PubMed Scopus (124) Google Scholar, 3Golenhofen N. Perng M.D. Quinlan R.A. Drenckhahn D. Histochem. Cell Biol. 2004; 122: 415-425Crossref PubMed Scopus (145) Google Scholar). It is now well established that αBC is a major player in the function of muscular tissues, for example it protects cardiomyocytes from adverse conditions such as ischemic stress (4Martin J.L. Bluhm W.F. He H. Mestril R. Dillmann W.H. Am. J. Physiol. 2002; 283: H85-H91Crossref PubMed Scopus (10) Google Scholar). αBC and sHsps in general are widely believed to act as molecular chaperones, preventing the aggregation and precipitation of damaged or misfolded proteins in an ATP-independent way in stress conditions (5Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 6Ganea E. Curr. Protein Pept. Sci. 2001; 2: 205-225Crossref PubMed Scopus (96) Google Scholar). Consistent with this property, sHsps accumulate in human degenerative diseases, particularly in diseases involving abnormal protein aggregation (7Sun Y. MacRae T.H. FEBS J. 2005; 272: 2613-2627Crossref PubMed Scopus (286) Google Scholar). Usually, sHsps exist as polydisperse hetero-oligomers that change in size and/or organization when interacting with substrates or upon stress exposure (8Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1745) Google Scholar, 9Carver J.A. Guerreiro N. Nicholls K.A. Truscott R.J. Biochim. Biophys. Acta. 1995; 1252: 251-260Crossref PubMed Scopus (100) Google Scholar, 10Ehrnsperger M. Lilie H. Gaestel M. Buchner J. J. Biol. Chem. 1999; 274: 14867-14874Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (656) Google Scholar, 12Wang K. Spector A. Eur. J. Biochem. 2000; 267: 4705-4712Crossref PubMed Scopus (69) Google Scholar). At the cellular level, sHsps protect cells from noxious conditions as diverse as toxicity promoted by aberrantly folded proteins, oxidative conditions, and proteasome inhibition. Distinct mechanisms have been proposed for the protective effects of sHsps, including chaperone-like activity, anti-apoptotic effects, or intracellular redox homeostasis (13Chavez Zobel A.T. Loranger A. Marceau N. Theriault J.R. Lambert H. Landry J. Hum. Mol. Genet. 2003; 12: 1609-1620Crossref PubMed Scopus (135) Google Scholar, 14Stamler R. Kappe G. Boelens W. Slingsby C. J. Mol. Biol. 2005; 353: 68-79Crossref PubMed Scopus (140) Google Scholar, 15den Engelsman J. Keijsers V. de Jong W.W. Boelens W.C. J. Biol. Chem. 2003; 278: 4699-4704Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 16Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 17Kamradt M.C. Lu M. Werner M.E. Kwan T. Chen F. Strohecker A. Oshita S. Wilkinson J.C. Yu C. Oliver P.G. Duckett C.S. Buchsbaum D.J. LoBuglio A.F. Jordan V.C. Cryns V.L. J. Biol. Chem. 2005; 280: 11059-11066Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 18Arrigo A.P. Virot S. Chaufour S. Firdaus W. Kretz-Remy C. Diaz-Latoud C. Antioxid. Redox. Signal. 2005; 7: 414-422Crossref PubMed Scopus (198) Google Scholar, 19Liu S. Li J. Tao Y. Xiao X. Biochem. Biophys. Res. Commun. 2007; 354: 109-114Crossref PubMed Scopus (104) Google Scholar). These distinct mechanisms are not exclusive and could occur concomitantly. Mutations in sHsps are associated with the development of several degenerative diseases. A number of mutations in αB-crystallin were identified that lead to the degeneration of distinct tissues, including the lens of the eye and/or cardiac and skeletal muscles (20Vicart P. Caron A. Guicheney P. Li Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (971) Google Scholar, 21Berry V. Francis P. Reddy M.A. Collyer D. Vithana E. MacKay I. Dawson G. Carey A.H. Moore A. Bhattacharya S.S. Quinlan R.A. Am. J. Hum. Genet. 2001; 69: 1141-1145Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 22Selcen D. Engel A.G. Ann. Neurol. 2003; 54: 804-810Crossref PubMed Scopus (231) Google Scholar, 23Pilotto A. Marziliano N. Pasotti M. Grasso M. Costante A.M. Arbustini E. Biochem. Biophys. Res. Commun. 2006; 346: 1115-1117Crossref PubMed Scopus (49) Google Scholar, 24Inagaki N. Hayashi T. Arimura T. Koga Y. Takahashi M. Shibata H. Teraoka K. Chikamori T. Yamashina A. Kimura A. Biochem. Biophys. Res. Commun. 2006; 342: 379-386Crossref PubMed Scopus (167) Google Scholar, 25Liu M. Ke T. Wang Z. Yang Q. Chang W. Jiang F. Tang Z. Li H. Ren X. Wang X. Wang T. Li Q. Yang J. Liu J. Wang Q.K. Investig. Ophthalmol. Vis. Sci. 2006; 47: 3461-3466Crossref PubMed Scopus (65) Google Scholar, 26Liu Y. Zhang X. Luo L. Wu M. Zeng R. Cheng G. Hu B. Liu B. Liang J.J. Shang F. Investig. Ophthalmol. Vis. Sci. 2006; 47: 1069-1075Crossref PubMed Scopus (122) Google Scholar). Which of the tissues actually is affected depends on the specific mutation, and currently it is not known what causes this clinical heterogeneity. In addition, two others sHsps, Hsp22 and Hsp27, have been associated with the human degenerative diseases Charcot-Marie-Tooth and distal hereditary motor neuron diseases (27Irobi J. Van Impe K. Seeman P. Jordanova A. Dierick I. Verpoorten N. Michalik A. De Vriendt E. Jacobs A. Van Gerwen V. Vennekens K. Mazanec R. Tournev I. Hilton-Jones D. Talbot K. Kremensky I. Van Den Bosch L. Robberecht W. Van Vandekerckhove J. Broeckhoven C. Gettemans J. De Jonghe P. Timmerman V. Nat. Genet. 2004; 36: 597-601Crossref PubMed Scopus (365) Google Scholar, 28Tang B. Liu X. Zhao G. Luo W. Xia K. Pan Q. Cai F. Hu Z. Zhang C. Chen B. Zhang F. Shen L. Zhang R. Jiang H. Arch. Neurol. 2005; 62: 1201-1207Crossref PubMed Scopus (68) Google Scholar, 29Tang B.S. Zhao G.H. Luo W. Xia K. Cai F. Pan Q. Zhang R.X. Zhang F.F. Liu X.M. Chen B. Zhang C. Shen L. Jiang H. Long Z.G. Dai H.P. Hum. Genet. 2005; 116: 222-224Crossref PubMed Scopus (166) Google Scholar, 30Zhang F.F. Tang B.S. Zhao G.H. Chen B. Zhang C. Luo W. Liu X.M. Xia K. Cai F. Hu Z.M. Yan X.X. Zhang R.X. Guo P. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005; 22: 361-363PubMed Google Scholar, 31Evgrafov O.V. Mersiyanova I. Irobi J. Van Den Bosch L. Dierick I. Leung C.L. Schagina O. Verpoorten N. Van Impe K. Fedotov V. Dadali E. Auer-Grumbach M. Windpassinger C. Wagner K. Mitrovic Z. Hilton-Jones D. Talbot K. Martin J.J. Vasserman N. Tverskaya S. Polyakov A. Liem R.K. Gettemans J. Robberecht W. De Jonghe P. Timmerman V. Nat. Genet. 2004; 36: 602-606Crossref PubMed Scopus (511) Google Scholar, 32Kijima K. Numakura C. Goto T. Takahashi T. Otagiri T. Umetsu K. Hayasaka K. J. Hum. Genet. 2005; 50: 473-476Crossref PubMed Scopus (82) Google Scholar, 33Liu X.M. Tang B.S. Zhao G.H. Xia K. Zhang F.F. Pan Q. Cai F. Hu Z.M. Zhang C. Chen B. Shen L. Zhang R.X. Jiang H. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005; 22: 510-513PubMed Google Scholar). The first discovered sHsp mutation is the missense mutation R120G in αBC (R120GαBC) (20Vicart P. Caron A. Guicheney P. Li Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (971) Google Scholar), and to date this is the best studied mutation. The R120G mutation in αBC results in dominant gain-of-function properties and causes a particular subtype of myofibrillar myopathy (desmin-related myopathy or αB-crystallinopathy) with associated cardiac involvement and cataract formation. The R120GαBC protein exhibits changes in its secondary, tertiary, and quaternary structural features (34Perng M.D. Muchowski P.J. van Den I.P. Wu G.J. Hutcheson A.M. Clark J.I. Quinlan R.A. J. Biol. Chem. 1999; 274: 33235-33243Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 35Kumar L.V. Ramakrishna T. Rao C.M. J. Biol. Chem. 1999; 274: 24137-24141Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 36Bova M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (341) Google Scholar, 37Treweek T.M. Rekas A. Lindner R.A. Walker M.J. Aquilina J.A. Robinson C.V. Horwitz J. Perng M.D. Quinlan R.A. Carver J.A. FEBS J. 2005; 272: 711-724Crossref PubMed Scopus (85) Google Scholar). It is more polydisperse than the wild-type αBC (WTαBC) (36Bova M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (341) Google Scholar) and is inherently unstable in solution (37Treweek T.M. Rekas A. Lindner R.A. Walker M.J. Aquilina J.A. Robinson C.V. Horwitz J. Perng M.D. Quinlan R.A. Carver J.A. FEBS J. 2005; 272: 711-724Crossref PubMed Scopus (85) Google Scholar). These structural disturbances correlate with a decreased in vitro chaperone-like activity (36Bova M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (341) Google Scholar). A recent study has established that R120GαBC directly promotes the aggregation of the desmin filament network and that desmin networks are differently affected, depending on the cellular backgrounds (38Perng M.D. Wen S.F. van den I.P. Prescott A.R. Quinlan R.A. Mol. Biol. Cell. 2004; 15: 2335-2346Crossref PubMed Scopus (95) Google Scholar). In various cell lines, independent of desmin levels, R120GαBC aggregates in a time-dependent process starting with the formation of multiple foci of insoluble proteins in the cytoplasm. Subsequently, these foci coalesce into large amorphous perinuclear aggregates in a microtubular network-dependent manner. Expression of R120GαBC leads to the formation of a cage of type III intermediate filament proteins such as vimentin, but also of a cage of type II intermediate filament proteins such as keratins, suggesting a general response of the intermediate filament networks to the aggregate formation. Nevertheless, these intermediate filament proteins have not been found as components of the aggregates by themselves, in contrast to desmin (13Chavez Zobel A.T. Loranger A. Marceau N. Theriault J.R. Lambert H. Landry J. Hum. Mol. Genet. 2003; 12: 1609-1620Crossref PubMed Scopus (135) Google Scholar). Moreover, R120GαBC and its pseudophosphorylated mutants are unable to confer resistance to differentiation-induced apoptosis during C2C12 myoblast differentiation because of their impaired capacity to inhibit the proteolytic activation of caspase-3 (39Kamradt M.C. Chen F. Sam S. Cryns V.L. J. Biol. Chem. 2002; 277: 38731-38736Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Overexpression of R120GαBC in cardiomyocytes of transgenic mice results in a 100% mortality by early adulthood in high expressing lines, whereas a modest expression level results in a strikingly similar phenotype to that observed in patients with R120GαBC-associated cardiomyopathies (40Wang X. Osinska H. Klevitsky R. Gerdes A.M. Nieman M. Lorenz J. Hewett T. Robbins J. Circ. Res. 2001; 89: 84-91Crossref PubMed Scopus (251) Google Scholar). In these transgenic mice, the desmin network, myofibril alignment, mitochondrial-sarcomere architecture, mitochondrial function, and the ubiquitin/proteasome system (UPS) were significantly impaired (41Maloyan A. Sanbe A. Osinska H. Westfall M. Robinson D. Imahashi K. Murphy E. Robbins J. Circulation. 2005; 112: 3451-3461Crossref PubMed Scopus (173) Google Scholar, 42Chen Q. Liu J.B. Horak K.M. Zheng H. Kumarapeli A.R. Li J. Li F. Gerdes A.M. Wawrousek E.F. Wang X. Circ. Res. 2005; 97: 1018-1026Crossref PubMed Scopus (139) Google Scholar). In addition, a hypertrophic response occurred, and the apoptotic pathways were activated (41Maloyan A. Sanbe A. Osinska H. Westfall M. Robinson D. Imahashi K. Murphy E. Robbins J. Circulation. 2005; 112: 3451-3461Crossref PubMed Scopus (173) Google Scholar). Taken together, it appears that all the known protective functions of αBC are impaired. The two other αBC mutations that are associated with myofibrillar myopathy are the nonsense mutation Q151X (Q151XαBC) and the frameshift mutation 464delCT (464αBC) (22Selcen D. Engel A.G. Ann. Neurol. 2003; 54: 804-810Crossref PubMed Scopus (231) Google Scholar). Both mutants caused the formation of cytoplasmic aggregates in skeletal muscles, without cardiac or eye lens involvement. No further information on these two mutants is available. Recently, several studies have investigated the potential of sHsp overexpression for the treatment of degenerative diseases. Hsp22, Hsp27, and WTαBC revealed a high capacity to dissociate the aggregates formed by R120GαBC expression in several cell lines (13Chavez Zobel A.T. Loranger A. Marceau N. Theriault J.R. Lambert H. Landry J. Hum. Mol. Genet. 2003; 12: 1609-1620Crossref PubMed Scopus (135) Google Scholar, 43Ito H. Kamei K. Iwamoto I. Inaguma Y. Tsuzuki M. Kishikawa M. Shimada A. Hosokawa M. Kato K. Cell. Mol. Life Sci. 2003; 60: 1217-1223Crossref PubMed Scopus (34) Google Scholar, 44Sanbe A. Osinska H. Villa C. Gulick J. Klevitsky R. Glabe C.G. Kayed R. Robbins J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13592-13597Crossref PubMed Scopus (106) Google Scholar, 45Sanbe A. Yamauchi J. Miyamoto Y. Fujiwara Y. Murabe M. Tanoue A. J. Biol. Chem. 2007; 282: 555-563Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Nevertheless, recent studies in cardiomyocytes suggest that the resulting hetero-oligomers of WTαBC/R120GαBC were more toxic for the cells than the homo-oligomers formed by the R120GαBC alone (44Sanbe A. Osinska H. Villa C. Gulick J. Klevitsky R. Glabe C.G. Kayed R. Robbins J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13592-13597Crossref PubMed Scopus (106) Google Scholar). In contrast, both Hsp22 and Hsp25 (murine equivalent of Hsp27) co-expression with R120GαBC rescued cell viability (45Sanbe A. Yamauchi J. Miyamoto Y. Fujiwara Y. Murabe M. Tanoue A. J. Biol. Chem. 2007; 282: 555-563Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). So far, there is no explanation for this differential effect of αBC and Hsp22 or Hsp27/Hsp25 to date. In this study we have determined properties of the three mutants of αBC (MTTαBC), R120GαBC, Q151XαBC, and of 464αBC, that are associated with myofibrillar myopathy. We show that all three mutant proteins form abnormal cytoplasmic aggregates in both cardiomyocytes and in COS-7 cells. Moreover, MTTαBC proteins expressed in COS-7 cells distribute into additional cell fractions as compared with WTαBC. We also show that all three mutant proteins are hyperphosphorylated in all the three known phosphorylation sites (serine residues 19, 45, and 59) when expressed in COS-7 cells. We also investigated the interaction properties of these three MTTαBC with themselves, with WTαBC, and with Hsp20, Hsp22, and Hsp27. These sHsps are known as interaction partners of αBC and are abundant in muscles (46Kato K. Goto S. Inaguma Y. Hasegawa K. Morishita R. Asano T. J. Biol. Chem. 1994; 269: 15302-15309Abstract Full Text PDF PubMed Google Scholar, 47Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 48Fontaine J.M. Sun X. Benndorf R. Welsh M.J. Biochem. Biophys. Res. Commun. 2005; 337: 1006-1011Crossref PubMed Scopus (86) Google Scholar). Using the yeast two-hybrid (TH) method, chemical cross-linking (CL), pulldown (PD) assays, and the quantitative fluorescence resonance energy transfer (qFRET) method in live mammalian cells, we show here that all three MTTαBC proteins are able to interact with themselves, with WTαBC, and with the other sHsps. We have identified a unique interaction pattern for each mutant with the other sHsps. It is expected that these identified abnormal properties of the three studied MTTαBC forms will contribute to a better understanding of the molecular processes that lead to the associated diseases and to the design of therapeutic strategies. Vector Constructs—TH vector constructs were made using the vectors pACT2 and pAS2 (Clontech). Cyan (CFP) and citrine (Cit) fluorescent fusion protein expression vectors were made using the vectors pECFP-N1, pECFP-C1 (both from Clontech), and pCit-N1 and pCit-C1 (49Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Myc/His-tagged and untagged protein constructs were made using the vectors pcDNA3.1(+)/myc-His B and pcDNA3, respectively (both from Invitrogen). A list of all used constructs and more detailed cloning information is given in supplemental Table 1 (construct numbers as used in this study are given in square braquets). Additional information on the constructs used is also given in previous publications (20Vicart P. Caron A. Guicheney P. Li Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (971) Google Scholar, 48Fontaine J.M. Sun X. Benndorf R. Welsh M.J. Biochem. Biophys. Res. Commun. 2005; 337: 1006-1011Crossref PubMed Scopus (86) Google Scholar, 50Sun X. Fontaine J.M. Rest J.S. Shelden E.A. Welsh M.J. Benndorf R. J. Biol. Chem. 2004; 279: 2394-2402Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 51Fontaine J.M. Sun X. Hoppe A.D. Simon S. Vicart P. Welsh M.J. Benndorf R. FASEB J. 2006; 20: 2168-2170Crossref PubMed Scopus (74) Google Scholar). Two-hybrid Method—Small scale sequential transformation of yeast strain AH109 was performed as described in the manufacturer's instructions (Clontech). Colonies were selected on -Trp, -Leu, -His medium for the phenotypes His+ (growth) and LacZ+ (blue color). The interaction assays were considered positive if both reporter genes were activated. For negative controls, yeast were transformed with each used vector alone and tested on -Trp, -Leu, and -His medium (not shown). Additionally, yeast were co-transformed with each vector and with the 'empty’ partner vector as indicated in the figure legends (C1–C11, cf. supplemental Figs. 1B, 2A, 3A, and 4A). In none of these controls were the reporter genes activated. Cell Culture and Transfections—COS-7 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen) and penicillin/streptomycin (Invitrogen) in a 5% CO2 humidified atmosphere. One day prior to transfections, cells were trypsinized and plated as specified below. Transfections were carried out using FuGENE 6 (Roche Diagnostics) with 0.75 μg (single construct) or 1.5 μg (two constructs) of vector DNA for CFP or Cit constructs or 2 μg of vector DNA for pcDNA3 or pcDNA3.1(+)/myc-His B constructs. Cardiomyocyte Isolation, Culture, and Infection—Rat neonatal ventricular myocytes were isolated by standard batch collagenase digestion and subsequently preplated to remove the fibroblasts. Cardiomyocytes were then counted and plated on gelatin-coated dishes in serum-free PC-1 medium (BioWhittaker). Twenty four hours after plating, the cells were washed, and the medium was changed to DMEM/M199 (4:1) maintenance medium. The cells were then transduced with adenovirus expressing the appropriate CFP fusion at an multiplicity of infection of 50. Two days later the cells were fixed in 2% paraformaldehyde solution, and rhodamine-phalloidin was stained before imaging. Immunofluorescence Microscopy—COS-7 cells were grown on coverslips, transfected, and 48 h later were washed twice with ice-cold PBS, fixed, and permeabilized at 4 °C for 5 min with cold methanol/acetone (7:3). Subsequently, cells were incubated for 1 h each at room temperature with primary polyclonal rabbit antibody (diluted 1:200 in PBS, 2% FCS) directed against the first 10 residues of αBC (Abcam) and with secondary goat anti-rabbit antibody (diluted 1:1000 in PBS, 2% FCS) coupled to AlexaFluor 11034 (Invitrogen). The coverslips were mounted on slides using Mowiol (Sigma). Images were collected using a fluorescent microscope (Leitz) equipped with a digital camera ORCA-ER (Hamamatsu) and processed using the Simple PCI 6.0 software (Compix Inc. Imaging Systems). Live Cell Imaging—COS-7 cells were grown in 6-well glass-bottom culture plates (MatTek Corp.). 48 h after transfection with the various CFP and Cit fusion protein vectors, cells were washed twice with PBS and kept in DMEM without phenol red (Invitrogen). For fluorescence microscopy an inverted epifluorescence microscope (Eclipse TE-2000 U; Nikon) was used equipped with a 100-watt mercury arc-lamp, exciter filters 430/25 and 500/20, a dichroic microscope filter 86002bs, and with a 505dcxr Dual View Micro Imager MSMI.DV.CC (Optical Insights) with the emission filters 470/30 and 535/30. Images were collected by a digital CoolSnap CCD camera (Photometrics) and processed using Metamorph image processing software version 6.2r5 (Molecular Devices). For determination of the fraction of cells with aggregates, microscopic fields were selected randomly using a Plan fluor ELWD ×40/0.6 objective lens (Nikon). At least 100 cells per sample group were included in these evaluations. Quantitative Fluorescence Resonance Energy Transfer Measurements in Live Cells—The qFRET method was applied for quantification of apparent fluorescence resonance energy transfer efficiencies (AAFE) as indicators of protein interaction. The configuration of the microscope was as described above for live cell imaging using a Fluor ELWD ×40/1.3 oil Dic H objective lens (Nikon). Maintenance and transfection of COS-7 cells with the various CFP and Cit fusion protein vectors was as described for live cell imaging. In each cell to be analyzed, three cytoplasmic areas without protein aggregates were selected for qFRET measurements. Images from at least 30 microscopic fields per sample group were acquired and background/shading-corrected prior to computation by the qFRET algorithm. The calculated output data were expressed as (EA + ED)/2 (EA is apparent acceptor efficiency calculated from sensitized emission and dependent on the fraction of acceptor in complex; ED is apparent donor efficiency calculated relative to donor fluorescence and dependent on the fraction of the donor in complex). More details concerning qFRET are given in earlier publications (49Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 51Fontaine J.M. Sun X. Hoppe A.D. Simon S. Vicart P. Welsh M.J. Benndorf R. FASEB J. 2006; 20: 2168-2170Crossref PubMed Scopus (74) Google Scholar). As negative controls, the cells were transfected with the “empty” CFP (peCFP-N1) and Cit (peCit-N1) vectors (Fig. 3D) or with the empty Cit (peCit-N1/C1) and the various CFP constructs (supplemental Figs. 2C, 3C, and 4D). AAFE values that were significantly different from the control signals indicated interaction. Quantitative data are expressed as the mean of AAFE values ±S.E. The data between groups were analyzed using one-way ANOVA. When overall significance was detected, a post hoc multiple group comparison was conducted using Tukey HSD adjustment. Differences between groups were considered statistically significant if p < 0.05. Analysis of the Phosphorylation Status of αB-crystallin—COS-7 cells were grown in 6-well plates. 48 h after transfection, cells were washed twice in ice-cold PBS and lysed using the ReadyPrep protein extraction kit (Bio-Rad) according to the manufacturer's instructions. One volume of buffer A (125 mm Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 400 mm dithiothreitol, 0.01% bromphenol blue) was added, and the samples were boiled for 3 min followed by SDS-PAGE/Western blotting. Equal loading of the samples was verified by visualization of vimentin on the same blots using a monoclonal anti-vimentin antibody diluted to 1:2000 (Sigma). Phosphorylation of the three known serine phosphorylation sites (Ser-19, Ser-45, and Ser-59) of αBC (52Kato K. Ito H. Kamei K. Inaguma Y. Iwamoto I. Saga S. J. Biol. Chem. 1998; 273: 28346-28354Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) was determined using phosphorylation site-specific polyclonal antibodies, diluted to 1:2000 (Stress-Gen). After electrotransfer of the proteins, the polyvinylidene difluoride membrane was blocked with bovine serum albumin free of IgG (Interchim). For immunodetection, secondary goat anti-mouse or anti-rabbit horseradish peroxidase-coupled secondary antibodies diluted to 1:10,000 (Pierce) were used. The degree of phosphorylation of the various αBC species was quantified on scanned images using ImageJ software (53Abramoff M.D. Magelhaes P.J. Ram S.J. Biophotonics International. 2004; 11: 36-42Google Scholar). The base-line signal was obtained from untransfected control cells. Protein Fractionation—COS-7 cells were grown in 10-cm cell culture dishes and transfected with vectors coding for Myc/His-tagged αBC species [5–7]. 48 h after transfection, ∼4 × 106 cells were harvested, and the cell proteins were differentially extracted yielding the fractions of cytosolic proteins, membrane/organelle proteins, nuclear proteins, and cytoskeletal proteins using the ProteoExtract subcellular proteome extraction kit (Calbiochem) according to the man" @default.
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