Talin 1 and 2 are required for myoblast fusion, sarcomere ... · fusion and sarcomere assembly....

3597RESEARCH ARTICLE

INTRODUCTIONSkeletal muscle development and function are dependent on 1integrins, a family of cell surface receptors that are formed byheterodimerization of the 1 subunit with different subunits(Hynes, 1992). In skeletal muscle, 1 integrins localize tocostameres and myotendinous junctions (MTJs), where theyestablish a link between the cytoskeleton and the extracellularmatrix (ECM) (Mayer, 2003). These connections are importantfor transmitting mechanical forces and for maintaining skeletalmuscle fibers. Accordingly, defects in integrin function lead tomuscle fiber degeneration: mutations in the gene encoding the 7integrin subunit cause congenital myopathy in humans (Hayashiet al., 1998), and genetic ablation of either the 5 or 7 integrinsubunits causes muscular dystrophy in mice (Mayer et al., 1997;Taverna et al., 1998). Integrins appear to be particularly importantat MTJs. Inactivation of the 7 integrin subunit leads todetachment of MTJs from the ECM (Miosge et al., 1999),whereas inactivation of integrin-linked kinase (ILK) and talin 1lead to detachment of integrin adhesion complexes from themuscle fiber cytoskeleton (Wang et al., 2008; Conti et al., 2008).Integrins also have important functions during skeletal muscledevelopment, as ablation of the murine 1 integrin subunit gene,which leads to loss of all 1 integrins, causes defects inmyoblast fusion and sarcomere assembly (Schwander et al.,2003). The mechanisms by which integrins carry out theirfunction in skeletal muscle are still incompletely understood.

Integrins assemble signaling complexes at the plasma membrane,which contain proteins that bind to the integrin cytoplasmic domainsor are recruited indirectly (Geiger et al., 2001; Liu et al., 2000).Several lines of evidence suggest that talin 1 is central for integrinsignaling. Talin 1 interacts with the cytoplasmic domain of 1integrins (as well as several other subunits) and with focaladhesion components such as focal adhesion kinase (FAK) andvinculin (Nayal et al., 2004). Talin 1 also binds to F-actin,establishing a link between 1 integrins and the cytoskeleton (Nayal

et al., 2004). The assembly of focal adhesions is regulated bymechanical force, which controls the recruitment of vinculin intofocal adhesions (Balaban et al., 2001; Choquet et al., 1997; Galbraithet al., 2002; Riveline et al., 2001). Talin 1 is required for the force-dependent recruitment of vinculin and strengthens the interactionsbetween integrins and the cytoskeleton (Giannone et al., 2003).Binding of talin 1 to the integrin cytoplasmic domain also enhancesthe strength of integrin adhesion to ligands (inside-out activation)(Nieswandt et al., 2007; Petrich et al., 2007; Tadokoro et al., 2003).However, it is less clear whether inside-out activation is essential forinteractions with insoluble ligands; integrins can be activateddirectly by binding to insoluble ligands (outside-in activation) (Duet al., 1991), and talin ablation in Drosophila causes detachment ofmyofibers from integrins without loss of adhesive contact with theECM (Brown et al., 2002).

Previously, we have shown that inactivation of the talin 1 gene(Tln1) in skeletal muscle leads to a progressive myopathy, caused bymechanical failure of MTJs (Conti et al., 2008). The phenotyperesembles the defect observed in mice with a mutation in the geneencoding the integrin 7 subunit (Itga7) (Mayer et al., 1997; Miosgeet al., 1999), suggesting that talin 1 mediates integrin 71 functionsat MTJs. The Tln1-deficient mice did not show the defects inmyoblast fusion and sarcomere assembly that have been observedin integrin 1-deficient mice (Schwander et al., 2003). Becausevertebrates contain two genes encoding two talins (talin 1 and 2)(McCann and Craig, 1997; McCann and Craig, 1999; Monkley etal., 2001), and because talin 1 and 2 have redundant functions inintegrin-mediated attachment of fibroblasts (Zhang et al., 2008), weargued that talin 2 might compensate for loss of talin 1 in skeletalmuscle. Talin 2 is expressed at higher levels in skeletal muscle thantalin 1 (Conti et al., 2008; Monkley et al., 2001; Senetar andMcCann, 2005), and talin 2 expression is upregulated duringmyotube formation (Senetar et al., 2007). Therefore, to determinethe function of talin 2 in skeletal muscle, we generated Tln2-deficient mice (referred to as Tln2-KO), and mice lacking both talin1 and 2 in skeletal muscle (referred to as Tln1/2-dKO). We showhere that ablation not only of the talin 1 gene but also of the talin 2gene leads to defects in the maintenance of MTJs, and we provideevidence that talin 1 and 2 mediate 1 integrin functions in myoblastfusion and sarcomere assembly.

Talin 1 and 2 are required for myoblast fusion, sarcomereassembly and the maintenance of myotendinous junctionsFrancesco J. Conti1, Sue J. Monkley2, Malcolm R. Wood1, David R. Critchley2 and Ulrich Müller1,*

Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we showthat MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion andsarcomere assembly, resembling defects in muscle lacking 1 integrins. Talin 1/2-deficient myoblasts express functionally active 1integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather bydisruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesioncomplexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial forskeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.

KEY WORDS: Integrin, Talin, Muscular dystrophy, Dystrophin, Mice

Development 136, 3597-3606 (2009) doi:10.1242/dev.035857

1The Scripps Research Institute, Department of Cell Biology and Institute ofChildhood and Neglected Diseases, La Jolla, CA 92037, USA. 2University of Leicester,Department of Biochemistry, Leicester LE1 9HN, UK.

*Author for correspondence ([email protected])

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MATERIALS AND METHODSGeneration of miceTln2-KO mice were generated by ablating the first coding exon of Tln2 (seeResults). Mice were genotyped by PCR using the following primers: (a) 5�-CAAACTGAATGAAGGCCCAACAG-3�; (b) 5�-TCTCCACTTACTC-CTTGCCC-3�; (c) 5�-GCCGAGGCTACATGGAGTCAGTAT-3�. Tln1/2-dKO mice and control mice were obtained by crossing Tln1flox/flox;Tln2+/–

mice with Tln1flox/+;Tln2+/–;HSA-CRE+/– mice. Tln1flox/flox and HSA-CREmice have been described previously (Conti et al., 2008; Leu et al., 2003).

Histology, western blotting, electron microscopy and CK levelsMuscle sections were stained with hematoxylin and eosin (H&E). Todetermine the number of fibers with central nuclei, random areas across themuscle were photographed and nuclei quantified. The number of fieldsanalyzed depended on the size of the muscle type (soleus, n3;gastrocnemius n9; tibialis anterioris, n6). Three mice per genotype andtime-point were analyzed. The mean ±s.d. was determined and a Student’st-test performed. Immunohistochemistry, analysis of cell proliferation andwestern blotting were carried out as described (Conti et al., 2008; Schwanderet al., 2003) using the following antibodies: (1) monoclonal against vinculin(Sigma), MHCf (Sigma), ILK (Li et al., 1999), tenascin C (Sigma), MHCs(Leica), CD9 (BD Pharmingen), sarcomeric -actinin (Sigma), BrdU(Pharmingen); (2) rabbit polyclonal against 7 integrin (kindly provided byU. Meyer, University of East Anglia, Norwich, UK), v integrin (Chemicon),1 integrin (Schwander et al., 2003), collagen type IV (Chemicon), laminin2 (Chemicon) and CX43 (Abcam). Polyclonal antibodies specific for talin1 and 2 have been described previously (Conti et al., 2008), and correspondto residues 1830-1850 (talin 1) and 940-957 (talin 2). Electron microscopyand Evans blue dye (EBD) uptake assays were performed as describedpreviously (Conti et al., 2008). Measurements of creatine kinase (CK) levelswere performed by Antech Diagnostics (Irvine, CA, USA).

Primary cultures of fetal myoblastsPrimary cultures of fetal myoblasts were prepared from hindlimb musclefrom E17.5 embryos as described previously (Schwander et al., 2003). Cellswere plated onto coverslips coated with 0.1% gelatin (Sigma) and grown inmedium consisting of 65% DMEM (Gibco), 25% Media 199 (Gibco) and10% fetal bovine serum (Gibco). Differentiation was induced by transferringthe cultures to medium consisting of 70% DMEM, 28% Media 199, 2%horse serum (Gibco) and 0.1 mg/ml insulin (Sigma). After 3 days, cells werefixed, permeabilized with 0.5% TX-100 and stained with antibodies to -actinin and MHCf and then with a secondary anti-mouse Alexa 488antibody. Myonuclei were stained with DAPI (Sigma). The fusion index wasdetermined as the ratio of myonuclei in cells with three or more nuclei to thetotal number of nuclei. To measure adhesion, 6�104 cells were plated onpoly-D-lysine, collagen type IV, laminin or fibronectin. After 90 minutes,cells were washed, fixed and myoblasts immunostained with antibodies to7 integrin or -actinin. Nuclei were stained with DAPI. The number ofcells adhering to each substrate was normalized to the number of cellsadhering to poly-D-lysine. The mean ±s.d. was determined and a Student’st-test performed. Cells were photographed using an Olympus AX70microscope and counted using Metamorph.

Flow cytometryMyoblasts were analyzed by fluorescence-activated cell sorting (FACS)based on 7 integrin subunit expression as described previously (Blanco-Bose et al., 2001). Cells were harvested and resuspended in PBS containing3% BSA. To detect 7 expression, cells (106/sample) were incubated withphycoeritrin (PE)-conjugated antibodies to 7 (MBL). To detect expressionof active 1 integrins, cells were then incubated with antibody 9EG7 (BDBiosciences), followed by a FITC-conjugated secondary antibody. Cellswere analyzed in a LSR II 2 flow cytometer (BD Biosciences).

RESULTSGeneration of Tln2-KO miceTo analyze talin 2 function during skeletal muscle development weinactivated Tln2 in mice. A gene-targeting vector was generated thatincluded, 5� of the first Tln2 coding exon, a neomycin (PGK-Neo)

cassette flanked by LoxP sites (Fig. 1A). A third LoxP site wasinserted between exons 2 and 3. Chimeric mice were generated thattransmitted the targeted Tln2 allele through the germline. Crossingof these mice with a CRE deleter mouse (Schwenk et al., 1995)generated three recombination events leading to mouse lines withdifferent Tln2 alleles: (1) mice lacking the neomycin cassette (rec);(2) mice lacking exon 2 (data not shown); and (3) mice lacking exon2 and the neomycin cassette (Tln2–; Fig. 1A). Mice homozygous forthe Tln2– allele were identified by genotyping using the PCRprimers indicated in Fig. 1A, which amplify a 569-base-pair bandspecific for Tln2-KO mice (Fig. 1B). Tln2-KO mice were viable andfertile and did not differ in general appearance from wild-type mice(Fig. 2A,B). To confirm that expression of talin 2 was ablated, weanalyzed protein expression by western blot using antibodiesspecific for talin 2 (Conti et al., 2008). Two bands corresponding tointact and cleaved talin 2 were detected in muscle extracts from 1-month-old wild-type but not Tln2-KO mice (Fig. 1C). We alsoimmunostained sections of gastrocnemius muscle in 1-month-oldmice. As reported previously (Conti et al., 2008), talin 2 wasconcentrated at MTJs in wild-type mice, but not in Tln2-KO musclefibers (Fig. 1D). We conclude that talin 2 expression was effectivelyablated in muscle from Tln2-KO mice.

Tln2-KO mice develop a myopathy with centrallynucleated fibersWe next analyzed histological sections from Tln2-KO mice forskeletal muscle defects. In several mouse models of musculardystrophy, muscle fibers undergo cycles of degeneration andregeneration. While the nuclei of healthy muscle fibers are locatedclose to the sarcolemma, regenerating fibers display centrallylocated nuclei, providing a useful readout for muscle defects

RESEARCH ARTICLE Development 136 (21)

Fig. 1. Generation of Tln2-KO mice. (A)Schematic representation ofthe targeting strategy. (B)PCR result from genotyping using the primersin (A) with DNA from 1-month-old wild-type (WT) and Tln2-KO mice(T2-KO). 325-bp and 569-bp bands indicative of wild-type and Tln2-alleles, respectively, were observed. (C)Protein extracts from 1-monthold gastrocnemius muscle were analyzed by western blot. Talin 2expression was ablated in Tln2-KO mice. Membranes were probed with-tubulin as a loading control. (D)Longitudinal sections of 1-month-oldgastrocnemius muscle were stained with antibodies against talin 2. Talin2 was undetectable in Tln2-KO muscle. Scale bar: 50mm. D

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(Pierson et al., 2005). At 1 month of age, skeletal muscles from Tln2-KO mice and controls were largely indistinguishable, but a slightincrease in the number of centrally nucleated fibers was noticeablein the mutants (Fig. 2C,D,I). By 7 months of age, the number ofcentrally located nuclei was drastically increased in Tln2-KO mice.Similar observations were made in gastrocnemius, soleus and tibialismuscles (Fig. 2E-I; data not shown). As observed in other mousemodels for muscular dystrophy (Straub et al., 1997), the severity ofthe phenotype showed differences between distinct muscles. InTln2-KO mice, a significantly higher percentage of centrally locatednuclei was observed in the soleus when compared with thegastrocnemius and tibialis muscles (gastrocnemius: wild-type1.27±0.93%, Tln2-KO9.55±0.31%; soleus: wild-type0.81±0.16%, Tln2-KO35.03±2.26%; tibialis: wild-type0.11±0.09%;Tln2-KO 9.27±1.34%; in all instances n3, values are mean ±s.d.).

The higher proportion of centrally nucleated fibers in the soleusmuscle suggests that deficiency of talin 2 might predominantly affectslow (type I) fibers. To determine whether this was the case, musclefibers were co-immunostained with DAPI and with antibodies toslow and fast myosin heavy chain isoforms (Fig. 2L-N, data notshown). Centrally located nuclei were found in both type I and typeII fibers. While central nuclei were found more frequently in slow

fibers, once the relative proportion of fast versus slow fibers in thesoleus muscle was taken into account, no significant difference wasobserved (Fig. 2R). Co-immunostaining for MHCf and talin 2showed that talin 2 was expressed at the MTJs of both fast and slowfibers (Fig. 2O-Q). Finally, analysis by western blot showed that,although talin 2 is expressed at higher levels in muscle than talin 1(Conti et al., 2008; Senetar and McCann, 2005), levels of talin 1 andtalin 2 did not differ between different muscles (Fig. 2S,T). Wetherefore conclude that deficiency of talin 2 equally affects slow andfast fiber types. The higher number of centrally nucleated fibers insoleus muscle could be explained by it being a postural muscle(Vandervoort and McComas, 1983), experiencing more stress thanthe gastrocnemius and tibialis muscles. Interestingly, muscles inTln1-KO mice, including the soleus, do not show centrally nucleatedmyofibers (Conti et al., 2008), which could reflect the fact that talin2 levels in muscle are higher than talin 1 levels.

No evidence for sarcolemmal damage in Tln2-KOmiceCentrally nucleated myofibers are frequently found in dystrophicmuscle fibers that also show defects in the stability of thesarcolemma, leading to efflux of proteins from muscle fibers. For

3599RESEARCH ARTICLETalin 1 and 2 in skeletal muscle development

Fig. 2. Tln2-KO mice develop a myopathy withcentrally nucleated fibers. (A,B)Normal appearance ofTln2-KO mice. (C-H)Sections of gastrocnemius musclewere stained with H&E. Centrally nucleated fibers wereevident in gastrocnemius (C-F) and soleus muscles (G,H) ofmice that were 1 (C,D) and 7 (E-H) months old.(I)Quantification of centrally nucleated fibers (CNF) ingastrocnemius and soleus muscles. The number of affectedfibers increased with the age of Tln2-KO mice (P0.006,gastrocnemius; *, P0.0007, soleus). (J)Serum creatinekinase (CK) levels were normal in 5-month-old Tln2-KOmice (n4-7 per genotype). (K)EBD was injected into 5-month-old mice. No dye incorporation was noted inmuscles of Tln2-KO mice. Occasionally, dye incorporationwas observed irrespective of genotype (control), validatingthe experimental set up (n4-7 per genotype). (L-N,R) Co-immunostaining of Tln2-KO muscle for MHCf (green) andDAPI (blue) revealed that fast and slow fibers wereaffected; laminin staining (red) highlights muscle fibercontours. (O-Q)Co-immunostaining for MHCf (red) andtalin 2 (green) revealed that talin 2 was expressed at theMTJs of fast and slow fibers. Yellow and white arrows pointto fast and slow fibers, respectively. (R)Quantification ofthe distribution of central nuclei (CNF) in slow and fastfibers (*, P0.02). (S,T) Expression of talin 1 and talin 2 wasevaluated by western blot. Equivalent expression levelswere observed in soleus and gastrocnemius muscles. Scalebars: 100mm.

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example, this phenotype is observed in patients affected byDuchenne muscular dystrophy and in the mdx mouse (Dmd –Mouse Genome Informatics), which bear mutations in thedystrophin gene (Carpenter and Karpati, 1979; Schmalbruch, 1975;Straub et al., 1997; Weller et al., 1990). By contrast, mutations inthe Itga7 and Tln1 genes cause muscle defects with little or noevidence of membrane damage, and the former but not the latterhave centrally nucleated myofibers (Conti et al., 2008; Hayashi etal., 1998; Rooney et al., 2006). To further evaluate whether talin 2-, talin 1- and integrin 7-deficient muscle fibers shared otherphenotypic features, we analyzed sarcolemmal damage bymeasuring creatine kinase (CK) levels in the plasma of 5-month-old mice. Tln2-KO mice presented no evidence for membranedamage (Fig. 2J). We also injected EBD in the tail vein.Occasionally, EBD-positive fibers (which by histology appeareddamaged; data not shown) were detected irrespective of thegenotype, validating the experimental set up (‘control’ in Fig. 2K);however, no obvious EBD accumulation was noted in Tln2-KOmice (Fig. 2K). Finally, while immune-cell infiltration and fibrosisare observed in mice with mutations that affect the dystrophincomplex (Stedman et al., 1991), these histopathologicalabnormalities were not present in muscle from Tln2-KO mice (datanot shown).

We conclude that Tln2-KO and 7 integrin mutant mice showsigns of skeletal muscle fiber degeneration that differ from thephenotype associated with mutations affecting the dystrophincomplex, providing evidence that these protein complexes regulatemuscle fiber maintenance in different ways.

Talin 2 is not essential for the assembly of themuscle fiber cytoskeletonTalin is essential for focal adhesion assembly and turnover (Francoet al., 2004; Priddle et al., 1998). However, sarcomere and costamereassembly were maintained when Tln1 was ablated in skeletal muscle(Conti et al., 2008). As talin 2 is expressed in muscle at higher levelsthan talin 1 (Conti et al., 2008; Senetar and McCann, 2005), wedetermined whether the muscle fiber cytoskeleton was abnormal inTln2-KO mice. Sarcomere integrity was evaluated by electronmicroscopy in 3-month-old mice. In wild-type muscle, the structureof the sarcomere was well maintained, with well-defined Z- and M-bands (Fig. 3A). In Tln2-KO muscle, necrotic material was observedwithin myofibers (Fig. 3B,C). Although the M-band (asterisk in Fig.3B) was not always evident in all areas of mutant muscle fibers (Fig.3C), the Z-line was present and the overall striation pattern wasmaintained. By contrast, major defects were observed at MTJs. Inwild-type muscle, actin filaments reached the sarcolemma at the endof muscle fibers (Fig. 3D,G). In Tln2-KOs, actin filaments detachedfrom the MTJs, and necrotic and membranous material localized inthe gap left by retracting myofilaments (Fig. 3E,H,I). Theperturbations at MTJs of Tln2-KO mice resemble the defects in micelacking Tln1 in skeletal muscle but were considerably more severeand prominent at an earlier age (3 instead of 6 months) (Conti et al.,2008). Unlike in Tln1-KO mice, lateral detachment of thecytoskeleton from the sarcolemma was also occasionally noted inTln2-KO mice (Fig. 3F). These data are consistent with talin 2 beingthe major talin isoform in skeletal muscle (Conti et al., 2008; Senetarand McCann, 2005).


Fig. 3. Talin 2 is required for MTJ integrity but notfor sarcomere organization. (A-C)EM micrographs ofgastrocnemius (A,B) and soleus (C) isolated from3-month-old mice. Disorganization was evident tovarying degrees in Tln2-KO muscle fibers, whichaccumulated necrotic material. Muscle fibers appearedcontracted, but the Z-line and A-band were evident(white arrows and asterisks, respectively). (D-I)Electronmicrographs of MTJ from soleus (D,E) and gastrocnemius(G-I) of 3-month-old mice. In wild-type mice,myofilaments reached the end of muscle fibers (D,G,arrowhead). In Tln2-KO mice, myofilaments weredetached from the MTJ, and necrotic materialaccumulated in the gaps (E,H,I, asterisks). Lateraldetachment of the cytoskeleton from the sarcolemmawas occasionally noted (F, arrowheads). (J-Q)Longitudinalsections of gastrocnemius muscle were immunostainedwith antibodies to 7 integrin (J,K), ILK (L,M), vinculin(Vn) (N,O) and talin 1 (Tln1) (P,Q). All proteins werelocalized at MTJs, but talin 1 staining was increased inthe mutants (arrow in Q). Scale bars: 2mm in A-C; 5mmin D-I; 100mm in J-Q.

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Assembly of integrin complexes and increasedrecruitment of talin 1 to the MTJ of Tln2-KO miceTo determine whether assembly of integrin complexes wascompromised in Tln2-KO mice, we evaluated the distribution ofintegrins and their effectors. In control muscle, 7 integrin andvinculin were localized at costameres and MTJs, whereas ILK wasonly found at MTJs. The distribution of these proteins was normalin Tln2-KO mice, although it appeared more diffuse, possiblyreflecting MTJ disorganization (Fig. 3J-O). Importantly, talin 1expression was undetectable or very low in wild-type mice (Fig. 3P)but was readily detectable at the MTJs of Tln2-KO mice (Fig. 3Q,arrow). Talin 1 accumulation at MTJs was likely to be caused byredistribution of talin 1 protein as western blot analysis showed thattotal talin 1 levels in muscle remained unchanged (see Fig. S1A inthe supplementary material). We conclude that talin 2 is not essentialfor the assembly of integrin complexes in skeletal muscle fibers, andthat talin 1 is likely to compensate for a lack of talin 2. However,defects at MTJs are observed in muscle lacking either talin 1 (Contiet al., 2008) or talin 2 (this study), suggesting that the two talinisoforms cannot completely compensate for each other or that areduction in the total amount of talin (1 and 2) protein caused thedefects at MTJs (see Discussion).

Severe defects in skeletal muscle development inTln1/2-dKO miceWe have previously shown that mice lacking 1 integrins in skeletalmuscle (refereed to as Itgb1-KOs) have a considerably more severephenotype than mice lacking either talin 1 or 2 (Conti et al., 2008;Schwander et al., 2003). This, together with the observed increasedlocalization of talin 1 at the MTJ in Tln2-KO mice (Fig. 3P,Q),prompted us to test whether the functions of talin 1 and 2 mightoverlap. We took advantage of our previous observation that aTln1flox allele can be effectively inactivated in developing skeletalmuscle by an HSA-CRE transgene (Conti et al., 2008). Using thesemice and the Tln2-KO mice described here, we generated Tln1/2-dKO mice, which lack both talin 1 and talin 2 in skeletal muscle (seeMaterials and methods). Similar to Itgb1-KO mice (Schwander etal., 2003), Tln1/2-dKO mice had a contracted posture (Fig. 4A) anddied shortly after birth. Immunohistochemical analysis confirmedthat expression of talin 2 was effectively ablated in muscle fromE17.5 Tln1/2-dKO embryos (Fig. 4B,C). While expression of talin1 in skeletal muscle was too low to be detected at this stage even inwild-type mice, the severity of the phenotype of the Tln1/2-dKOmice compared with single mutants suggests that Tln1 waseffectively inactivated. In addition, PCR analysis of forelimbmuscles confirmed recombination of the Tln1flox/flox allele (see Fig.S1C in the supplementary material).

Histological analysis of embryos at embryonic day 17.5 (E17.5)revealed severe defects in muscle development throughout the body.Although muscles could still be detected, they had an abnormalmorphology similar to the phenotype of Itgb1-KO mice (Schwanderet al., 2003). In wild-type embryos, muscle fibers were welldeveloped and possessed a uniform size (Fig. 4D,F). In Tln1/2-dKOmice, a general disorganization of the muscles was evident, withstriking variations in fiber size (Fig. 4G,H,I,K).

We next determined whether skeletal muscle defects wereaccompanied by changes in cell proliferation or differentiation. Toevaluate proliferation, we carried out BrdU labeling experiments.Proliferating cells were located in several muscle groups, includingintercostals and semispinalis muscles, and were identified by co-immunostaining for BrdU and desmin (see Fig. S2A,B,E in thesupplementary material; data not shown). No difference in the

number of proliferating cells per unit area (mm2) muscle tissue wasobserved between control and Tln1/2-dKO embryos. Next, weimmunostained E16.5-E17.5 embryos with antibodies to desmin(see Fig. S2A-D in the supplementary material, red), -actinin (seeFig. S2G,H in the supplementary material) and MHCf (see Fig. S2I,Jin the supplementary material). These markers were normallyexpressed in Tln1/2-dKO embryos, indicating that the differentiationof myoblasts was not affected in the mutants. We thereforehypothesized that, similar to mice lacking 1 integrins (Schwanderet al., 2003), defects in skeletal muscle development in Tln1/2-dKOmice might be a consequence of perturbations in myoblast fusionand sarcomere assembly.

Defects in sarcomere organization in Tln1/2-dKOmiceAlthough no alterations in sarcomere organization were noted inTln1-KO or Tln2-KO mice (Fig. 3) (Conti et al., 2008), simultaneousinactivation of Tln1 and Tln2 caused severe defects. In control


Fig. 4. Tln1/2-dKO mice die at birth with skeletal muscle defects.(A)Tln1/2-dKO embryos (dKO) had a contracted posture comparedwith that of wild-type (WT) embryos. (B,C)Immunostaining of sectionsfrom E18.5 wild-type and Tln1/2-dKO muscle showed that talin 2 waseffectively ablated from costameres and MTJs (white arrows).(D-K)Sections showing intracostal muscle in E18.5 wild-type (D,F) andTln1/2-dKO embryos (E,G,H-K) stained with H&E. Myofibers fromTln1/2-dKO mice had abnormal morphology and variation in fiber size(black arrows in G-K). Scale bars: 50mm in B,C; 100mm in D-E; 50mmin F-K.

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embryos, vinculin was evenly localized at the sarcolemma (Fig. 5A).By contrast, vinculin distribution was strongly reduced and patchyin the disorganized and short muscle fibers of Tln1/2-dKO mice(Fig. 5B). ECM proteins such as collagen type IV (data not shown)and laminin were deposited around Tln1/2-deficient muscle fibersbut showed signs of disorganization and discontinuity (Fig. 5C,D).Tenascin C expression is associated with processes of degenerationand regeneration in dystrophic muscle (Settles et al., 1996; Tavernaet al., 1998). In control muscle fibers, tenascin C was confined at theperiosteum and the tendon (Fig. 5E). In Tln1/2-dKO muscle fibers,tenascin C was expressed in areas distal from the MTJs (Fig. 5F).Severe defects in sarcomere organization were noted at theultrastructural level. In control embryos, myofilaments and thestriation of sarcomeres were well defined (Fig. 5G). In Tln1/2-dKOmuscle, the organization of myofilaments was disrupted, and theassembly of Z-bands appeared rudimentary (Fig. 5H,I). Amorphousfilamentous material was abundant between myofilaments. Weconclude that the skeletal muscle fiber cytoskeleton is severelydisrupted in Tln1/2-dKO muscle.

Defects in the assembly of integrin adhesioncomplexes in Tln1/2-dKO miceImmunohistochemical evaluation of the expression andlocalization of components of integrin adhesion complexes inskeletal muscle of E17.5 embryos revealed severe defects inTln1/2-dKO mice. In wild-type embryos, 7-, v- and 1-integrinswere clustered at MTJs (Fig. 6A,C; data not shown), whereas none

of these proteins was localized at MTJs in Tln1/2-dKO mice (Fig.6B,D; data not shown). Likewise, the localization of vinculin andILK was compromised (Fig. 6E-H, see also Fig. 5A,B), indicatingthat talin 1 and 2 are essential for the assembly and clustering ofintegrin adhesion complexes at MTJs. Consistent with these data,MTJs were rarely detected in Tln1/2-dKO mice by ultrastructuralanalysis. When present, they appeared abnormal: myofilamentsand the electron-dense plaque at the muscle terminus were absent(arrows in Fig. 6I,J). Notably, these defects at MTJs differ fromthose in muscle from Itgb1-KO mice, where MTJs developnormally (Schwander et al., 2003). A possible explanation for thisdifference is the presence of the integrin v subunit at MTJs(Hirsch et al., 1994). Unlike the integrin 7 subunit, vheterodimerizes with several integrin subunits in addition to 1(Hynes, 1992). The expression and localization of v were notaffected in muscle from Itgb1-KO mice, but v was no longerpresent at MTJs from Tln1/2-dKO mice (Fig. 6C,D), suggestingthat the v subunit with a heterodimeric partner other than 1 issufficient to direct the assembly of MTJs. Taken together, our dataindicate that, in Tln1/2-dKO mice, the formation of integrinadhesion complexes in skeletal muscle was affected, leading todefects in the formation of the MTJs and the assembly of themuscle fiber cytoskeleton.

Impaired fusion of Tln1/2-dKO myoblastsMyoblast fusion depends on the alignment of the membranes ofmyoblasts, the formation of prefusion complexes characterized byelectron-dense plaques, and the subsequent breakdown of theplasma membrane. In wild-type mice, it is difficult to capturemyoblasts containing the electron-dense plaques because prefusioncomplexes are rapidly resolved (Schwander et al., 2003). Bycontrast, we could readily observe in developing skeletal musclefrom Tln1/2-dKO embryos unfused myoblasts containing theelectron-dense plaques (Fig. 7A-C), suggesting that fusion wasperturbed.

To evaluate directly whether fusion was affected, we establishedprimary cultures of fetal myoblasts from the hindlimbs of E17.5embryos and analyzed myotube formation in vitro. After 3 days inculture, myoblasts isolated from wild-type embryos formednumerous long myotubes (Fig. 7D,F). By contrast, Tln1/2-dKOmyoblasts attached to the underlying fibroblast layer but failed tofuse (Fig. 7E,F). Myotubes formed occasionally, but they were shortand assembled a rudimentary cytoskeleton: -actinin and MHCfwere recruited in a striated pattern in wild-type myotubes, but theirlocalization was altered in double mutants (Fig. 7G-J).

The defects in sarcomere assembly and myoblast fusion inTln1/2-dKO mice were similar to those observed in Itgb1-KO mice(Schwander et al., 2003). In other cells types, such as fibroblasts andplatelets, talin mediates the assembly of integrin complexes andintegrin activation (Nieswandt et al., 2007; Petrich et al., 2007;Tadokoro et al., 2003). Defects in either of these processes (or both)could affect myoblast fusion. We therefore evaluated 1 integrinexpression and activation in primary cultures of fetal myoblasts fromTln1/2-dKO mice by FACS analysis using the 9EG7 antibody, whichspecifically recognizes an exposed epitope when 1 integrins are inan active conformation (Bazzoni et al., 1995). To distinguishmyoblasts from contaminating fibroblasts, we used an antibody to7-integrin, which is specifically expressed by myoblasts (Blanco-Bose et al., 2001) (Fig. 7K). 7-integrin expression levels werenormal in myoblasts from double-mutant mice (Fig. 7L,N),excluding that defects in muscle fiber development were caused bya reduction of the amount of integrin expressed at the cell surface of


Fig. 5. Defective sarcomere assembly in Tln1/2-dKO muscle.(A-F)Sections from E18.5 embryos were immunostained withantibodies against vinculin (Vn) (A,B), laminin (Lm) (C,D) and tenascin C(Tn C) (E,F). Expression levels of vinculin were reduced in Tln1/2-dKOembryos. Laminin localized around myofibers but appeareddisorganized in mutants (arrow in D). Tenascin C was exclusivelylocalized at the MTJ and in periosteum in controls (E) but was expressedin Tln1/2-dKOs in extrajunctional areas as well (arrow in F).(G-I)Electron micrographs of intercostal muscles from wild-type (G) andTln1/2-dKO embryos (H,I). The cytoskeletal structure appearedimmature and disorganized in Tln1/2-dKO embryos. Disorganizedfilamentous material accumulated throughout the myofiber, andZ-bands appeared to be incompletely assembled (arrows in H,I)compared with those of controls (arrow in G). Scale bars: 25mm in A-D;50mm in G,H; 2mm in I.

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myoblasts. To our surprise, labeling by the 9EG7 antibody of Tln1/2-dKO myoblasts was also comparable to that of controls (Fig. 7L,M;myoblasts are represented by the gated population), indicting thatintegrin activation was not affected. To confirm these findingsfurther, we seeded myoblasts on collagen type IV, laminin andfibronectin. No significant difference in adhesion was noted on anyof the tested substrates (Fig. 7O). We conclude that defects indevelopment of Tln1/2-dKO muscle are not likely to be caused byan inability of 1 integrins to interact with ligands.

To investigate the mechanisms that might cause the fusiondefect in Tln1/2-dKO myoblasts further, we analyzed theexpression of connexin 43 (CX43; also known as Gja1) and thetetraspanin CD9, which have been shown to regulate cell fusion.CX43 is upregulated in myoblasts preceding fusion (Araya et al.,2005; Gorbe et al., 2007) and was expressed in control and mutantmyoblasts (Fig. 7P,Q). Although the localization of CD9 wasaffected in 1 integrin mutant myoblasts (Schwander et al., 2003),it was normally expressed in myoblasts isolated from Tln1/2-dKOmice (Fig. 7R,S). It is interesting to note that CD9 and 7 integrinwere detected, although not exclusively, at the contact surfacebetween talin1/2-deficient myoblasts (Fig. 7P-S, arrows),indicating that their recruitment was not dependent on talin1/2.Talin 1/2 are therefore likely required at a subsequent step inmyoblast fusion, potentially by linking integrins to the actincytoskeleton. Unfortunately, analysis of the organization of the F-actin cytoskeleton of myoblasts using phalloidin was notinformative because of the small size and rounded morphology ofthese cells (data not shown).

DISCUSSIONWe show here that talin 1 and 2 are essential for skeletal muscledevelopment and function. Tln2-KO mice are viable and fertile butdevelop a myopathy with centrally located nuclei that is associatedwith defects in the maintenance of MTJs. When talin 1 and 2 areinactivated simultaneously, severe defects in myoblast fusion andsarcomere assembly are observed that are not present in the singlemutants. The defects in skeletal muscle development in Tln1/2-dKOmice closely resemble the phenotype of muscle lacking 1 integrins.As talin1/2-deficient myoblasts expressed functionally active 1integrins, defects in muscle development are likely not primarilycaused by lack of an ability of 1 integrins to bind to ECM ligands

but by the disruption of their interaction with the cytoskeleton.Consistent with this finding, recruitment of integrin effectors isperturbed in the remaining small muscle fibers of talin1/2-deficientmice.

Previous studies in invertebrates have shown that talin is requiredfor the attachment of skeletal muscle fibers (Brown et al., 2002;Cram et al., 2003). The findings presented here and in our previousreport (Conti et al., 2008) extend these findings and show that talinhas an evolutionarily conserved function in skeletal muscleattachment. Consistent with the higher expression levels of talin 2in skeletal muscle compared with those of talin 1 (Conti et al., 2008;Senetar and McCann, 2005), Tln2-KO mice developed a moresevere myopathy than talin 1 mutants, which is characterized bycentrally nucleated myofibers and prominent MTJ defects.Nevertheless, MTJ defects were also present in muscle lacking talin1 (Conti et al., 2008). This result could be explained by twomechanisms. First, talin protein levels might be important. Talin 1was redistributed to MTJs in Tln2-KO mice, but overall talin 1 levelswere not changed. Therefore, a reduction of total talin levels due toloss of talin 2 might have caused MTJ instability. Alternatively, talin1 and 2 at MTJs might not be entirely interchangeable. Althoughtalin 1 shares 74% identity with talin 2, differences in the remainingamino acids could possibly affect protein function. This model isconsistent with previous findings. Although myoblasts expressintegrin 1A, muscle fibers express the 1D isoform, which bindswith higher affinity to F-actin (Belkin et al., 1997; Belkin et al.,1996; van der Flier et al., 1997). The I/LWEQ module of talin 2binds with higher affinity to muscle -actin than the correspondingmodule in talin 1, which in turn binds with higher affinity to non-muscle -actin (Senetar et al., 2004). These data delineate a modelwhereby the expression of 1D-integrin and talin 2 might beimportant to confer a strong mechanical link between integrins andthe cytoskeleton. As ILK-deficient mice also show defects at MTJs(Wang et al., 2008), it appears that several integrin effectorscooperate in this process. Although speculative, talin 1 might bemore important for dynamic connections in other cells such asfibroblasts and platelets.

Our studies also provide evidence that talin 1 and 2 cooperate toregulate muscle fiber development. Tln1/2dKO mice show defectsin myoblast fusion and sarcomere assembly similar to 1 integrin-deficient mice (Schwander et al., 2003), suggesting that talin1/2 are


Fig. 6. Compromised assembly of integrincomplexes in Tln1/2-dKO mice. (A-H)Sections fromE18.5 embryos were immunostained with antibodiesagainst 7 and v-integrins and to vinculin (Vn) and ILK.The localization of integrins and their effectors at MTJswas disrupted in Tln1/2-dKO muscle. Dotted lineshighlight the location of the MTJ in Tln1/2-dKO muscle.(I,J)Electron micrographs of the MTJ of intercostalmuscles from E18.5 wild-type (I, arrows) and Tln1/2-dKO (J, arrows) embryos. Muscle fibers close to MTJs inTln1/2-dKO were disorganized (asterisk in J). Scale bars:50mm in A-H; 2mm in I,J.

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required for integrin functions in muscle. Previous studies haveshown that talin1/2 regulate both integrin activation and theirlinkage to the cytoskeleton. Although defects in either of theseprocesses could lead to the skeletal muscle defects in Tln1/2dKOmice, our data suggest that defects in cytoskeletal linkage are thecause for the phenotype in the mutant mice. Consistent with thismodel, 1 integrin expression and activation were not affected inprimary cultures of fetal myoblasts from Tln1/2-dKO mice. Similarto our findings, it has previously been shown that talin-mediatedintegrin activation is also not essential for the initial adhesion offibroblasts to ECM substrates (Zhang et al., 2008). Althoughintegrin activation by talin is essential in other cell types such asplatelets (Petrich et al., 2007; Nieswandt et al., 2007), it might beless important in myoblasts. Kindlin-3 synergizes with talin inregulating integrin activation in platelets (Moser et al., 2009;Svensson et al., 2009), and other proteins, such as ILK, regulate thisprocess (Honda et al., 2009). Therefore, the ligand binding activityof integrins might be regulated by different mechanisms in a cell-type-specific manner.

Instead, our findings suggest that defects in Tln1/2-deficientskeletal muscle fibers are caused by defects in the interaction ofintegrins with the cytoskeleton. In support of this model, weobserved in the mutants detachment of the muscle fibercytoskeleton from MTJs and defects in the linkage of integrinadhesion complexes to costameres. The fact that integrins in talin1/2-deficient myoblasts effectively bound to ligands suggests thatsteps subsequent to adhesion also led to defects in myoblastfusion. Fusion defects in mice lacking 1 integrins areaccompanied by reduced recruitment of the tetraspanin CD9 tothe site of fusion (Schwander et al., 2003). However, CD9 wasstill recruited normally in talin1/2-deficient myoblasts, suggestingthat talin1/2 act at a subsequent step. Interestingly, severalproteins that regulate integrin function and actin dynamics areimplicated in myoblast fusion. For example, the guanine-nucleotide exchange factors (GRFs) Dock180 and Brag2/GEP100

are required for the fusion of myoblasts and macrophages (Laurinet al., 2008; Pajcini et al., 2008); genetic ablation of Dock180 inmice leads to impaired myoblast fusion (Laurin et al., 2008);


Fig. 7. Defective fusion but normal integrin activation in myoblasts from Tln1/2-dKO mice. (A-C)Electron micrographs of muscle fromE18.5 Tln1/2-dKO embryos revealed myoblasts at intermediate stages of fusion. Plasma membranes were aligned (arrows in A,B) and electron-dense adhesion plaques were evident (arrows in C). (D-J)Cell fusion was evaluated in primary cultures of fetal myoblasts. (D,E)Cultures wereimmunostained with antibodies against -actinin to label myotubes and myoblasts. In cultures from Tln1/2-dKO mice, myoblast fusion wasimpaired; only a few short, dysmorphic myotubes were detected. (F)The fusion index was determined (number of nuclei in myoblasts/total numberof nuclei) (n3 mice per genotype) (*P0.035). (G-J)Immunostaining with antibodies against -actinin (G,H) and MHCf (I,J) revealed that thecytoskeleton in myofibers from Tln1/2-dKO remained immature. Arrowheads in G,I refer to costameres. (K-O)Analysis of integrin expression andactivation by FACS. (K)Cell surface expression of 7 integrin was used to distinguish myoblasts from fibroblasts (bracket indicates 7-integrin-positive population). (L)Representative dot plots of FACS sorted myoblasts from wild-type and Tln1/2-dKO mice analyzed for 7 integrin expressionand presence of the 9EG7 epitope (detecting activated 1-integrins). Gated area represents myoblasts. (M)Histogram representing frequency of the9EG7 epitope on myoblasts. No significant difference was observed between myoblasts from wild-type and Tln1/2-dKO mice. (N)Surface expressionlevels of 7-integrin were normal in Tln1/2-dKOs (n3 controls, 5 double mutants). (O)Adhesion to collagen type IV (Coll IV), laminin (Lm) andfibronectin (Fn) was evaluated in primary cultures of fetal myoblasts. No adhesion defects of Tln1/2-dKO myoblasts were observed (n2 pergenotype). The mean ± s.d. are indicated. (P-S)Myoblasts from E17.5 embryos were transferred to differentiation medium and stained for 7integrin (red), CX43 (P,Q) and CD9 (R,S). The expression levels of CX43 and CD9 were normal in Tln1/2-dKO cells. 7 integrin, CD9 and occasionallyCX43, were localized at the interface of fusing myoblasts (arrows). Scale bars: 5mm in A; 500 nm in B; 250 nm in C; 20mm in P-S.

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ablation of filamin C leads to defects in myogenesis (Dalkilic etal., 2006); furthermore, ablation of FAK affects cell fusion andmyofiber regeneration (Quach et al., 2009). This raises theinteresting possibility that integrins, and their effectors such astalin1/2, FAK and filamin C, might cooperate to regulate actinreorganization during myoblast fusion.

Centrally nucleated skeletal muscle fibers, as observed in Tln2-KO mice, are also present in several myopathies (Pierson et al.,2005). In patients and mice with mutations that affect thedystrophin complex, centrally nucleated fibers are an indicationof fiber degeneration and regeneration that is caused by plasmamembrane breakdown (Straub et al., 1997). By contrast, centrallynucleated fibers in Tln2-KO mice accumulated without noticeableplasma membrane breakdown. This resembles the situation inmice and humans with mutations in the gene encoding the integrin7 subunit (Hayashi et al., 1998; Mayer et al., 1997). The findingssuggest that mechanisms associated with defects in the dystrophinand integrin adhesion complexes differ. Interestingly, mutationsin genes encoding proteins that are indirectly involved in actinreorganization or membrane trafficking, such as myotubularin 1(Mtm1), dynamin 2 (Dnm2) and g-actin, also lead to centrallynucleated skeletal muscle fibers without plasma membranebreakdown (Bitoun et al., 2005; Buj-Bello et al., 2002;Sonnemann et al., 2006). The Tln2-KO mice presented hereprovide a useful model for studying the molecular mechanismsthat lead to fiber degeneration in the absence of plasma membranedamage. Our data also suggest that it will be important tosequence the TLN1 and TLN2 genes in patients affected withgenetically uncharacterized congenital myopathies.

AcknowledgementsWe thank Heather Elledge for technical assistance and members of thelaboratory for comments on the manuscript. This work was funded by NIHgrants NS046456 and MH078833 (U.M.). Deposited in PMC for release after12 months.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/21/3597/DC1

ReferencesAraya, R., Eckardt, D., Maxeiner, S., Kruger, O., Theis, M., Willecke, K. and

Saez, J. C. (2005). Expression of connexins during differentiation andregeneration of skeletal muscle: functional relevance of connexin43. J. Cell Sci.118, 27-37.

Balaban, N. Q., Schwarz, U. S., Riveline, D., Goichberg, P., Tzur, G., Sabanay,I., Mahalu, D., Safran, S., Bershadsky, A., Addadi, L. et al. (2001). Force andfocal adhesion assembly: a close relationship studied using elasticmicropatterned substrates. Nat. Cell Biol. 3, 466-472.

Bazzoni, G., Shih, D. T., Buck, C. A. and Hemler, M. E. (1995). Monoclonalantibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligandand manganese, but inhibited by calcium. J. Biol. Chem. 270, 25570-25577.

Belkin, A. M., Zhidkova, N. I., Balzac, F., Altruda, F., Tomatis, D., Maier, A.,Tarone, G., Koteliansky, V. E. and Burridge, K. (1996). Beta 1D integrindisplaces the beta 1A isoform in striated muscles: localization at junctionalstructures and signaling potential in nonmuscle cells. J. Cell Biol. 132, 211-226.

Belkin, A. M., Retta, S. F., Pletjushkina, O. Y., Balzac, F., Silengo, L., Fassler,R., Koteliansky, V. E., Burridge, K. and Tarone, G. (1997). Muscle beta1Dintegrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesivefunction by alternative splicing. J. Cell Biol. 139, 1583-1595.

Bitoun, M., Maugenre, S., Jeannet, P. Y., Lacene, E., Ferrer, X., Laforet, P.,Martin, J. J., Laporte, J., Lochmuller, H., Beggs, A. H. et al. (2005).Mutations in dynamin 2 cause dominant centronuclear myopathy. Nat. Genet.37, 1207-1209.

Blanco-Bose, W. E., Yao, C. C., Kramer, R. H. and Blau, H. M. (2001).Purification of mouse primary myoblasts based on alpha 7 integrin expression.Exp. Cell Res. 265, 212-220.

Brown, N. H., Gregory, S. L., Rickoll, W. L., Fessler, L. I., Prout, M., White, R.A. and Fristrom, J. W. (2002). Talin is essential for integrin function inDrosophila. Dev. Cell 3, 569-579.

Buj-Bello, A., Laugel, V., Messaddeq, N., Zahreddine, H., Laporte, J.,Pellissier, J. F. and Mandel, J. L. (2002). The lipid phosphatase myotubularin isessential for skeletal muscle maintenance but not for myogenesis in mice. Proc.Natl. Acad. Sci. USA 99, 15060-15065.

Carpenter, S. and Karpati, G. (1979). Duchenne muscular dystrophy: plasmamembrane loss initiates muscle cell necrosis unless it is repaired. Brain 102, 147-161.

Choquet, D., Felsenfeld, D. P. and Sheetz, M. P. (1997). Extracellular matrixrigidity causes strengthening of integrin-cytoskeleton linkages. Cell 88, 39-48.

Conti, F. J., Felder, A., Monkley, S., Schwander, M., Wood, M. R., Lieber, R.,Critchley, D. and Müller, U. (2008). Progressive myopathy and defects in themaintenance of myotendinous junctions in mice that lack talin 1 in skeletalmuscle. Development 135, 2043-2053.

Cram, E. J., Clark, S. G. and Schwarzbauer, J. E. (2003). Talin loss-of-functionuncovers roles in cell contractility and migration in C. elegans. J. Cell Sci. 116,3871-3878.

Dalkilic, I., Schienda, J., Thompson, T. G. and Kunkel, L. M. (2006). Loss ofFilaminC (FLNc) results in severe defects in myogenesis and myotube structure.Mol. Cell. Biol. 26, 6522-6534.

Du, X. P., Plow, E. F., Frelinger, A. L., O’Toole, T. E., Loftus, J. C. and Ginsberg,M. H. (1991). Ligands “activate” integrin alpha IIb beta 3 (platelet GPIIb-IIIa).Cell 65, 409-416.

Franco, S. J., Rodgers, M. A., Perrin, B. J., Han, J., Bennin, D. A., Critchley, D.R. and Huttenlocher, A. (2004). Calpain-mediated proteolysis of talin regulatesadhesion dynamics. Nat. Cell Biol. 6, 977-983.

Galbraith, C. G., Yamada, K. M. and Sheetz, M. P. (2002). The relationshipbetween force and focal complex development. J. Cell Biol. 159, 695-705.

Geiger, B., Bershadsky, A., Pankov, R. and Yamada, K. M. (2001).Transmembrane crosstalk between the extracellular matrix-cytoskeletoncrosstalk. Nat. Rev. Mol. Cell Biol. 2, 793-805.

Giannone, G., Jiang, G., Sutton, D. H., Critchley, D. R. and Sheetz, M. P.(2003). Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation. J. Cell Biol. 163, 409-419.

Gorbe, A., Krenacs, T., Cook, J. E. and Becker, D. L. (2007). Myoblastproliferation and syncytial fusion both depend on connexin43 function intransfected skeletal muscle primary cultures. Exp. Cell Res. 313, 1135-1148.

Hayashi, Y. K., Chou, F. L., Engvall, E., Ogawa, M., Matsuda, C., Hirabayashi,S., Yokochi, K., Ziober, B. L., Kramer, R. H., Kaufman, S. J. et al. (1998).Mutations in the integrin alpha7 gene cause congenital myopathy. Nat. Genet.19, 94-97.

Hirsch, E., Gullberg, D., Balzac, F., Altruda, F., Silengo, L. and Tarone, G.(1994). Alpha v integrin subunit is predominantly located in nervous tissue andskeletal muscle during mouse development. Dev. Dyn. 201, 108-120.

Honda, S., Shirotani-Ikejima, H., Tadokoro, S., Maeda, Y., Kinoshita, T.,Tomiyama, Y. and Miyata, T. (2009). Integrin-linked kinase associated withintegrin activation. Blood 113, 5304-5313.

Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in celladhesion. Cell 69, 11-25.

Laurin, M., Fradet, N., Blangy, A., Hall, A., Vuori, K. and Cote, J. F. (2008). Theatypical Rac activator Dock180 (Dock1) regulates myoblast fusion in vivo. Proc.Natl. Acad. Sci. USA 105, 15446-15451.

Leu, M., Bellmunt, E., Schwander, M., Farinas, I., Brenner, H. R. and Muller,U. (2003). Erbb2 regulates neuromuscular synapse formation and is essential formuscle spindle development. Development 130, 2291-2301.

Li, F., Zhang, Y. and Wu, C. (1999). Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesionlocalization of integrin-linked kinase is regulated by the PINCH-binding ANKrepeats. J. Cell Sci. 112, 4589-4599.

Liu, S., Calderwood, D. A. and Ginsberg, M. H. (2000). Integrin cytoplasmicdomain-binding proteins. J. Cell Sci. 113, 3563-3571.

Mayer, U. (2003). Integrins: redundant or important players in skeletal muscle? J.Biol. Chem. 278, 14587-14590.

Mayer, U., Saher, G., Fassler, R., Bornemann, A., Echtermeyer, F., von derMark, H., Miosge, N., Poschl, E. and von der Mark, K. (1997). Absence ofintegrin alpha 7 causes a novel form of muscular dystrophy. Nat. Genet. 17,318-323.

McCann, R. O. and Craig, S. W. (1997). The I/LWEQ module: a conservedsequence that signifies F-actin binding in functionally diverse proteins from yeastto mammals. Proc. Natl. Acad. Sci. USA 94, 5679-5684.

McCann, R. O. and Craig, S. W. (1999). Functional genomic analysis reveals theutility of the I/LWEQ module as a predictor of protein:actin interaction. Biochem.Biophys. Res. Commun. 266, 135-140.

Miosge, N., Klenczar, C., Herken, R., Willem, M. and Mayer, U. (1999).Organization of the myotendinous junction is dependent on the presence ofalpha7beta1 integrin. Lab. Invest. 79, 1591-1599.

Monkley, S. J., Pritchard, C. A. and Critchley, D. R. (2001). Analysis of themammalian talin2 gene TLN2. Biochem. Biophys. Res. Commun. 286, 880-885.


DEVELO

PMENT

3606

Moser, M., Bauer, M., Schmid, S., Ruppert, R., Schmidt, S., Sixt, M., Wang, H.V., Sperandio, M. and Fassler, R. (2009). Kindlin-3 is required for beta2 integrin-mediated leukocyte adhesion to endothelial cells. Nat. Med. 15, 300-305.

Nayal, A., Webb, D. J. and Horwitz, A. F. (2004). Talin: an emerging focal pointof adhesion dynamics. Curr. Opin. Cell Biol. 16, 94-98.

Nieswandt, B., Moser, M., Pleines, I., Varga-Szabo, D., Monkley, S.,Critchley, D. and Fassler, R. (2007). Loss of talin1 in platelets abrogatesintegrin activation, platelet aggregation, and thrombus formation in vitro and invivo. J. Exp. Med. 204, 3113-3118.

Pajcini, K. V., Pomerantz, J. H., Alkan, O., Doyonnas, R. and Blau, H. M.(2008). Myoblasts and macrophages share molecular components thatcontribute to cell-cell fusion. J. Cell Biol. 180, 1005-1019.

Petrich, B. G., Marchese, P., Ruggeri, Z. M., Spiess, S., Weichert, R. A., Ye, F.,Tiedt, R., Skoda, R. C., Monkley, S. J., Critchley, D. R. et al. (2007). Talin isrequired for integrin-mediated platelet function in hemostasis and thrombosis. J.Exp. Med. 204, 3103-3111.

Pierson, C. R., Tomczak, K., Agrawal, P., Moghadaszadeh, B. and Beggs, A.H. (2005). X-linked myotubular and centronuclear myopathies. J. Neuropathol.Exp. Neurol. 64, 555-564.

Priddle, H., Hemmings, L., Monkley, S., Woods, A., Patel, B., Sutton, D.,Dunn, G. A., Zicha, D. and Critchley, D. R. (1998). Disruption of the talin genecompromises focal adhesion assembly in undifferentiated but not differentiatedembryonic stem cells. J. Cell Biol. 142, 1121-1133.

Quach, N. L., Biressi, S., Reichardt, L. F., Keller, C. and Rando, T. A. (2009).Focal Adhesion Kinase Signaling Regulates the Expression of Caveolin 3 andbeta1 Integrin, Genes Essential for Normal Myoblast Fusion. Mol. Biol. Cell 20,3422-3435.

Riveline, D., Zamir, E., Balaban, N. Q., Schwarz, U. S., Ishizaki, T., Narumiya,S., Kam, Z., Geiger, B. and Bershadsky, A. D. (2001). Focal contacts asmechanosensors: externally applied local mechanical force induces growth offocal contacts by an mDia1-dependent and ROCK-independent mechanism. J.Cell Biol. 153, 1175-1186.

Rooney, J. E., Welser, J. V., Dechert, M. A., Flintoff-Dye, N. L., Kaufman, S. J.and Burkin, D. J. (2006). Severe muscular dystrophy in mice that lackdystrophin and alpha7 integrin. J. Cell Sci. 119, 2185-2195.

Schmalbruch, H. (1975). Segmental fiber breakdown and defects of theplasmalemma in diseased human muscles. Acta Neuropathol. 33, 129-141.

Schwander, M., Leu, M., Stumm, M., Dorchies, O. M., Ruegg, U. T., Schittny,J. and Muller, U. (2003). Beta1 integrins regulate myoblast fusion andsarcomere assembly. Dev. Cell 4, 673-685.

Schwenk, F., Baron, U. and Rajewsky, K. (1995). A cre-transgenic mouse strainfor the ubiquitous deletion of loxP-flanked gene segments including deletion ingerm cells. Nucleic Acids Res. 23, 5080-5081.

Senetar, M. A. and McCann, R. O. (2005). Gene duplication and functionaldivergence during evolution of the cytoskeletal linker protein talin. Gene 362,141-152.

Senetar, M. A., Foster, S. J. and McCann, R. O. (2004). Intrasteric inhibitionmediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, andHip12 with actin. Biochemistry 43, 15418-15428.

Senetar, M. A., Moncman, C. L. and McCann, R. O. (2007). Talin2 is inducedduring striated muscle differentiation and is targeted to stable adhesioncomplexes in mature muscle. Cell Motil. Cytoskeleton 64, 157-173.

Settles, D. L., Cihak, R. A. and Erickson, H. P. (1996). Tenascin-C expression indystrophin-related muscular dystrophy. Muscle Nerve 19, 147-154.

Sonnemann, K. J., Fitzsimons, D. P., Patel, J. R., Liu, Y., Schneider, M. F.,Moss, R. L. and Ervasti, J. M. (2006). Cytoplasmic gamma-actin is not requiredfor skeletal muscle development but its absence leads to a progressivemyopathy. Dev. Cell 11, 387-397.

Stedman, H. H., Sweeney, H. L., Shrager, J. B., Maguire, H. C., Panettieri, R.A., Petrof, B., Narusawa, M., Leferovich, J. M., Sladky, J. T. and Kelly, A.M. (1991). The mdx mouse diaphragm reproduces the degenerative changes ofDuchenne muscular dystrophy. Nature 352, 536-539.

Straub, V., Rafael, J. A., Chamberlain, J. S. and Campbell, K. P. (1997). Animalmodels for muscular dystrophy show different patterns of sarcolemmaldisruption. J. Cell Biol. 139, 375-385.

Svensson, L., Howarth, K., McDowall, A., Patzak, I., Evans, R., Ussar, S.,Moser, M., Metin, A., Fried, M., Tomlinson, I. et al. (2009). Leukocyteadhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrinactivation. Nat. Med. 15, 306-312.

Tadokoro, S., Shattil, S. J., Eto, K., Tai, V., Liddington, R. C., de Pereda, J. M.,Ginsberg, M. H. and Calderwood, D. A. (2003). Talin binding to integrin betatails: a final common step in integrin activation. Science 302, 103-106.

Taverna, D., Disatnik, M. H., Rayburn, H., Bronson, R. T., Yang, J., Rando, T.A. and Hynes, R. O. (1998). Dystrophic muscle in mice chimeric for expressionof alpha5 integrin. J. Cell Biol. 143, 849-859.

van der Flier, A., Gaspar, A. C., Thorsteinsdottir, S., Baudoin, C., Groeneveld,E., Mummery, C. L. and Sonnenberg, A. (1997). Spatial and temporalexpression of the beta1D integrin during mouse development. Dev. Dyn. 210,472-486.

Vandervoort, A. A. and McComas, A. J. (1983). A comparison of the contractileproperties of the human gastrocnemius and soleus muscles. Eur. J. Appl. Physiol.Occup. Physiol. 51, 435-440.

Wang, H. V., Chang, L. W., Brixius, K., Wickstrom, S. A., Montanez, E.,Thievessen, I., Schwander, M., Muller, U., Bloch, W., Mayer, U. et al.(2008). Integrin-linked kinase stabilizes myotendinous junctions and protectsmuscle from stress-induced damage. J. Cell Biol. 180, 1037-1049.

Weller, B., Karpati, G. and Carpenter, S. (1990). Dystrophin-deficient mdxmuscle fibers are preferentially vulnerable to necrosis induced by experimentallengthening contractions. J. Neurol. Sci. 100, 9-13.

Zhang, X., Jiang, G., Cai, Y., Monkley, S. J., Critchley, D. R. and Sheetz, M. P.(2008). Talin depletion reveals independence of initial cell spreading fromintegrin activation and traction. Nat. Cell Biol. 10, 1062-1068.


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