Title: Bin1 Src Homology 3 Domain Acts as a Scaffold for Myofiber Sarcomere Assembly
Abstract: In skeletal muscle development, the genes and regulatory factors that govern the specification of myocytes are well described. Despite this knowledge, the mechanisms that regulate the coordinated assembly of myofiber proteins into the functional contractile unit or sarcomere remain undefined. Here we explored the hypothesis that modular domain proteins such as Bin1 coordinate protein interactions to promote sarcomere formation. We demonstrate that Bin1 facilitates sarcomere organization through protein-protein interactions as mediated by the Src homology 3 (SH3) domain. We observed a profound disorder in myofiber size and structural organization in a murine model expressing the Bin1 SH3 region. In addition, satellite cell-derived myogenesis was limited despite the accumulation of skeletal muscle-specific proteins. Our experiments revealed that the Bin1 SH3 domain formed transient protein complexes with both actin and myosin filaments and the pro-myogenic kinase Cdk5. Bin1 also associated with a Cdk5 phosphorylation domain of titin. Collectively, these observations suggest that Bin1 displays protein scaffold-like properties and binds with sarcomeric factors important in directing sarcomere protein assembly and myofiber maturation. In skeletal muscle development, the genes and regulatory factors that govern the specification of myocytes are well described. Despite this knowledge, the mechanisms that regulate the coordinated assembly of myofiber proteins into the functional contractile unit or sarcomere remain undefined. Here we explored the hypothesis that modular domain proteins such as Bin1 coordinate protein interactions to promote sarcomere formation. We demonstrate that Bin1 facilitates sarcomere organization through protein-protein interactions as mediated by the Src homology 3 (SH3) domain. We observed a profound disorder in myofiber size and structural organization in a murine model expressing the Bin1 SH3 region. In addition, satellite cell-derived myogenesis was limited despite the accumulation of skeletal muscle-specific proteins. Our experiments revealed that the Bin1 SH3 domain formed transient protein complexes with both actin and myosin filaments and the pro-myogenic kinase Cdk5. Bin1 also associated with a Cdk5 phosphorylation domain of titin. Collectively, these observations suggest that Bin1 displays protein scaffold-like properties and binds with sarcomeric factors important in directing sarcomere protein assembly and myofiber maturation. Skeletal muscle differentiation is a highly orchestrated phenomenon. The transition from cycling myoblasts to mature myofibers is dependent on a coordinated response involving up-regulation of muscle-specific transcription factors, engagement of a defined gene expression program, followed by an ordered assembly of muscle structural proteins to form the basic contractile units known as sarcomeres. The key molecular genetic features of this skeletal muscle differentiation program are well understood (1Sabourin L.A. Rudnicki M.A. Clin. Genet. 2000; 57: 16-25Crossref PubMed Scopus (565) Google Scholar, 2Buckingham M. Bajard L. Chang T. Daubas P. Hadchouel J. Meilhac S. Montarras D. Rocancourt D. Relaix F. J. Anat. 2003; 202: 59-68Crossref PubMed Scopus (651) Google Scholar, 3Zhao P. Hoffman E.P. Dev. Dyn. 2004; 229: 380-392Crossref PubMed Scopus (158) Google Scholar). Nevertheless, the regulatory networks that control and integrate sarcomeric assembly in developing myofibers remain comparatively unknown. The sarcomere is composed of thick myosin and thin actin myofilaments together with the giant sarcomeric proteins titin and nebulin. The actin and myosin filaments are anchored at the Z-line and M-line, respectively. Titin has been coined a “molecular ruler” of the thick filament because it mediates an ordered and repetitive series of interactions with myosin and with several proteins at the Z- and M-lines that include the sarcomeric protein complex (4Au Y. Cell. Mol. Life Sci. 2004; 61: 3016-3033Crossref PubMed Scopus (45) Google Scholar, 5Lange S. Ehler E. Gautel M. Trends Cell Biol. 2006; 16: 11-18Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Similarly, nebulin has also been coined the molecular ruler of the thin filament for its ordered assembly of actin (6Labeit S. Gibson T. Lakey A. Leonard K. Zeviani M. Knight P. Wardale J. Trinick J. FEBS Lett. 1991; 282: 313-316Crossref PubMed Scopus (167) Google Scholar, 7McElhinny A.S. Schwach C. Valichnac M. Mount-Patrick S. Gregorio C.C. J. Cell Biol. 2005; 170: 947-957Crossref PubMed Scopus (74) Google Scholar), and recent evidence indicates that nebulin also mediates protein interactions of the sarcomere (reviewed in Refs. 4Au Y. Cell. Mol. Life Sci. 2004; 61: 3016-3033Crossref PubMed Scopus (45) Google Scholar, 8Sinz A. Wang K. Biochemistry. 2001; 40: 7903-7913Crossref PubMed Scopus (65) Google Scholar). The large number of protein interactions that initiate and establish the mature sarcomere implies that one or more protein structural motifs may be critical to the assembly process. Surprisingly, many of these proteins contain Src homology 3 domains (SH3), 3The abbreviations used are: SH3Src homology 3HAhemagglutinindpcdays post-coitumGFPgreen fluorescent proteinMS/MStandem mass spectrometryLCliquid chromatographyPIpropidium iodide. a well characterized protein-protein interaction domain (reviewed in Refs. 9Kaneko T. Li L. Li S.S. Front. Biosci. 2008; 13: 4938-4952Crossref PubMed Scopus (146) Google Scholar, 10Li S.S. Biochem. J. 2005; 390: 641-653Crossref PubMed Scopus (21) Google Scholar). Titin contains numerous SH3 domains, many of which affect its function. Similarly, nebulin function and incorporation into the mature sarcomere appear to be dependent on an endogenous SH3 domain (4Au Y. Cell. Mol. Life Sci. 2004; 61: 3016-3033Crossref PubMed Scopus (45) Google Scholar, 11Ma K. Forbes J.G. Gutierrez-Cruz G. Wang K. J. Biol. Chem. 2006; 281: 27539-27556Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Ma K. Wang K. FEBS Lett. 2002; 532: 273-278Crossref PubMed Scopus (59) Google Scholar, 13Politou A.S. Millevoi S. Gautel M. Kolmerer B. Pastore A. J. Mol. Biol. 1998; 276: 189-202Crossref PubMed Scopus (35) Google Scholar). Titin-associated proteins such as obscurin have SH3 motifs that appear to modulate the G-protein-coupled signal transduction pathways. Notably, a stretch of prolines representative of an SH3 binding region resides within the Rho guanine nucleotide exchange factor domain of obscurin (14Young P. Ehler E. Gautel M. J. Cell Biol. 2001; 154: 123-136Crossref PubMed Scopus (237) Google Scholar, 15Bang M.L. Centner T. Fornoff F. Geach A.J. Gotthardt M. McNabb M. Witt C.C. Labeit D. Gregorio C.C. Granzier H. Labeit S. Circ. Res. 2001; 89: 1065-1072Crossref PubMed Scopus (513) Google Scholar). These observations suggest that SH3 adaptor protein(s) play a pivotal role in the construction and stabilization of the sarcomere. Src homology 3 hemagglutinin days post-coitum green fluorescent protein tandem mass spectrometry liquid chromatography propidium iodide. Once assembled, the sarcomere must be stabilized with other structures in the developing myofiber. Paramount among these components is the sarcolemma/t-tubule system. The sarcolemma is a highly specialized membrane with numerous involutions (t-tubules) that couple the external signal for contraction to the basic contractile unit, the sarcomere. As such, it is reasonable to hypothesize that sarcomere assembly and sarcolemmal biogenesis may be facilitated by an overlapping set of proteins. However, invoking such a model will be dependent on the identification of a modular protein that utilizes distinct domains to influence each of these disparate activities. Within this context, one candidate factor that has emerged is the tumor suppressor protein Bin1 (bridging integrator protein 1). Bin1 was initially characterized as a c-Myc interacting protein, capable of repressing c-Myc transcriptional activation (16Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (313) Google Scholar, 17Elliott K. Sakamuro D. Basu A. Du W. Wunner W. Staller P. Gaubatz S. Zhang H. Prochownik E. Eilers M. Prendergast G.C. Oncogene. 1999; 18: 3564-3573Crossref PubMed Scopus (100) Google Scholar). Bin1 retains distinct modular features that include a mid-body c-Myc binding domain, a C-terminal SH3 domain with unique structural features not shared with SH3 regions of sequence-related proteins, and an N-terminal domain (referred to as the BAR domain) with sequence similarity to a larger family of synaptic vesicle/clathrin-interacting factors, exemplified by the neuron-enriched protein amphiphysin (18Wechsler-Reya R. Sakamuro D. Zhang J. Duhadaway J. Prendergast G.C. J. Biol. Chem. 1997; 272: 31453-31458Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 19Kojima C. Hashimoto A. Yabuta I. Hirose M. Hashimoto S. Kanaho Y. Sumimoto H. Ikegami T. Sabe H. EMBO J. 2004; 23: 4413-4422Crossref PubMed Scopus (60) Google Scholar, 20Ren G. Vajjhala P. Lee J.S. Winsor B. Munn A.L. Microbiol. Mol. Biol. Rev. 2006; 70: 37-120Crossref PubMed Scopus (157) Google Scholar). Bin1 has been implicated in regulating striated muscle function across a variety of model systems. Overexpression of Bin1 in a myoblast cell line inhibits cell growth and results in a more rapid onset of differentiation following growth factor withdrawal (21Wechsler-Reya R.J. Elliott K.J. Prendergast G.C. Mol. Cell. Biol. 1998; 18: 566-575Crossref PubMed Scopus (95) Google Scholar). Generation of mice with a null mutation in bin1 leads to a severe disruption in cardiomyocyte function through an undetermined mechanism (22Muller A.J. Baker J.F. DuHadaway J.B. Ge K. Farmer G. Donover P.S. Meade R. Reid C. Grzanna R. Roach A.H. Shah N. Soler A.P. Prendergast G.C. Mol. Cell. Biol. 2003; 23: 4295-4306Crossref PubMed Scopus (103) Google Scholar). Null mutations of the Drosophila bin1 homologue have revealed that Bin1 is required for maintenance of excitation-contraction coupling in skeletal muscle (23Razzaq A. Robinson I.M. McMahon H.T. Skepper J.N. Su Y. Zelhof A.C. Jackson A.P. Gay N.J. O'Kane C.J. Genes Dev. 2001; 15: 2967-2979Crossref PubMed Scopus (188) Google Scholar, 24Zelhof A.C. Bao H. Hardy R.W. Razzaq A. Zhang B. Doe C.Q. Development. 2001; 128: 5005-5015Crossref PubMed Google Scholar). Regulation of the contractile response was attributed to an ability of the Bin1 BAR domain to enhance sarcolemmal membrane curvature, influencing t-tubule assembly and maturation (25Lee E. Marcucci M. Daniell L. Pypaert M. Weisz O.A. Ochoa G.C. Farsad K. Wenk M.R. De Camilli P. Science. 2002; 297: 1193-1196Crossref PubMed Scopus (328) Google Scholar). Interestingly, a recent study has demonstrated that patients suffering from centronuclear myopathy have homozygous mutations in bin, at the regions encoding either the BAR or SH3 domain (26Nicot A.S. Toussaint A. Tosch V. Kretz C. Wallgren-Pettersson C. Iwarsson E. Kingston H. Garnier J.M. Biancalana V. Oldfors A. Mandel J.L. Laporte J. Nat. Genet. 2007; 39: 1134-1139Crossref PubMed Scopus (285) Google Scholar). A representative tissue culture model of the individual BAR domain or SH3 domain mutations displayed obvious defects in membrane tubulation events. This myopathy also displayed a phenotype consistent with sarcomere disorder, yet the significance of the Bin1 BAR and SH3 domains was not tested in this regard. Here we explored the hypothesis that distinct modular domains of Bin1 separately influence the key events associated with skeletal muscle differentiation. Specifically, we propose that the Bin1 SH3 domain is a sarcomere-organizing protein. We demonstrate that transgenic overexpression of the Bin1 SH3 domain results in a profound perturbation of skeletal muscle ultrastructure, characterized by increased myofiber size with sarcomeric disorganization. This phenotypic outcome derives in part from a disruption in the endogenous interaction between the Bin1 SH3 domain and a number of contractile proteins, including sarcomeric actin and myosin. The sarcomere disruption in this model is also influenced by a loss in the endogenous interaction between the Bin1 SH3 domain and Cdk5, a pro-myogenic kinase that promotes sarcomeric assembly in part by phosphorylating a serine-responsive region of titin. Collectively, these observations suggest that Bin1 is a crucial adaptor protein that acts to promote skeletal muscle differentiation through domain-specific assembly of the mature sarcomere. The SH3 region (encoding amino acids 361–434) of the murine bin1 gene (gene identifier) was produced through PCR amplification. To direct systemic expression of the Bin1 SH3 construct, the sequence-verified PCR product was cloned in-frame into the pCAGGS expression vector containing a modified cytomegalovirus enhancer/promoter with a rabbit β-globin poly(A) (27Niwa H. Yamamura K. Miyazaki J. Gene. 1991; 108: 193-199Crossref PubMed Scopus (4616) Google Scholar, 28Lobe C.G. Koop K.E. Kreppner W. Lomeli H. Gertsenstein M. Nagy A. Dev. Biol. 1999; 208: 281-292Crossref PubMed Scopus (452) Google Scholar). Injection and derivation of transgenic mice were performed as described previously (29Guy L.G. Kothary R. DeRepentigny Y. Delvoye N. Ellis J. Wall L. EMBO J. 1996; 15: 3713-3721Crossref PubMed Scopus (58) Google Scholar). A total of 12 founder lines were generated, of which 5 were subject to further characterization. Tissues were removed and fixed in 10% formalin for 4–5 days (skeletal muscle, heart, mammary gland, prostate, skin, liver, lung, brain). Fixed tissues were then embedded in paraffin, sectioned at 10 μm, stained, and counter-stained with hematoxylin and eosin. Muscle fiber diameters were assessed on hindlimb muscle groups from multiple founder lines, with a minimum of four individual muscles measured per group. Ultrastructure examination of wild type and bin1SH3 gastrocnemius was performed as described previously (30Franzini-Armstrong C. Dev. Biol. 1991; 146: 353-363Crossref PubMed Scopus (124) Google Scholar). Longitudinal semi-thin (0.3–0.5 μm) sections were used to visualize the muscle ultrastructure under an accelerating voltage of 100 kV. Embryos were collected between 8.5 and 12.5 days post-coitum (dpc) and prepared as described previously (31Sassoon D. Lyons G. Wright W.E. Lin V. Lassar A. Weintraub H. Buckingham M. Nature. 1989; 341: 303-307Crossref PubMed Scopus (492) Google Scholar) A 5′-portion of the murine bin1 cDNA was used as a probe for whole mount in situ hybridizations. In situ detection of the pCAGGS transgene was performed with a riboprobe generated against the full-length rabbit β-globin poly(A) region. The myogenin riboprobe used was as described previously (32Wilkinson D.G. Nieto M.A. Methods Enzymol. 1993; 225: 361-373Crossref PubMed Scopus (724) Google Scholar). Digoxigenin-labeled sense and antisense riboprobes were generated as per the manufacturer's instructions (Roche Applied Science). For in situ hybridization, murine embryos were treated as described (31Sassoon D. Lyons G. Wright W.E. Lin V. Lassar A. Weintraub H. Buckingham M. Nature. 1989; 341: 303-307Crossref PubMed Scopus (492) Google Scholar). Skeletal myoblast c2c12 cells were cultured as described previously (33Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11025-11030Crossref PubMed Scopus (446) Google Scholar). All reagents for c2c12 culture were obtained from Invitrogen. Primary myoblast cell lines (muscle satellite cells) were isolated as described previously (33Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11025-11030Crossref PubMed Scopus (446) Google Scholar). Immunolocalization of skeletal muscle differentiation markers in cultured cells was performed as described previously (33Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11025-11030Crossref PubMed Scopus (446) Google Scholar). Cells were fixed in 4% paraformaldehyde and stained with anti-myosin heavy chain MF20 hybridoma and visualized by counterstaining with a fluorescein isothiocyanate-conjugated secondary antibody. Adenoviral expression vectors were generated using the AdEasy adenoviral vector system (Stratagene). The AdHA-SH3-hrGFP-2 construct was generated using the bin1 SH3 region (exons 15 and 16). The KSP region of titin (cDNA gift from Siegfried Labeit, Mannheim, Germany) was used to generate the AdHA-TiKSP-GFP. Annexin-V-propidium iodide (PI) analyses of apoptotic and necrotic cells were performed using the annexin-V-FLUOS staining kit (Roche Applied Science) on a Beckman-Coulter ALTRA flow cytometer. Cell growth assays were evaluated in both cycling cells and in cells after 2 days in low serum media (differentiation media) using a 5-bromo-2-deoxyuridine cell proliferation assay kit (Roche Applied Science). Myoblast fusion was evaluated by determining the number of myosin heavy chain-positive myotubes containing two or more nuclei relative to the total number of myosin heavy chain-positive myotubes. Cell and tissue protein lysates were prepared in modified radioimmunoprecipitation assay (RIPA) buffer (33Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11025-11030Crossref PubMed Scopus (446) Google Scholar). Immunoblots assays were performed as described previously (33Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11025-11030Crossref PubMed Scopus (446) Google Scholar) using antibodies against myogenin, p38, MEF2C, skeletal α-actin and myosin, Cdk5, and Bin199D (all from Santa Cruz Biotechnology), myosin heavy chain MF20 hybridoma, and M-cadherin and HA tag (Sigma). Lysates from c2c12 cultured cells were prepared as above, and 800–1000 μg of total cell lysate was loaded onto an equilibrated Superose 6HR 10/30 column. Samples were run in 0.05 m phosphate buffer with 0.15 m NaCl using an ÄKTA 10 Explorer FPLC. Co-immunoprecipitation assays were performed as described previously (33Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11025-11030Crossref PubMed Scopus (446) Google Scholar). GST-pulldown assays were performed using recombinant GST and GST-SH3 purified on glutathione beads with 150 μg of total protein from tissue culture lysates. Protein kinase analyses were performed as described previously (33Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11025-11030Crossref PubMed Scopus (446) Google Scholar). A two-dimensional nano-liquid chromatography MS/MS approach was used to identify Bin1 SH3-interacting proteins. Following GST pulldown assays, the tagged protein complexes were subjected to on-bead tryptic digests, and peptides were bound to a cation exchange column on a CapLC capillary LC system (Waters). Peptides were eluted with a gradient ammonium acetate solution (2, 5, 10, 25, 50 100, and 200 mm) and separated through a C18 reverse phase column followed by electrospray ionization and quadrupole/time-of-flight MS/MS on a Q-TOF Ultima hybrid mass spectrometer. Peptide MS/MS spectra were searched against the NCBInr data base using the MASCOT searching algorithm. The modular domain structure of Bin1 indicated that this protein may serve as an ideal adaptor protein for the assembly and maintenance of the mature myofiber. However, the post-natal distribution of Bin1 has been reported to be ubiquitous with an enriched expression pattern in neural and cardiac tissues and in adult skeletal muscle (16Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (313) Google Scholar, 18Wechsler-Reya R. Sakamuro D. Zhang J. Duhadaway J. Prendergast G.C. J. Biol. Chem. 1997; 272: 31453-31458Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 34Butler M.H. David C. Ochoa G.C. Freyberg Z. Daniell L. Grabs D. Cremona O. De Camilli P. J. Cell Biol. 1997; 137: 1355-1367Crossref PubMed Scopus (216) Google Scholar). Therefore, to address the function of Bin1 in skeletal muscle as a probable maturation regulatory factor, we examined its distribution during murine embryogenesis using an antisense riboprobe specific to the murine bin1 cDNA (16Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (313) Google Scholar). bin1 expression was not detectable in developing embryos prior to 8.5 dpc. By 10–10.5 dpc, bin1 transcripts were readily visible within the branchial arches, somites, and the ependymal region of the developing forebrain and hindbrain (Fig. 1, A–D). Somites represent the tissue reservoirs from which most skeletal muscle is derived. The myogenic stem cells originating from the somites provide the myoblast pool from which all trunk and limb musculature arises, whereas the branchial arch myoblasts contribute to components of head musculature (35Buckingham M. Curr. Opin. Genet. Dev. 2006; 16: 525-532Crossref PubMed Scopus (336) Google Scholar). Histological sections of embryos at 10.5 dpc confirmed that bin1 expression was concentrated in the differentiated regions of the somite (myotome portion) and ependymal layers of the developing brain (Fig. 1, C and D). In addition, embryos revealed elevated bin1 expression in both the epicardial region of ventricular trabeculae and within the developing optic cup (data not shown), although the predominant expression appeared to be within the skeletal muscle primordia. At later stages of embryogenesis (12.5 dpc), the expression of bin1 remained elevated within tissues of the skeletal muscle lineage, including the developing pre-muscle masses of the limbs, head, and scapular regions (Fig. 1E). However, closer examination of these embryos revealed a divergence in the skeletal muscle expression pattern of bin1 in that the highest expression was found in regions where differentiation had recently initiated such as in limb and scapular muscle groups (Fig. 1, E and F). Conversely, bin1 expression declined in areas of more established muscle development, such as the primordia of body wall muscle at 12.5 dpc (Fig. 1F). The enriched expression of bin1 in regions specific to skeletal muscle development suggested a site-specific function for this protein. Interestingly, accumulation of differentially spliced bin1 variants in neural, cardiac, testicular, and other tissues may also suggest site-specific functions of bin1 (reviewed in Ref. 20Ren G. Vajjhala P. Lee J.S. Winsor B. Munn A.L. Microbiol. Mol. Biol. Rev. 2006; 70: 37-120Crossref PubMed Scopus (157) Google Scholar). Indeed, other members of the BAR domain family have been assigned a variety of cellular functions, including transcription, apoptosis, endocytosis, tumor suppression, and cell growth control (reviewed in Ref. 20Ren G. Vajjhala P. Lee J.S. Winsor B. Munn A.L. Microbiol. Mol. Biol. Rev. 2006; 70: 37-120Crossref PubMed Scopus (157) Google Scholar). Conceivably, such divergent roles for Bin1 may be explained by invoking a model whereby separable domains of the Bin1 protein manage domain-specific cellular activities. Therefore, to directly test this hypothesis, we created a transgenic strain that overexpressed the SH3 domain of Bin1. The SH3 domain remains the most conserved feature of all the Bin1 splice variants (36Owen D.J. Wigge P. Vallis Y. Moore J.D. Evans P.R. McMahon H.T. EMBO J. 1998; 17: 5273-5285Crossref PubMed Scopus (142) Google Scholar). Moreover, the Bin1 SH3 domain appears to be structurally unique across a wide variety of SH3 motifs suggesting a distinct or limited function for this region of the Bin1 protein (19Kojima C. Hashimoto A. Yabuta I. Hirose M. Hashimoto S. Kanaho Y. Sumimoto H. Ikegami T. Sabe H. EMBO J. 2004; 23: 4413-4422Crossref PubMed Scopus (60) Google Scholar, 36Owen D.J. Wigge P. Vallis Y. Moore J.D. Evans P.R. McMahon H.T. EMBO J. 1998; 17: 5273-5285Crossref PubMed Scopus (142) Google Scholar, 37Pineda-Lucena A. Ho C.S. Mao D.Y. Sheng Y. Laister R.C. Muhandiram R. Lu Y. Seet B.T. Katz S. Szyperski T. Penn L.Z. Arrowsmith C.H. J. Mol. Biol. 2005; 351: 182-194Crossref PubMed Scopus (75) Google Scholar). As such, we anticipated that the overexpression of this domain would disrupt endogenous Bin1 interactions and thereby impair Bin1 function with little to no effect on other SH3-containing proteins. We utilized a ubiquitously expressed transgene (pCAGGS expression vector) as a targeted delivery system for overexpression of the Bin1 SH3 domain (27Niwa H. Yamamura K. Miyazaki J. Gene. 1991; 108: 193-199Crossref PubMed Scopus (4616) Google Scholar) (Fig. 2A). The enhancer promoter combination of this transgene has frequently demonstrated a global expression pattern in transgenic mice (28Lobe C.G. Koop K.E. Kreppner W. Lomeli H. Gertsenstein M. Nagy A. Dev. Biol. 1999; 208: 281-292Crossref PubMed Scopus (452) Google Scholar). Importantly, the use of a ubiquitous promoter allowed us to address whether the SH3 domain of Bin1 performed tissue-specific functions or whether this region of the Bin1 protein conveyed non-muscle cellular functions, in addition to the anticipated role in skeletal muscle. We performed numerous rounds of pronuclear injections to collect and analyze founder embryos (transient transgenics) that might display profound changes in vivo and create founder lines for breeding to homozygosity. As expected, a ubiquitous expression of Bin1 SH3 gene product was detected in embryos at 10.5 days with a riboprobe designed against the β-globin poly(A) sequence from the pCAGGS targeting construct (Fig. 2B). Transgenic mice expressing the SH3 region (bin1SH3) were characteristically smaller than littermate controls (Fig. 2C). Despite the ubiquitous expression of bin1SH3 in young pups, we observed an enriched accumulation of the Bin1 SH3 transgene in both the heart and skeletal muscles of adult mice. The pronounced expression of the SH3 domain in skeletal muscle may indicate a bias for expression of the transgene/enhancer combination in this tissue type. Nevertheless, robust expression was noted in several other cell types. To investigate putative pathological effects caused by the overexpression of the Bin1 SH3 domain, we performed an extensive histologic examination of the Bin1 SH3 gene product in mice. The loss of Bin1 function has been associated with malignant transformation in a variety of tissues and cells (38Tajiri T. Liu X. Thompson P.M. Tanaka S. Suita S. Zhao H. Maris J.M. Prendergast G.C. Hogarty M.D. Clin. Cancer Res. 2003; 9: 3345-3355PubMed Google Scholar, 39Ge K. Minhas F. Duhadaway J. Mao N.C. Wilson D. Buccafusca R. Sakamuro D. Nelson P. Malkowicz S.B. Tomaszewski J. Prendergast G.C. Int. J. Cancer. 2000; 86: 155-161Crossref PubMed Scopus (82) Google Scholar, 40Hogarty M.D. Liu X. Thompson P.M. White P.S. Sulman E.P. Maris J.M. Brodeur G.M. Med. Pediatr. 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Prendergast G.C. Cancer Res. 2007; 67: 100-107Crossref PubMed Scopus (33) Google Scholar), suggesting that altered expression of the Bin1 SH3 domain may result in pleitrophic pathology. We inspected the progeny of five independent bin1SH3 transgenic lines (>200 mice) during the natural lifespan of these animals. Overt tumor formation was never observed in any progeny even up to 1 year of age. Moreover, with the exception of skeletal muscle, histologic inspection of target organs in aged mice showed normal cellular architecture with no evidence of hyperplasia despite the robust expression of bin1SH3 (Fig. 3A). For example, sections of heart, lung, liver, prostate, and skin revealed no overt alteration in cellular architecture or in gross morphology (Fig. 3A). As noted above, the absence of pathology in these tissues is in contrast to studies demonstrating tumor formation with bin1 disruption. Bin1 and its homologue in yeast have also been implicated as integral signal components in apoptotic programs (38Tajiri T. Liu X. Thompson P.M. Tanaka S. Suita S. Zhao H. Maris J.M. Prendergast G.C. Hogarty M.D. Clin. Cancer Res. 2003; 9: 3345-3355PubMed Google Scholar, 42Ge K. DuHadaway J. Du W. Herlyn M. Rodeck U. Prendergast G.C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9689-9694Crossref PubMed Scopus (161) Google Scholar, 45Ramalingam A. Farmer G.E. Stamato T.D. Prendergast G.C. Cell Cycle. 2007; 6: 1914-1918Crossref PubMed Scopus (13) Google Scholar, 46Telfer J.F. Urquhart J. Crouch D.H. Cell. Signal. 2005; 17: 701-708Crossref PubMed Scopus (12) Google Scholar, 47Elliott K. Ge K. Du W. Prendergast G.C. Oncogene. 2000; 19: 4669-4684Crossref PubMed Scopus (95) Google Scholar). To further evaluate the consequences of Bin1 SH3 overexpression on cellular homeostasis, we performed comparative whole mount terminal dUTP nick-end label