Developmental Biology 382 (2013) 160–171

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Developmental Biology

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The N-BAR domain protein, Bin3, regulates Rac1- andCdc42-dependent processes in myogenesis

Adriana Simionescu-Bankston a,b, Giovanna Leoni c, Yanru Wang d, Peter P. Phamb,Arivudainambi Ramalingam e, James B. DuHadaway f, Victor Faundez g, Asma Nusrat c,George C. Prendergast f, Grace K. Pavlath b,n

a Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, Atlanta, GA 30322, USAb Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USAc Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit,Emory University School of Medicine, Atlanta, GA 30322, USAd Department of Cell & Molecular Physiology, Loyola University Chicago, Maywood, IL 60153, USAe Hayward Genetics Center, Tulane University School of Medicine, New Orleans, LA 70112, USAf Lankenau Institute for Medical Research, Wynnewood, PA 19096, USAg Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA

a r t i c l e i n f o

Article history:Received 29 April 2013Received in revised form1 July 2013Accepted 5 July 2013Available online 16 July 2013

Keywords:BAR domainMyogenesisRac1Cdc42F-actinMuscle regeneration

06/$ - see front matter & 2013 Elsevier Inc. Ax.doi.org/10.1016/j.ydbio.2013.07.004

viations: BAR, Bin-Amphiphysin-Rvs; Bin, Bril area; DM, Differentiation media; eMyHC, EmFilamentous actin; GAP, GTPase-activating prhange factor; GM, Growth media; GRAF1, GTPhesion kinase-1; KO, Knockout; PAK, p21-activ, domain; RV, Retrovirus; WT, Wild-type.esponding author. Fax: +1 404 727 3590.ail address: gpavlat@emory.edu (G.K. Pavlath)

a b s t r a c t

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BARdomain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletalmuscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3),during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo.During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizeswith F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPasesinvolved in actin polymerization, which are known to be essential for myotube formation. Importantly, aBin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells.Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes inmyogenesis, and further implicate BAR domain proteins in muscle growth and repair.

& 2013 Elsevier Inc. All rights reserved.

Introduction

Skeletal muscle growth and repair occur by the process ofmyogenesis, in which myogenic progenitor cells differentiate,migrate and fuse with one another to form multinucleatedmyofibers (Abmayr and Pavlath, 2012). The plasma membranesof myogenic cells undergo dynamic changes to facilitate variousstages of myogenesis (Abramovici and Gee, 2007; Kim et al., 2008;Mukai et al., 2009; Stadler et al., 2010; Yoon et al., 2007), includinginward membrane invaginations occurring with endocytosis, andoutward membrane protrusions during cell migration (Suetsugu

ll rights reserved.

dging integrator; CSA, Cross-bryonic myosin heavy chain;otein; GEF, Guanine nucleo-ase regulator associated withated protein kinase; PBDp21-

.

et al., 2010). Coordinated changes in actin polymerization at theplasma membrane provide the force for these dynamic membranealterations. A key question is how changes in the plasma mem-brane and rearrangements of the actin cytoskeleton are regulatedand coordinated at different stages of myogenesis.

The Bin-Amphiphysin-Rvs (BAR) domain superfamily of pro-teins regulates both membrane and actin dynamics via BARdomains, crescent shaped dimers that bind to membranes andcan either sense or induce membrane curvature (Habermann,2004). The inward or outward direction of membrane bending isgenerally dependent on the particular class of BAR domain, such asclassical BAR, N-terminal amphipathic helix-BAR (N-BAR), Fes/CIP4 Homology-BAR/FCH-BAR (F-BAR), BAR-Pleckstrin Homology(BAR-PH), PhoX-BAR (PX-BAR) or Inverse-BAR/IMD-BAR/IRSp53-MIM Homology domain (I-BAR) (Frost et al., 2009; Habermann,2004; Quinones and Oro, 2010; Suetsugu, 2010; Suetsuguet al., 2010). Many BAR domain proteins also regulate Rho GTPasesand/or other actin regulatory proteins (de Kreuk and Hordijk,2012), and therefore may link membrane dynamics to rearrange-ments of the actin cytoskeleton. Based on these functions, BAR

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domain proteins would be predicted to be key regulators ofmyogenesis; however, these proteins have been little studied inskeletal muscle.

The best characterized BAR domain protein in mammalianskeletal muscle is GTPase Regulator Associated with Focal Adhe-sion Kinase-1 (GRAF1), a BAR-PH domain protein containing a RhoGTPase-activating protein (RhoGAP) domain and an SH3 domain(Doherty et al., 2011b; Hildebrand et al., 1996). GRAF1 regulatesdifferentiation and fusion in the mouse muscle cell line C2C12, andis critical for muscle development in Xenopus laevis embryos(Doherty et al., 2011b). During myogenesis, GRAF1 is localized tothe tips of elongating myoblasts, where it is proposed to locallylimit the polymerization of filamentous actin (F-actin) (Dohertyet al., 2011b). Additional functions ascribed to GRAF1 include cellspreading and migration in HeLa cells (Doherty et al., 2011b) andfluid-phase endocytosis in fibroblasts (Lundmark et al., 2008). Theonly other BAR domain protein studied in mammalian skeletalmuscle is Bridging integrator 1 (Bin1), an N-BAR domain proteinwith an SH3 domain, which regulates differentiation and fusion inC2C12 cells (Wechsler-Reya et al., 1998) and in primary myoblastsin vitro (Fernando et al., 2009), and also facilitates sarcomereorganization in muscles of mice in vivo (Fernando et al., 2009).These studies highlight the importance of BAR domain proteins inmuscle differentiation and fusion, but raise questions about theinterplay between BAR domain proteins of various classes inregulating myogenesis.

We studied the role of Bridging integrator 3 (Bin3), a ubiquitouslyexpressed (Prendergast et al., 2009) and evolutionarily conserved(Ren et al., 2006) N-BAR domain protein in skeletal muscle. Incontrast to the previously studied BAR domain proteins in myogen-esis, Bin3 contains only the N-BAR domain (Ren et al., 2006). Both thebudding and fission yeast orthologs of Bin3, Rvs161p and Hob3p,respectively, have critical roles in F-actin localization in yeast (Renet al., 2006). The ability of Hob3p to modulate actin dynamics hasbeen proposed to result from its interaction with the Rho GTPaseCdc42 (Coll et al., 2007; Routhier et al., 2001). Interestingly, Rvs161palso regulates endocytosis and cell-cell fusion (Ren et al., 2006), twocellular processes intimately associated with myotube formation(Abmayr and Pavlath, 2012; Doherty et al., 2008; Posey et al.,2011). Loss of Bin3 in mice leads to juvenile cataracts with a neartotal loss of F-actin in lens fiber cells (Ramalingam et al., 2008).However, the role of Bin3 in regulating endocytosis, cell–cell fusionand actin dynamics during myogenesis is unknown.

Using Bin3 null mice, we show Bin3 is required for properformation of multinucleated muscles both in vivo and in vitro.Defects in lamellipodia formation and cell migration were noted inthe absence of Bin3 in differentiated muscle cells prior to myotubeformation. Importantly, the levels of active Rac1 and Cdc42 weregreatly decreased in the absence of Bin3. As Rac1 and Cc42 areimportant for actin dynamics in muscle cells in vitro (Vasyutinaet al., 2009), and are essential for muscle cell fusion both in vitroand in vivo (Vasyutina et al., 2009), these studies identify a majorrole for a Bin3-dependent signaling pathway in regulating Rac1-and Cdc42- dependent processes during myotube formation.

Results

Muscle regeneration defects occur in Bin3 KO mice

We observed that the steady-state levels of Bin3 were transi-ently increased at early stages of muscle regeneration whenmyogenic progenitor cells are differentiating, migrating and fusingto form small myofibers (Fig. 1A). These results suggested apotential role for Bin3 in regulating muscle regeneration. Todetermine the functional role of Bin3 during muscle regeneration,

the growth of regenerating myofibers in tibialis anterior musclesof wild-type (WT) and Bin3 null (KO) mice was analyzed at varioustimepoints after injury (Fig. 1B). No difference in myofiber cross-sectional area (CSA) was observed between WT and Bin3 KOmuscles prior to injury (Fig. 1C). In contrast, myofiber CSA wastransiently decreased by 28% in Bin3 KO muscles at 10 days postinjury (Fig. 1D), indicating a delay in regeneration in the absenceof Bin3.

Further analyses of regenerating muscles revealed a patternsuggestive of myofiber branching, an abnormal regenerative out-come associated with severe injury and muscular dystrophy(Pavlath, 2010). In branched myofibers, the plasma membrane ofthe parent myofiber is contiguous with several smaller myofibers(Pavlath, 2010). To analyze the function of Bin3 in regulatingmyofiber branching during severe injury, individual myofiberswere isolated from the gastrocnemius muscles of WT and Bin3KO mice 21 days following the second of two injuries. Whilemyofiber branching was increased in both WT and Bin3 KOmuscles after injury, Bin3 KO muscles exhibited an 18% increasein the percentage of branched myofibers (Fig. 2A). However, thepercentage of regenerated myofibers, which could affect theoverall percentage of branched myofibers, did not differ betweenWT and Bin3 KO muscles (Fig. 2B). To gain a deeper understandingof the myofiber branching observed, we examined both thenumber and type of branches in WT and Bin3 KO muscles. Wefound that Bin3 KO muscles exhibited a 27% increase in thepercentage of myofibers with two or more branches after injury(Fig. 2C). Furthermore, we noted three distinct patterns ofbranched myofibers: bifurcated, split and process (Fig. S1); how-ever, the percentage of regenerated myofibers with these branch-ing patterns did not differ between WT and Bin3 KO muscles(Fig. 2D). In contrast, we observed a 2.7 fold increase in thepercentage of regenerated Bin3 KO myofibers exhibiting a mix ofthese different patterns (Fig. 2D and E), suggesting more complexmyofiber branching. Together, these data highlight a function forBin3 in muscle growth and myofiber branching during muscleregeneration.

Bin3 is necessary for myotube formation in vitro

The regeneration defects observed in Bin3 KO muscles in vivocould result from impairments in multiple cell types contributingto the repair process. To distinguish between cell-autonomousand non-autonomous effects of Bin3 on myogenesis, satellite cellswere isolated from hindlimb muscles of WT and Bin3 KO miceand analyzed in vitro in the absence of other cell types. Duringin vitro myogenesis, satellite cell-derived myoblasts differentiateinto myocytes, which then migrate and fuse to one another toform nascent myotubes, small myotubes with few nuclei; subse-quently, more myocytes fuse in with nascent myotubes, givingrise to mature myotubes, large myotubes containing many nuclei(Abmayr and Pavlath, 2012). Immunoblotting analyses revealedthat muscle cells expressed Bin3 at all stages of differentiation(Fig. 3A). Similarly, by immunostaining, Bin3 was present in allmuscle cells in the culture (Fig. 3B). To test whether Bin3regulates myotube formation, WT and Bin3 KO muscle cells weredifferentiated into nascent myotubes for 24 h, or into maturemyotubes for 40 h, and immunostained for embryonic myosinheavy chain (eMyHC), a marker of differentiation. eMyHC stain-ing revealed that Bin3 KO myotubes were smaller at both stages(Fig. 3C). Quantitative analyses subsequently showed that Bin3KO myotubes exhibited a 33% decrease in the fusion index at 24 h(Fig. 3D), and contained 20% fewer nuclei at both 24 and 40 h(Fig. 3E). In addition, Bin3 KO myotubes were 12% thinner at both24 and 40 h (Fig. 3F) and 19% shorter at 24 h (Fig. 3G). However,the number of nuclei analyzed per field (Fig. 3H), which could

Fig. 1. Bin3 is required for muscle regeneration. (A) Bin3 protein levels were transiently increased in gastrocnemius muscles of WT mice at 2 and 4 days post injury.Specificity of the Bin3 antibody was demonstrated by lack of antibody reaction in Bin3 KO muscles. Hsp90 was used as a loading control. (B) Hematoxylin and eosin stainedsections are shown from tibialis anterior (TA) muscles of WT and Bin3 KO mice at 0, 5, 10 and 21 days after injury. Bar, 50 μm. (C) No difference in the cross-sectional area(CSA) of uninjured TA myofibers was observed between genotypes. (D) The CSA of regenerated TA myofibers was transiently decreased in Bin3 KO mice at 10 days post injury(*Po0.05). Data are mean7s.e.m., n¼4–7 mice for each genotype per timepoint.

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affect the extent of myotube formation, did not differ betweenWT and Bin3 KO cultures. Moreover, the steady-state levels ofmyogenin (Fig. S2A and B) and eMyHC (Fig. S2C and D), early andlate markers of differentiation, respectively, did not differbetween WT and Bin3 KO muscle cells. Overall, these dataindicate that Bin3-dependent processes within muscle cells arenecessary for proper myotube formation.

Functional redundancy by other N-BAR domain proteinsduring myogenesis may exist and could diminish the effects ofBin3 loss on myotube formation. Therefore, we examined mRNAlevels of Bin3 and the most closely related N-BAR domainproteins of the Amphiphysin/Bin family (Table S1) by real-timePCR. Besides Bin3, only Bin1 was expressed in primary musclecells (Table S2). However, since loss of Bin1 results in differ-entiation defects early in myogenesis (Wechsler-Reya et al.,1998), we could not test whether Bin1 can partially compensatefor Bin3 at the later stages of myogenesis analyzed in ourstudies.

We also assessed whether retroviral-mediated overexpressionof recombinant HA-Bin3 in WT cells (Fig. S3A) was sufficient toenhance myotube size. By eMyHC staining, myotubes did notappear larger at either 24 or 40 h of differentiation (Fig. S3B).Subsequent quantification revealed slight but statistically insignif-icant increases in the fusion index (Fig. S3C) and the number ofnuclei per myotube (Fig. S3D), likely due to a small increase in thenumber of nuclei per field (Fig. S3F) following Bin3 overexpression.The differentiation index was not altered following Bin3 over-expression (Fig. S3E). The inability of Bin3 overexpression to

enhance myotube formation is likely due to downstreammediatorsof Bin3 action being rate-limiting.

Endocytosis defects are not observed in Bin3 KO muscle cells

One of the yeast orthologs of Bin3 regulates endocytosis (Renet al., 2006), a process likely to be important for myogenesis. Indeed,molecules regulating endocytosis have recently been implicated inmyotube formation (Doherty et al., 2008; Leikina et al., 2013; Poseyet al., 2011). Thus, we hypothesized that an endocytic defect couldcontribute to impaired myotube formation in the absence of Bin3.Since many BAR domain proteins regulate receptor-mediated endo-cytosis, the most common and well-studied pathway utilized forinternalization (Qualmann et al., 2011), we tested whether Bin3regulates this process in muscle cells using fluorescently labeledtransferrin. We labeled WT and Bin3 KO myocytes (18 h in DM) withAlexa-594 conjugated transferrin to allow (37 1C) or prevent (4 1C)transferrin internalization, in the presence or absence of an acid washto remove the cell surface transferrin and permit analysis of only theinternalized fraction (Fig. S4A). No difference in the internalizedtransferrin fractionwas observed betweenWTand Bin3 KOmyocytes(Fig. S4B). To ensure that we could detect a difference in transferrininternalization, WT myocytes were treated with Dynasore, aninhibitor of dynamin-dependent endocytosis (Macia et al., 2006),prior to performing the internalization assay. Dynasore treatmentcaused a reduction in transferrin internalization in WT myocytes at37 1C (Fig. S4C and D). Together, these data suggest Bin3 is notnecessary for receptor-mediated endocytosis in myocytes.

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Fig. 2. Bin3 regulates myofiber branching. (A) Bin3 KO muscles contained a greater percentage of branched myofibers after injury (I) than WT muscles. Minimal branchingwas observed in uninjured muscles (U) regardless of genotype (*Po0.05). (B) No difference in the percentage of regenerated myofibers was observed between genotypes.(C) Bin3 KO muscles exhibited a greater percentage of myofibers with 2 or more branches after injury (*Po0.05). (D) Bin3 KO muscles contained a greater percentage ofregenerated myofibers with a mix of branching patterns (*Po0.05). Bifurc¼Bifurcated; Proc¼Process. (E) Myofibers after injury visualized with phase contrast microscopyand DAPI are shown. Myofiber with a mix of branching patterns (arrowheads) is shown in comparison to an unbranched myofiber. Bar, 150 μm. Data are mean7s.e.m., n¼4mice for each genotype.

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Bin3 plays a role in myocyte migration

Cell migration is another process critical for myotube forma-tion (Bae et al., 2008; Bondesen et al., 2007; Jansen and Pavlath,2006; Mylona et al., 2006; O′Connor et al., 2007). BAR domainproteins of different classes, including the N-BAR domain

protein Bridging integrator 2 (Bin2) (Sánchez-Barrena et al.,2012), have been implicated in regulating cell migration (deKreuk et al., 2011; Doherty et al., 2011a; Guerrier et al., 2009;Pichot et al., 2010; Quinones et al., 2010; Tsuboi et al., 2009). Asmigration of both myoblasts and myocytes is important formyotube formation, we analyzed the role of Bin3 in regulating the

Fig. 3. Myotube formation in vitro is altered in the absence of Bin3. (A) Bin3 protein was expressed in WT muscle cells throughout differentiation. Specificity of the Bin3antibody was demonstrated by lack of antibody reaction in Bin3 KO muscle cells. Hsp90 was used as a loading control. (B) Immunostaining of differentiated WT muscle cellswith an anti-Bin3 antibody revealed Bin3 was expressed in all muscle cells in the culture. Nuclei were counterstained with DAPI. Bar, 150 μm. (C) WT and Bin3 KO musclecells immunostained for eMyHC are shown at 24 and 40 h in DM. Bar, 150 μm. (D) Fusion index (*Po0.001), (E) myonuclear number (*Po0.001), (F) myotube diameter(*Po0.01) and (G) myotube length (*Po0.05) were decreased in Bin3 KO muscle cells . No differences were noted in the number of nuclei per field (H) between WT and Bin3KO muscle cells. Data are mean7s.e.m., n¼4 independent isolates for each genotype.

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migration of both cell types using time-lapse microscopy. The migra-tory cell paths of WT and Bin3 KO myoblasts were only slightlydifferent (Fig. S5A), and their average velocity did not differ signifi-cantly (Fig. S5B and C). In contrast, Bin3 KOmyocytes migrated shorterdistances than WT cells (Fig. 4A), and their average velocity was

decreased by 30% (Fig. 4B and C). Retroviral-mediated expression ofrecombinant HA-Bin3 in Bin3 KO cells (Fig. 4D) rescued their migra-tion defect and restored the average velocity to WT levels (Fig. 4E).These results show Bin3 is required specifically for migration ofmyocytes during myogenesis.

Fig. 4. Bin3 plays a role in myocyte migration. (A) Time-lapse microscopy revealed that Bin3 KO myocytes migrated shorter distances than WT myocytes. The migratorypaths of 30 individual cells per genotype are shown. (B) Histogram illustrating the absence of a large population of rapidly moving cells in Bin3 KO myocytes. (C) Cell velocitywas decreased in Bin3 KO myocytes (*Po0.05). (D) Immunoblot demonstrating expression of recombinant HA-Bin3 (Bin3 RV) in Bin3 KO myocytes after retroviral infection.Ctrl RV¼empty vector. Tubulin was used as a loading control. (E) Retroviral-mediated expression of HA-Bin3 (Bin3 RV) in Bin3 KO myocytes resulted in increased cell velocitycompared to empty vector control (Ctrl RV) (*Po0.05). In panels B, C and E, 60 individual cells were analyzed per genotype with n¼3 independent isolates for each genotype.Data in panels C and E are mean7s.e.m.

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Bin3 is involved in lamellipodia formation in myocytes

Actin polymerization is harnessed for cell motility anddrives the forward extension of lamellipodia, broad actin-based protrusions associated with motility (Le Clainche andCarlier, 2008; Ridley, 2011; Small et al., 2002). Interestingly,the two yeast orthologs of Bin3 both regulate F-actin localiza-tion (Ren et al., 2006), and Bin3 KO mice exhibit loss of F-actinin lens fiber cells (Ramalingam et al., 2008). Therefore, wereasoned that Bin3 may also be important for actin-dependentprocesses in muscle cells. We visualized F-actin in WT and Bin3KO myocytes at 18 and 24 h of differentiation using FITC-

phalloidin. We observed fewer Bin3 KO myocytes with lamel-lipodia at these timepoints (arrowheads and insets, Fig. 5A).Following quantification, depending on the timepoint, 33–57%fewer Bin3 KO myocytes exhibited lamellipodia (Fig. 5B). Sub-sequently, we found that Bin3 and F-actin colocalized inlamellipodia of myocytes (arrowheads, Fig. 6A). Due to lowlevels of endogenous Bin3 in muscle cells, we retrovirallyexpressed recombinant HA-Bin3 and performed HA immunos-taining to better examine the localization of Bin3. HA-Bin3 alsocolocalized with F-actin in lamellipodia of myocytes (arrow-heads, Fig. 6B). Together, these data demonstrate that Bin3regulates lamellipodia formation in myocytes.

Fig. 5. Bin3 is involved in lamellipodia formation in myocytes. (A) Lamellipodia were visualized by examining F-actin localization (FITC-phalloidin) in WT and Bin3 KOmyocytes at 18 and 24 h in DM (arrowheads). Nuclei were counterstained with DAPI. Insets indicate lamellipodia at higher magnification (red box). Bar, 50 μm. (B) Bin3 KOmyocytes exhibited a lower percentage of cells with lamellipodia (*Po0.01) in DM. Data are mean7s.e.m., n¼3 independent isolates for each genotype.

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Decreased levels of active Rac1 and Cdc42 in Bin3 KO myocytes

Rho GTPases play critical roles in regulating actin dynamicsduring cell migration (Ridley, 2011). In particular, Rac1 and Cdc42are associated with actin regulation in lamellipodia (Ridley, 2011).Based on the function of Bin3 in regulating lamellipodia formationin myocytes, we hypothesized that Bin3 may regulate the activityof Rac1 and Cdc42. Pull-down of active Rac1 and Cdc42 usingbeads coated with the p21-binding domain (PBD) of p21-activatedprotein kinase 1 (PAK1), termed PAK1-PBD, followed by immuno-blotting, showed a major decrease in the active levels of these RhoGTPases (Fig. 7A) in Bin3 KO myocytes. Quantification of immuno-blots revealed decreases of approximately 70% in the active levelsof both Rho GTPases (Fig. 7B) in Bin3 KO myocytes. Retroviral-mediated expression of recombinant HA-Bin3 in Bin3 KO cells(Fig. 7C) led to a 2.4 fold increase in the levels of active Rac1 and a3.3 fold increase in the levels of active Cdc42 (Fig. 7D). In addition,HA-Bin3 was detected in a complex with active Rac1 and Cdc42(Fig. 7C) in Bin3 KO myocytes. These data indicate that a Bin3-dependent pathway is a key regulator of Rac1 and Cdc42 activityin myocytes.

Discussion

Myofiber formation during myogenesis is key to muscle regen-eration. Our data provide insights into the mechanisms by whichdynamic rearrangements of the actin cytoskeleton are regulated

during myogenesis. We show the N-BAR domain protein, Bin3, isimportant for myogenesis both in vitro and in vivo.

Muscle regeneration is a complex process requiring interplaybetween myogenic and non-myogenic cells (Chazaud et al., 2003;Saclier et al., 2013; Sonnet et al., 2006). Absence of Bin3, aubiquitously expressed protein (Prendergast et al., 2009), resultedin a transient delay in the growth of regenerating myofibers afterinjury. Muscle-intrinsic functions of Bin3 likely contributed in partto the growth phenotype, as myocyte migration and myotubeformation in vitro were impaired in the absence of Bin3. Poten-tially Bin3 function may also be required in non-myogenic cellsduring regeneration, and the absence of Bin3 in these cells mayhave further contributed to the observed growth phenotype. Thetransient delay in myofiber growth during regeneration may bedue to compensation by other molecules that regulate this process.Functional compensation may also explain in part the fact thatmyofiber size did not differ between WT and Bin3 KO muscles inthe absence of injury.

In the absence of Bin3 we also observed abnormal branchedmyofibers. Although myofiber branching has been studied formany years, Bin3 is the only molecule found to regulate thisprocess besides the G protein coupled receptor, mouse olfactoryreceptor 23 (MOR23) (Griffin et al., 2009). While the mechanismsunderlying myofiber branching during muscle regeneration areunknown, interestingly both MOR23 and Bin3 regulate myocytemigration and myotube formation in vitro (Griffin et al., 2009),suggesting aberrations in these processes may contribute to theformation of branched myofibers.

Fig. 6. Bin3 and F-actin colocalize in lamellipodia of myocytes. (A) Bin3 and F-actin colocalized in lamellipodia of WT myocytes at 18 h in DM (arrowheads). Bar, 50 μm.(B) Retrovirally-expressed recombinant HA-Bin3 also colocalized with F-actin in lamellipodia of myocytes (arrowheads, bottom row). The specificity of the HA antibody wasdemonstrated by lack of antibody reaction in cells with the empty vector control (top row). Bar, 50 μm. Nuclei were counterstained with DAPI.

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Since muscle cell migration plays a crucial role in myotubeformation (Bae et al., 2008; Bondesen et al., 2007; Jansen andPavlath, 2006; Mylona et al., 2006; O′Connor et al., 2007), weinitially investigated the role of Bin3 in migration. We found Bin3was required only for myocyte migration, but not for myoblastmigration. Bin3 likely interacts with different downstream mole-cules in these two cell types, leading to the specificity in regulatingstage-specific cellular migration. Bin3 is the first example of acytoplasmic protein which controls muscle cell migration, as todate only secreted molecules and transmembrane proteins havebeen shown to regulate this process (Simionescu and Pavlath,2011). Interestingly, Bin2, an N-BAR domain protein with aC-terminal tail containing acidic and serine/proline-rich segments(Ge and Prendergast, 2000; Sánchez-Barrena et al., 2012), hasrecently been implicated in monocyte migration (Sánchez-Barrenaet al., 2012). Together, these data associate N-BAR domain proteinswith cell migration, a process requiring the formation of outwardmembrane protrusions, which N-BAR domains are not classicallylinked with (Suetsugu, 2010).

Dynamic changes in the actin cytoskeleton are critical for cellsto extend protrusions to sense their environment and subsequently

migrate towards a target. Migrating cells extend filopodia, thinexploratory extensions from the plasma membrane containingparallel bundles of actin filaments (Ridley, 2011) or lamellipodia,broad protrusions containing branched actin filaments (Le Claincheand Carlier, 2008; Ridley, 2011; Small et al., 2002). Many BARdomain proteins positively regulate actin polymerization and theformation of outward membrane protrusions (de Kreuk andHordijk, 2012). Some BAR domain proteins are enriched, and cancolocalize with F-actin, in these protrusions (de Kreuk and Hordijk,2012). Indeed, the N-BAR domain protein Bin3 colocalized withF-actin in lamellipodia, and was important for lamellipodia forma-tion in myocytes. In contrast, the BAR-PH domain protein GRAF1localizes to sites devoid of F-actin in muscle cells (Doherty et al.,2011b). These data suggest that BAR domain proteins of differentclasses may have complementary roles in regulating actindynamics in myogenesis.

Myotube formation requires Rac1- and Cdc42-dependent actinpolymerization (Vasyutina et al., 2009), but the upstream signalscontrolling the activity of these Rho GTPases in differentiatedmuscle cells are unknown. The levels of active Rac1 and Cdc42were greatly decreased in myocytes in the absence of Bin3,

Fig. 7. Decreased levels of active Rac1 and Cdc42 in Bin3 KO myocytes. (A) Active Rac1 and Cdc42 were pulled down fromWT and Bin3 KO myocytes using beads coated withthe p21-binding domain of PAK1 (PAK1-PBD). Decreased levels of active Rac1 and Cdc42 were detected in Bin3 KO myocytes. (B) The active/total ratios for Rac1 and Cdc42were decreased in Bin3 KO myocytes (*Po0.05). (C) Active Rac1 and Cdc42 were pulled down from Bin3 KO myocytes retrovirally-expressing recombinant HA-Bin3 (Bin3 RV)or empty vector (Ctrl RV) using PAK1-PBD beads. Increased amounts of active Rac1 and Cdc42 were observed in Bin3 KO myocytes following Bin3 overexpression. Moreover,HA-Bin3 was detected in a complex with active Rac1 and Cdc42. (D) The active/total ratios for Rac1 and Cdc42 were increased following Bin3 overexpression. HAimmunostaining showed an average of 89% HA+ cells. Data in panel B are mean7s.e.m., n¼3 independent isolates for each genotype. Data in panel D are mean7s.e.m., n¼2independent isolates for each condition.

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suggesting Bin3 is a major positive regulator of Rac1 and Cdc42 inmuscle cells. The fusion defect observed in Bin3 KO myotubesin vitro was less severe than that seen in Rac1 or Cdc42 KO cells(Vasyutina et al., 2009), possibly due to the residual low levels ofthese GTPases in Bin3 KO myocytes. In contrast, the BAR-PHdomain protein, GRAF1, is a negative regulator of RhoA activityin C2C12 muscle cells but does not affect Cdc42 activity in L6myoblasts (Doherty et al., 2011b), suggesting that BAR domainproteins of different classes show specificity in controlling GTPaseactivity during myogenesis. As we did not observe a visibledifference in actin stress fibers between WT and Bin3 KO myo-cytes, we suggest that levels of active RhoA, a major regulator ofstress fiber formation (Pellegrin and Mellor, 2007), are unlikely tobe affected in Bin3 KO myocytes.

We detected recombinant Bin3 in a complex with active Rac1or Cdc42 in myocytes. How could Bin3, a protein with only anN-BAR domain, regulate the activity of Rac1 and Cdc42 in musclecells? Some BAR domain proteins modulate the activity of RhoGTPases via a RhoGAP or Rho Guanine nucleotide exchange factor(RhoGEF) domain (de Kreuk and Hordijk, 2012). In contrast, BARdomain proteins that lack a RhoGAP/GEF domain regulate RhoGTPases by recruiting other proteins with RhoGAP/GEF activity. Forexample, one of the Bin3 yeast orthologs, Hob3p, facilitates theinteraction between Gef-1, a Cdc42GEF, and Cdc42 (Coll et al.,2007). Bin3 may similarly interact with an unknown RhoGAP/GEFprotein to modulate the activity of Rac1 and Cdc42 in muscle cells.Moreover, Bin3 could heterodimerize with another BAR domainprotein (Ren et al., 2006), which ultimately serves as the link tomodulating RhoGAP/GEF activity. Additional studies are necessaryto define Bin3 interacting partners to better understand themechanisms by which Bin3 may regulate Rac1 and Cdc42 activity

in myocytes. Understanding these mechanisms may help explainthe myofiber branching phenotype observed in the absence ofBin3 in vivo, and whether Rac1 and Cdc42 are also implicated inthis process. In addition, Bin3 may also regulate myogenesisthrough Rho GTPase-independent mechanisms.

The study of BAR domain proteins is an emerging area inskeletal muscle. Our studies extend previous work in this field(Doherty et al., 2011b; Wechsler-Reya et al., 1998) and suggest thatmultiple classes of BAR domain proteins will prove critical forregulating important cellular processes during myogenesis. Under-standing the role of BAR domain proteins in muscle growth andrepair will likely impact treatments for muscle diseases in whichthese processes are impaired.

Materials and methods

Animals

Wild-type (WT) and Bin3 null (KO) mice were maintained on amixed C57BL/6J-129/SvJ genetic background (Ramalingam et al.,2008). Mice were age- and sex- matched in experiments and usedbetween 4 and 23 weeks of age for in vitro experiments andbetween 10 and 34 weeks of age for in vivo experiments inaccordance with the IACUC guidelines of Emory University.

Muscle injuries

For analyses of myofiber cross-sectional area, muscle injurywas induced by a single injection of 50 μL of 1.2% BaCl2 in PBSinto the tibialis anterior muscles of mice as described previously

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(O′Connor et al., 2007). Muscles were collected at various time-points after injury and histological sections were prepared andimaged as described previously (Jansen and Pavlath, 2006). Myo-fiber cross-sectional area was quantified using ImageJ. A total of480–1200 myofibers from 4 to 7 mice were analyzed per genotypefor each timepoint.

For analyses of myofiber branching, severe muscle injury wasinduced by two consecutive injections of 50 μL of 1.2% BaCl2 in PBStwo days apart into the gastrocnemius muscles of mice. Muscleswere collected 21 days after the second injury. Subsequently,muscles were enzymatically digested for 1 h 20 min with gentleagitation and single myofibers were isolated as described pre-viously (Kafadar et al., 2009), fixed with 3.7% formaldehyde in PBSand stained with 4′,6-diamidino-2-phenylindole (DAPI) to identifynuclei. Regenerated myofibers, identified by the presence ofcentral nuclei, were analyzed for the number and type of branches.A total of 300–450 myofibers from 4 mice were analyzed pergenotype.

Differentiation and fusion assays

Primary myoblasts were isolated from the hindlimb muscles ofWT and Bin3 KO mice as previously described with the exceptionof a Percoll gradient (Bondesen et al., 2004). Cells were cultured ingrowth media (GM; Ham′s F10, 20% fetal bovine serum (FBS),100 U/mL penicillin G, 100 μg/mL streptomycin, 5 ng/mL FGF) oncollagen-coated plates. Cultures were 495% myogenic as definedby MyoD immunostaining.

To induce differentiation, primary mouse myoblasts wereplated on entactin-collagen IV-laminin (ECL; Millipore) in GMand after 1 h switched to differentiation media (DM; DMEM,100 U/mL penicillin G, 100 μg/mL streptomycin, 1% Insulin-Transferrin-Selenium-A supplement (ITS; Gibco) for 24 or 40/48 h. Cells were then fixed with 3.7% formaldehyde and immu-nostained with an antibody (F1.652; Developmental StudiesHybridoma Bank) against embryonic myosin heavy chain asdescribed previously (Horsley et al., 2001).

The differentiation index was determined by dividing the totalnumber of nuclei in eMyHC+ cells by the total number of nucleicounted (Jansen and Pavlath, 2006). The fusion index was deter-mined by dividing the total number of nuclei in myotubes by thetotal number of nuclei counted (Jansen and Pavlath, 2006). Theaverage myonuclear number was determined by dividing the totalnumber of nuclei in myotubes (≥2 nuclei) by the total number ofmyotubes counted (Jansen and Pavlath, 2006). A total of 2500–5400 nuclei were analyzed per genotype or condition for eachtimepoint. To quantify myotube diameter using ImageJ, a range of3–30 measurements were taken perpendicular to the axis of themyotube at equal distances along the axis of the myotube,depending on the length of the myotube. To quantify myotubelength using ImageJ, a line was drawn along the entire myotubeand only myotubes visible in their entirety were analyzed. Three tofour independent isolates were analyzed per genotype orcondition.

Endocytosis assays

Myoblasts were differentiated into myocytes for 18 h in DMand switched to serum-free media lacking transferrin (DMEM,10 mM Hepes pH 7.4, 0.2% BSA) at 37 1C for 3 h prior to the assay.Cells were cooled to 4 1C to inhibit endocytic internalization,followed by incubation with Alexa-594 conjugated transferrin(Invitrogen) in the presence or absence of Dynasore (Abcam), toallow both transferrin and Dynasore to equilibrate at the cellsurface. Subsequently, in the continuous presence or absence ofDynasore, cells were either incubated at 37 1C for 35 min to allow

transferrin internalization, or kept at 4 1C to prevent it. All cellswere then returned to 4 1C in the presence or absence of an acidicbuffer (0.5 M NaCl, 15 mM MES (2-(N-Morpholino) ethanesulfonicacid hydrate (Sigma-Aldrich) pH 4.5 in PBS), to remove cell surfacetransferrin. Finally, while still at 4 1C, all cells were fixed with 4%paraformaldehyde, and stained with DAPI. Intensities were mea-sured with MetaMorph software version 6.1 (Molecular Devices,Sunnyvale, CA) as integrated pixel intensity, and the ratio (inter-nalized/total) was plotted for some experiments. A total of 20–90cells were analyzed per genotype or condition. One to threeindependent isolates were analyzed per genotype or condition.

Cell migration assays

Cell migration experiments were performed as previouslydescribed (Jansen and Pavlath, 2006). Myoblasts or myocytes(18 h in DM) were visualized using an Axiovert 200M microscopewith a 0.3 NA 10X Plan-Neofluar objective (Carl Zeiss MicroIma-ging, Inc.), and images were recorded using a camera (QImaging)and OpenLab 5.5.2 (Improvision) software every 5 min for 3 husing time-lapse microscopy. Using ImageJ, the migratory paths of60 individual cells were analyzed per genotype and mean cellvelocities were calculated. Three to four independent isolates wereanalyzed per genotype.

Phalloidin staining

Differentiated muscle cells at 18 h in DM were fixed with 3.7%formaldehyde and incubated in blocking buffer (0.1% Triton-X 100,1% BSA in PBS) for 20 min, followed by FITC-phalloidin (Enzo LifeSciences) in PBS with 1% BSA for 20 min. Nuclei were counter-stained with DAPI. The percentage of cells with lamellipodia wasquantified. A total of 130–160 cells were analyzed per genotype foreach timepoint. Three independent isolates were analyzed pergenotype.

Immunostaining

Differentiated muscle cells at 18 h or 24 h in DM were fixed with3.7% formaldehyde and incubated in blocking buffer (5% donkeyserum, 0.1% or 0.25% Triton X-100, 0.5% or 1% BSA in PBS), followedby an overnight incubation at 4 1C with Bin3 hybridoma 3A4(Ramalingam et al., 2008) or HA (Covance) primary antibodies.Primary antibodies were detected using biotin-conjugated donkey-anti mouse IgG (Jackson ImmunoResearch), HRP-conjugated strep-tavidin (PerkinElmer) and the tyramide signal amplification redreagent (Tyramide Signal Amplifcation (TSA) kit, Perkin Elmer).Nuclei were counterstained with DAPI.

Retroviral infection

Mouse Bin3 cDNA (Open Biosystems) with 3xHA tags at theC-terminus was cloned into the pBABE retroviral vector(Morgenstern and Land, 1990) together with an IRES-EGFP marker.A control vector with an IRES-EGFP marker was also generated. WTand Bin3 KO myoblasts were subjected to 2–4 rounds of retroviralinfection (Abbott et al., 1998) using either the HA-Bin3 vector or thecontrol vector. Infected cells were subsequently grown in GM underpuromycin selection at 0.75 μg/mL for a minimum of 48 h. Theinfection efficiency was 495% based on cell survival in the presenceof puromycin.

Immunoblotting and pull-down assays

Samples were lysed in RIPA-2 buffer (50 mM Tris–HCl, pH 8.0,150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) containing

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protease inhibitors (Mini Complete; Roche) and centrifuged at21,000� g for 15 min. The pellet was then discarded and thesupernatant was subjected to SDS-PAGE and immunoblotting. Anequal amount of protein from each sample was loaded onto 10% or12% SDS-polyacrylamide gels and transferred to either a 0.2 μm or a0.45 μm nitrocellulose membrane (Bio-Rad Laboratories). Afterblocking non-specific binding, the membranes were incubated withthe appropriate primary antibodies. Primary antibodies used were asfollows: Bin3 (3A4; Ramalingam et al., 2008), Cdc42 (Santa Cruz),EF1α (Millipore), eMyHC (F1.652; Developmental Studies HybridomaBank), HA (Covance), Hsp90 (Santa Cruz), Myogenin (F5D; Develop-mental Studies Hybridoma Bank), Rac1 (BD Biosciences) and Tubulin(Sigma-Aldrich), followed by incubation with the appropriate horse-radish peroxidase (HRP)-conjugated IgG (Jackson ImmunoResearch)secondary antibodies.

To perform Rac1 and Cdc42 activity assays, myocytes werelysed in supplied magnesium lysis buffer (MLB; Millipore) contain-ing protease/phosphatase inhibitors (Sigma), and centrifuged at14,000� g for 10 min. The pellet was then discarded and thesupernatant was incubated for 30 min with 10 mM EDTA. Thereaction was stopped by the addition of 60 mM MgCl2. An equalamount of protein from each sample was incubated with 20 μLPAK1-PBD beads (Millipore) for 1.5 h at 4 1C. The beads were thenwashed, spun quickly and the supernatants were subjected to SDS-PAGE and immunoblotting. The relative amounts of active Rac1and Cdc42 were determined by densitometric analysis. Two tothree independent isolates were analyzed per genotype orcondition.

Real-time PCR

Total RNA was isolated from WT myocytes using TRIzol reagent(Life Technologies) according to the manufacturer′s instructions,followed by treatment with DNaseI (Life Technologies). DNaseI-treated RNA (2 mg) was reverse transcribed using random primers(Invitrogen) and M-MLV reverse transcriptase (Invitrogen). mRNAlevels were determined by real-time PCR using the SYBR SelectMaster Mix (Invitrogen), the StepOnePlus Real-Time PCR System andStepOne Software version 2.2.2 (Applied Biosystems, Life Technolo-gies). All reactions were run in duplicate. Primers for mouse AMPH(PPM30542A), BIN1 (PPM25097A), BIN2 (PPM66366A) and BIN3(PPM26566A) were obtained from SABiosciences. Three independentisolates were analyzed. Mouse gastrocnemius muscle or brain wasused as a positive control.

Image acquisition

For the analysis of myofiber branching in vivo, the differentia-tion and fusion assays in vitro, as well as the Bin3 immunostainingin differentiated cells, images were obtained using an Axiovert200M microscope (Carl Zeiss MicroImaging) with a 0.3 NA 10XPlan-Neofluar objective (Carl Zeiss MicroImaging) and wererecorded using a camera (QImaging) and OpenLab 5.5.2 (Improvi-sion) software. For all other experiments, images were obtainedusing an Axioplan microscope (Carl Zeiss MicroImaging) witheither a 0.3 NA 10X Plan-Neofluar objective (Carl Zeiss MicroIma-ging) or with a 0.8 NA 25X Plan-Neofluar objective (Carl ZeissMicroImaging) and were recorded with a camera (Carl ZeissMicroImaging) and Scion Image 1.63 (Scion Corporation) software.All images were assembled using Adobe Photoshop CS5.1 forMacintosh (Adobe) and equally processed for size, color levels,brightness, and contrast.

Statistical analysis

Statistical analysis to determine significance between twogroups was performed using a Student′s t test. One-way or two-way ANOVA with Bonferroni′s posttest was used for comparisonsbetween multiple groups as appropriate. All statistical analyseswere performed using GraphPad Prism 5.0 for Macintosh (Graph-Pad Software). Differences were considered to be statisticallysignificant at Po0.05.

Acknowledgements

We thank the Emory University Custom Cloning Core Facilityfor generating the HA-Bin3 and control constructs. This work wassupported by grants from the National Institutes of Health(AR-04731408 to G.K.P.; DK59888 to A.N.; GM077569 and NS42599to V.F.) and from the Emory University Research Committee to V.F.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ydbio.2013.07.004.

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