The N-BAR Domain Protein, Bin3, Regulates Rac1- and Cdc42-Dependent Processes in Myogenesis

Abstract

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. 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 colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-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 in myogenesis, and further implicate BAR domain proteins in muscle growth and repair.

Introduction

Skeletal muscle growth and repair occur by the process of myogenesis, in which myogenic progenitor cells differentiate, migrate and fuse with one another to form multinucleated myofibers (Abmayr and Pavlath, 2012). The plasma membranes of myogenic cells undergo dynamic changes to facilitate various stages of myogenesis (Abramovici and Gee, 2007; Kim et al., 2008; Mukai et al., 2009; Stadler et al., 2010; Yoon et al., 2007), including inward membrane invaginations occurring with endocytosis, and outward membrane protrusions during cell migration (Suetsugu et al., 2010). Coordinated changes in actin polymerization at the plasma membrane provide the force for these dynamic membrane changes. A key question is how changes in the plasma membrane and rearrangements of the actin cytoskeleton are regulated and coordinated at different stages of myogenesis.

The Bin-Amphiphysin-Rvs (BAR) domain superfamily of proteins regulates both membrane and actin dynamics via BAR domains, crescent shaped dimers that bind to membranes and can either sense or induce membrane curvature (Habermann, 2004). The inward or outward direction of membrane bending is generally dependent on the particular class of BAR domain, such as classical 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; Suetsugu et al., 2010). Many BAR domain proteins also regulate Rho GTPases and/or other actin regulatory proteins (de Kreuk and Hordijk, 2012), and therefore may link membrane dynamics to rearrangements of the actin cytoskeleton. Based on these functions, BAR domain proteins would be predicted to be key regulators of myogenesis; however, these proteins have been little studied in skeletal muscle.

The best characterized BAR domain protein in mammalian skeletal muscle is GTPase Regulator Associated with Focal Adhesion Kinase-1 (GRAF1), a BAR-PH domain protein containing a Rho GTPase-activating protein (RhoGAP) domain and an SH3 domain (Doherty et al., 2011b; Hildebrand et al., 1996). GRAF1 regulates differentiation and fusion in the mouse muscle cell line C2C12, and is critical for muscle development in Xenopus laevis embryos (Doherty et al., 2011b). During myogenesis, GRAF1 is localized to the tips of elongating myoblasts, where it is proposed to locally limit the polymerization of filamentous actin (F-actin) (Doherty et al., 2011b). Additional functions ascribed to GRAF1 include cell spreading and migration in HeLa cells (Doherty et al., 2011b) and fluid-phase endocytosis in fibroblasts (Lundmark et al., 2008). The only other BAR domain protein studied in mammalian skeletal muscle is Bridging integrator 1 (Bin1), an N-BAR domain protein with an SH3 domain, which regulates differentiation and fusion in C2C12 cells (Wechsler-Reya et al., 1998) and in primary myoblasts in vitro (Fernando et al., 2009), and also facilitates sarcomere organization in muscles of mice in vivo (Fernando et al., 2009). These studies highlight the importance of BAR domain proteins in muscle differentiation and fusion, but raise questions about the interplay between BAR domain proteins of various classes in regulating myogenesis.

We studied the role of Bridging integrator 3 (Bin3), a ubiquitously expressed (Prendergast et al., 2009) and evolutionarily conserved (Ren et al., 2006) N-BAR domain protein in skeletal muscle. In contrast to the previously studied BAR domain proteins in myogenesis, Bin3 contains only the N-BAR domain (Ren et al., 2006). Both the budding and fission yeast orthologs of Bin3, Rvs161p and Hob3p, respectively, have critical roles in F-actin localization in yeast (Ren et al., 2006). The ability of Hob3p to modulate actin dynamics has been proposed to result from its interaction with the Rho GTPase Cdc42 (Coll et al., 2007; Routhier et al., 2001). Interestingly, Rvs161p also regulates endocytosis and cell-cell fusion (Ren et al., 2006), two cellular 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 near total loss of F-actin in lens fiber cells (Ramalingam et al., 2008). However, the role of Bin3 in regulating endocytosis, cell-cell fusion and actin dynamics during myogenesis is unknown.

Using Bin3 null mice, we show Bin3 is required for proper formation of multinucleated muscles both in vivo and in vitro. Defects in lamellipodia formation and cell migration were noted in the absence of Bin3 in differentiated muscle cells prior to myotube formation. Importantly, the levels of active Rac1 and Cdc42 were greatly decreased in the absence of Bin3. As Rac1 and Cc42 are important for actin dynamics in muscle cells in vitro (Vasyutina et al., 2009), and are essential for muscle cell fusion both in vitro and in vivo (Vasyutina et al., 2009), these studies identify a major role 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 transiently increased at early stages of muscle regeneration when myogenic progenitor cells are differentiating, migrating and fusing to form small myofibers (Fig. 1A). These results suggested a potential role for Bin3 in regulating muscle regeneration. To determine the functional role of Bin3 during muscle regeneration, the growth of regenerating myofibers in tibialis anterior muscles of wild-type (WT) and Bin3 null (KO) mice was analyzed at various timepoints after injury (Fig. 1B). No difference in myofiber cross-sectional area (CSA) was observed between WT and Bin3 KO muscles prior to injury (Fig. 1C). In contrast, myofiber CSA was transiently decreased by 28% in Bin3 KO muscles at 10 days post injury (Fig. 1D), indicating a delay in regeneration in the absence of Bin3.

Figure 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 stained sections 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 regenerating TA myofibers was transiently decreased in Bin3 KO mice at 10 days post injury (*P<0.05). Data are mean ± s.e.m., n=4-7 mice for each genotype per timepoint.

Further analyses of regenerating muscles revealed a pattern suggestive of myofiber branching, an abnormal regenerative outcome associated with severe injury and muscular dystrophy (Pavlath, 2010). In branched myofibers, the plasma membrane of the parent myofiber is contiguous with several smaller myofibers (Pavlath, 2010). To analyze the function of Bin3 in regulating myofiber branching during severe injury, individual myofibers were isolated from the gastrocnemius muscles of WT and Bin3 KO mice 21 days following the second of two injuries. While myofiber branching was increased in both WT and Bin3 KO muscles after injury, Bin3 KO muscles exhibited an 18% greater increase in the percentage of branched myofibers (Fig. 2A). However, the percentage of regenerated myofibers, which could affect the overall percentage of branched myofibers, did not differ between WT and Bin3 KO muscles (Fig. 2B). To gain a deeper understanding of the myofiber branching observed, we examined both the number and type of branches in WT and Bin3 KO muscles. We found that Bin3 KO muscles exhibited a 27% increase in the percentage of myofibers with two or more branches after injury (Fig. 2C). Furthermore, we noted three distinct patterns of branched myofibers: bifurcated, split and process (Fig. S1); however, the percentage of regenerating myofibers with these branching patterns did not differ between WT and Bin3 KO muscles (Fig. 2D). In contrast, we observed a 2.7 fold increase in the percentage of regenerating Bin3 KO myofibers exhibiting a mix of these different patterns (Fig. 2D,E), suggesting more complex myofiber branching. Together, these data highlight a function for Bin3 in muscle growth and myofiber branching during muscle regeneration.

Figure 2. Bin3 regulates myofiber branching.

(A) Bin3 KO muscles contained a greater percentage of branched myofibers after injury (I) than WT muscles. Minimal branching was observed in uninjured muscles (U) regardless of genotype (*P<0.05). (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 (*P<0.05). (D) Bin3 KO muscles contained a greater percentage of regenerated myofibers with a mix of branching patterns (*P<0.05). Bifurc = Bifurcated; Proc = Process. (E) Myofibers after injury visualized with phase contrast microscopy and DAPI are shown. Myofiber with a mix of branching patterns (arrowheads) is shown in comparison to an unbranched myofiber. Bar, 150 μm. Data are mean ± s.e.m., n=4 mice for each genotype.

Bin3 is necessary for myotube formation in vitro

The regeneration defects observed in Bin3 KO muscles in vivo could result from impairments in multiple cell types contributing to the repair process. To distinguish between cell-autonomous and non-autonomous effects of Bin3 on myogenesis, satellite cells were isolated from hindlimb muscles of WT and Bin3 KO mice and analyzed in vitro in the absence of other cell types. During in vitro myogenesis, satellite cell-derived myoblasts differentiate into myocytes, which then migrate and fuse to one another to form nascent myotubes, small myotubes with few nuclei; subsequently, more myocytes fuse in with nascent myotubes, giving rise to mature myotubes, large myotubes containing many nuclei (Abmayr and Pavlath, 2012). Immunoblotting analyses revealed that muscle cells expressed Bin3 at all stages of differentiation (Fig. 3A). Similarly, by immunostaining, Bin3 was present in all muscle cells in the culture (Fig. 3B). To test whether Bin3 regulates myotube formation, WT and Bin3 KO muscle cells were differentiated into nascent myotubes for 24 hrs, or into mature myotubes for 40 hrs, and immunostained for embryonic myosin heavy chain (eMyHC), a marker of differentiation. eMyHC staining revealed that Bin3 KO myotubes were smaller at both stages (Fig. 3C). Quantitative analyses subsequently showed that Bin3 KO myotubes exhibited a 33% decrease in the fusion index at 24 hrs (Fig. 3D), and contained 20% fewer nuclei at both 24 and 40 hrs (Fig. 3E). In addition, Bin3 KO myotubes were 12% thinner at both 24 and 40 hrs (Fig. 3F) and 19% shorter at 24 hrs (Fig. 3G). However, the number of nuclei analyzed per field (Fig. 3H), which could affect the extent of myotube formation, did not differ between WT and Bin3 KO cultures. Moreover, the steady-state levels of myogenin (Fig. S2A,B) and eMyHC (Fig. S2C,D), early and late markers of differentiation, respectively, did not differ between WT and Bin3 KO muscle cells. Overall, these data indicate that Bin3-dependent processes within muscle cells are necessary for proper myotube formation.

Figure 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 Bin3 antibody 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 cells with 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 muscle cells immunostained for eMyHC are shown at 24 and 40 hrs in DM. Bar, 150 μm. (D) Fusion index (*P<0.001), (E) myonuclear number (*P<0.001), (F) myotube diameter (*P<0.01) and (G) myotube length (*P<0.05) were decreased in Bin3 KO muscle cells in DM. No differences were noted in the number of nuclei per field (H) between WT and Bin3 KO muscle cells. Data are mean ± s.e.m., n=4 independent isolates for each genotype.

Functional redundancy by other N-BAR domain proteins during myogenesis may exist and could diminish the effects of Bin3 loss on myotube formation. Therefore, we examined mRNA levels of Bin3 and the most closely related N-BAR domain proteins of the Amphiphysin/Bin family (Table S1) by real-time PCR. Only Bin1 was expressed in primary muscle cells (Table S2). However, since loss of Bin1 results in differentiation defects early in myogenesis (Wechsler-Reya et al., 1998), we could not test whether Bin1 can partially compensate for Bin3 at the later stages of myogenesis analyzed in our studies.

We also assessed whether retroviral-mediated overexpression of recombinant HA-Bin3 in WT cells (Fig. S3A) was sufficient to enhance myotube size. By eMyHC staining, myotubes did not appear larger at either 24 or 40 hrs of differentiation (Fig. S3B). Subsequent quantification revealed slight but statistically insignificant increases in the fusion index (Fig. S3C) and the number of nuclei per myotube (Fig. S3D), likely due to a small increase in the number of nuclei per field (Fig. S3F) following Bin3 overexpression. The differentiation index was not altered following Bin3 overexpression (Fig. S3E). The inability of Bin3 overexpression to enhance myotube formation is likely due to downstream mediators of Bin3 action being rate-limiting.

Endocytosis defects are not observed in Bin3 KO muscle cells

One of the yeast orthologs of Bin3 regulates endocytosis (Ren et al., 2006), a process likely to be important for myogenesis. Indeed, molecules regulating endocytosis have recently been implicated in myotube formation (Doherty et al., 2008; Leikina et al., 2013; Posey et al., 2011). Thus, we hypothesized that an endocytic defect could contribute to impaired myotube formation in the absence of Bin3. Since many BAR domain proteins regulate receptor-mediated endocytosis, the most common and well-studied pathway utilized for internalization (Qualmann et al., 2011), we tested whether Bin3 regulates this process in muscle cells using fluorescently labeled transferrin. We labeled WT and Bin3 KO myocytes (18 hrs) with Alexa-594 conjugated transferrin to allow (37°C) or prevent (4°C) transferrin internalization, in the presence or absence of an acid wash to remove the cell surface transferrin and permit analysis of only the internalized fraction (Fig. S4A). No difference in the internalized transferrin fraction was observed between WT and Bin3 KO myocytes (Fig. S4B). To ensure that we could detect a difference in transferrin internalization, WT myocytes were treated with Dynasore, an inhibitor of dynamin-dependent endocytosis (Macia et al., 2006), prior to performing the internalization assay. Dynasore treatment caused a reduction in transferrin internalization in WT myocytes at 37°C (Fig. S4C,D). Together, these data suggest Bin3 is not necessary for receptor-mediated endocytosis in myocytes.

Bin3 plays a role in myocyte migration

Cell migration is another process critical for myotube formation (Bae et al., 2008; Bondesen et al., 2007; Jansen and Pavlath, 2006; Mylona et al., 2006; O’Connor et al., 2007). BAR domain proteins 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 (de Kreuk et al., 2011; Doherty et al., 2011a; Guerrier et al., 2009; Pichot et al., 2010; Quinones et al., 2010; Tsuboi et al., 2009). As migration of both myoblasts and myocytes is important for myotube formation, we analyzed the role of Bin3 in the migration of both cell types using time-lapse microscopy. The migratory cell paths of WT and Bin3 KO myoblasts were only slightly different (Fig. S5A), and their average velocity did not differ significantly (Fig. S5B,C). In contrast, Bin3 KO myocytes migrated shorter distances than WT cells (Fig. 4A), and their average velocity was decreased by 30% (Fig. 4B,C). Retroviral-mediated expression of recombinant HA-Bin3 in Bin3 KO cells (Fig. 4D) rescued their migration defect and restored their average velocity to WT levels (Fig. 4E). These results show Bin3 is required specifically for migration of myocytes during myogenesis.

Figure 4. Bin3 plays a role in myocyte migration.

(A) Time-lapse microscopy revealed that Bin3 KO myocytes migrated shorter distances than WT myocytes. The migratory paths 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 velocity was decreased in Bin3 KO myocytes (*P<0.05). (D) Immunoblot demonstrating expression of recombinant HA-Bin3 (Bin3 RV) in Bin3 KO myocytes after retroviral infection. Ctrl RV = empty 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 velocity compared to empty vector control (Ctrl RV) (*P<0.05). In panels B, C and E, 60 individual cells were analyzed per genotype with n=3 independent isolates for each genotype. Data in panels C and E are mean ± s.e.m.

Bin3 is involved in lamellipodia formation in myocytes

Actin polymerization is harnessed for cell motility and drives the forward extension of lamellipodia, broad actin-based protrusions associated with motility (Le Clainche and Carlier, 2008; Ridley, 2011; Small et al., 2002). Interestingly, the two yeast orthologs of Bin3 both regulate F-actin localization (Ren et al., 2006), and Bin3 KO mice exhibit loss of F-actin in lens fiber cells (Ramalingam et al., 2008). Therefore, we reasoned that Bin3 may also be important for actin-dependent processes in muscle cells. We visualized F-actin in WT and Bin3 KO myocytes at 18 and 24 hrs of differentiation using FITC-phalloidin. We observed fewer Bin3 KO myocytes with lamellipodia at these timepoints (arrowheads and insets, Fig. 5A). Following quantification, depending on the timepoint, 33-57% fewer Bin3 KO myocytes exhibited lamellipodia (Fig. 5B). Subsequently, we found that Bin3 and F-actin colocalized in lamellipodia of myocytes (arrowheads, Fig. 6A). Due to low levels of endogenous Bin3 in muscle cells, we retrovirally expressed recombinant HA-Bin3 and performed HA immunostaining to better examine the localization of Bin3. HA-Bin3 also colocalized with F-actin in lamellipodia of myocytes (arrowheads, Fig. 6B). Together, these data demonstrate that Bin3 regulates lamellipodia formation in myocytes.

Figure 5. Bin3 is involved in lamellipodia formation in myocytes.

(A) Lamellipodia were visualized by examining F-actin localization (FITC-phalloidin) in WT and Bin3 KO myocytes at 18 and 24 hrs in DM (arrowheads). Nuclei were counterstained with DAPI. Insets indicate lamellipodia at higher magnification (red box). Bar, 50 μm. (B) Bin3 KO myocytes exhibited a lower percentage of cells with lamellipodia (*P<0.01) in DM. Data are mean ± s.e.m., n=3 independent isolates for each genotype.

Figure 6. Bin3 and F-actin colocalize in lamellipodia of myocytes.

(A) Bin3 and F-actin colocalized in lamellipodia of WT myocytes at 18 hrs 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 was demonstrated by lack of antibody reaction in cells with the empty vector control (top row). Bar, 50 μm. Nuclei were counterstained with DAPI.

Decreased levels of active Rac1 and Cdc42 in Bin3 KO myocytes

Rho GTPases play critical roles in regulating actin dynamics during cell migration (Ridley, 2011). In particular, Rac1 and Cdc42 are associated with actin regulation in lamellipodia (Ridley, 2011). Based on the function of Bin3 in regulating lamellipodia formation in myocytes, we hypothesized that Bin3 may regulate the activity of Rac1 and Cdc42. Pull-down of active Rac1 and Cdc42 using beads coated with the p21 binding domain (PBD) of p21-activated protein kinase 1 (PAK1), termed PAK1-PBD, followed by immunoblotting, showed a major decrease in the active levels of these Rho GTPases (Fig. 7A) in Bin3 KO myocytes. Quantification of immunoblots revealed decreases of approximately 70% in the active levels of 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 a 3.3 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 activity in myocytes.

Figure 7. Decreased levels of active Rac1 and Cdc42 in Bin3 KO myocytes.

(A) Active Rac1 and Cdc42 were pulled down from WT and Bin3 KO myocytes using beads coated with the 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 Cdc42 were decreased in Bin3 KO myocytes (*P<0.05). (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. HA immunostaining showed an average of 89% HA⁺ cells. Data in panel B are mean ± s.e.m., n=3 independent isolates for each genotype. Data in panel D are mean ± s.e.m., n=2 independent isolates for each condition.

Discussion

Myofiber formation during myogenesis is key to muscle regeneration. Our data provide insights into the mechanisms by which dynamic rearrangements of the actin cytoskeleton are regulated during myogenesis. We show the N-BAR domain protein, Bin3, is important for myogenesis both in vitro and in vivo.

Muscle regeneration is a complex process requiring interplay between myogenic and non-myogenic cells (Chazaud et al., 2003; Saclier et al., 2012; Sonnet et al., 2006). Absence of Bin3, a ubiquitously expressed protein (Prendergast et al., 2009), resulted in a transient delay in the growth of regenerated myofibers after injury. Muscle-intrinsic functions of Bin3 likely contributed in part to the growth phenotype, as myocyte migration and myotube formation in vitro were impaired in the absence of Bin3. Potentially Bin3 function may also be required in non-myogenic cells during regeneration, and the absence of Bin3 in these cells may have contributed to the observed growth phenotype. The transient delay in myofiber growth during regeneration may be due to compensation by other molecules that regulate this process. Functional compensation may also explain in part the fact that myofiber size did not differ between WT and Bin3 KO muscles in the absence of injury.

In the absence of Bin3 we also observed abnormal branched myofibers. Although myofiber branching has been studied for many years, Bin3 is the only molecule found to regulate this process besides the G protein coupled receptor, mouse olfactory receptor 23 (MOR23) (Griffin et al., 2009). While the mechanisms underlying myofiber branching during muscle regeneration are unknown, interestingly both MOR23 and Bin3 regulate myocyte migration and myotube formation in vitro (Griffin et al., 2009), suggesting aberrations in these processes may contribute to the formation of branched myofibers.

Since muscle cell migration plays a crucial role in myotube formation (Bae et al., 2008; Bondesen et al., 2007; Jansen and Pavlath, 2006; Mylona et al., 2006; O’Connor et al., 2007), we initially investigated the role of Bin3 in migration. We found Bin3 was required only for myocyte migration, but not for myoblast migration. Bin3 likely interacts with different downstream molecules in these two cell types, leading to the specificity in regulating stage-specific cellular migration. Bin3 is the first example of a cytoplasmic protein which controls muscle cell migration, as to date only secreted molecules and transmembrane proteins have been shown to regulate this process (Simionescu and Pavlath, 2011). Interestingly, Bin2, an N-BAR domain protein with a C-terminal tail containing acidic and serine/proline-rich segments (Ge and Prendergast, 2000; Sánchez-Barrena et al., 2012), has recently been implicated in monocyte migration (Sánchez-Barrena et al., 2012). Together, these data associate N-BAR domain proteins with cell migration, a process requiring the formation of outward membrane protrusions, which N-BAR domains are not classically linked with (Suetsugu, 2010).

Dynamic changes in the actin cytoskeleton are critical for cells to extend protrusions to sense their environment and subsequently migrate towards a target. Migrating cells extend filopodia, thin exploratory extensions from the plasma membrane containing parallel bundles of actin filaments (Ridley, 2011) or lamellipodia, broad protrusions containing branched actin filaments (Le Clainche and Carlier, 2008; Ridley, 2011; Small et al., 2002). Many BAR domain proteins positively regulate actin polymerization and the formation of outward membrane protrusions (de Kreuk and Hordijk, 2012). Some BAR domain proteins are enriched, and can colocalize with F-actin, in these protrusions (de Kreuk and Hordijk, 2012). Indeed, the N-BAR domain protein Bin3 colocalized with F-actin in lamellipodia and was important for lamellipodia formation in myocytes. In contrast, the BAR-PH domain protein GRAF1 localizes to sites devoid of F-actin in muscle cells (Doherty et al., 2011b). These data suggest that BAR domain proteins of different classes may have complementary roles in regulating actin dynamics in myogenesis.

Myotube formation requires Rac1 and Cdc42-dependent actin polymerization (Vasyutina et al., 2009), but the upstream signals controlling the activity of these Rho GTPases in differentiated muscle cells are unknown. The levels of active Rac1 and Cdc42 were greatly decreased in myocytes in the absence of Bin3, suggesting Bin3 is a major positive regulator of Rac1 and Cdc42 in muscle cells. The fusion defect observed in Bin3 KO myotubes in vitro was less severe than seen in Rac1 or Cdc42 KO cells (Vasyutina et al., 2009), possibly due to the residual low levels of these GTPases in Bin3 KO myocytes. In contrast, the BAR-PH domain protein, GRAF1, is a negative regulator of RhoA activity in C2C12 muscle cells but does not affect Cdc42 activity in L6 myoblasts (Doherty et al., 2011b), suggesting that BAR domain proteins of different classes show specificity in controlling GTPase activity in myogenesis. As we did not observe a visible difference in actin stress fibers between WT and Bin3 KO myocytes, we suggest that levels of active RhoA, a major regulator of stress fiber formation (Pellegrin and Mellor, 2007), are unlikely to be affected in Bin3 KO myocytes.

We detected recombinant Bin3 in a complex with active Rac1 or Cdc42 in myocytes. How could Bin3, a protein with only an N-BAR domain, regulate the activity of Rac1 and Cdc42 in muscle cells? Some BAR domain proteins modulate the activity of Rho GTPases via a RhoGAP or Rho Guanine nucleotide exchange factor (RhoGEF) domain (de Kreuk and Hordijk, 2012). In contrast, BAR domain proteins that lack a RhoGAP/GEF domain regulate Rho GTPases by recruiting other proteins with RhoGAP/GEF activity. For example, one of the Bin3 yeast orthologs, Hob3p, facilitates the interaction between Gef-1, a Cdc42GEF, and Cdc42 (Coll et al., 2007). Bin3 may similarly interact with an unknown RhoGAP/GEF protein to modulate the activity of Rac1 and Cdc42 in muscle cells. Moreover, Bin3 could heterodimerize with another BAR domain protein (Ren et al., 2006), which ultimately serves as the link to modulating RhoGAP/GEF activity. Additional studies are necessary to define Bin3 interacting partners to better understand the mechanisms by which Bin3 may regulate Rac1 and Cdc42 activity in myocytes. Understanding these mechanisms may help explain the myofiber branching phenotype observed in the absence of Bin3 in vivo, and whether Rac1 and Cdc42 are also implicated in this process. In addition, Bin3 may also regulate myogenesis through RhoGTPase-independent mechanisms.

The study of BAR domain proteins is an emerging area in skeletal muscle. Our studies extend previous work in this field (Doherty et al., 2011b; Wechsler-Reya et al., 1998) and suggest that multiple classes of BAR domain proteins will prove critical for regulating important cellular processes myogenesis. Understanding the role of BAR domain proteins in muscle growth and repair will likely impact treatments for muscle diseases in which these processes are impaired.

Materials and Methods

Animals

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

Muscle injuries

For analyses of myofiber cross-sectional area, muscle injury was induced by a single injection of 50 μl of 1.2% BaCl₂ in PBS into the tibialis anterior muscles of mice as described previously (O’Connor et al., 2007). Muscles were collected at various timepoints after injury and histological sections were prepared and imaged as described previously (Jansen and Pavlath, 2006). Myofiber cross-sectional area was quantified using ImageJ. A total of 480-1200 myofibers from 4-7 mice were analyzed per genotype for each timepoint.

For analyses of myofiber branching, severe muscle injury was induced by two consecutive injections of 50 μl of 1.2% BaCl₂ in PBS two days apart into the gastrocnemius muscles of mice. Muscles were collected 21 days after the second injury. Subsequently, muscles were enzymatically digested for 1 hr 20 minutes with gentle agitation and single myofibers were isolated as described previously (Kafadar et al., 2009), fixed with 3.7% formaldehyde in PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei. Regenerated myofibers, identified by the presence of central nuclei, were analyzed for the number and type of branches. A total of 300-450 myofibers from 4 mice were analyzed per genotype.

Differentiation and fusion assays

Primary myoblasts were isolated from the hindlimb muscles of WT and Bin3 KO mice as previously described with the exception of a Percoll gradient (Bondesen et al., 2004). Cells were cultured in growth media (GM; Ham’s F10, 20% fetal bovine serum (FBS), 100 U/mL penicillin G, 100 μg/mL streptomycin, 5 ng/mL FGF) on collagen-coated plates. Cultures were >95% myogenic as defined by MyoD immunostaining.

To induce differentiation, primary mouse myoblasts were plated on entactin-collagen IV-laminin (ECL; Millipore) in GM and after 1 hr 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 hrs. Cells were then fixed with 3.7% formaldehyde and immunostained with an antibody (F1.652; Developmental Studies Hybridoma Bank) against embryonic myosin heavy chain as described previously (Horsley et al., 2001).

The differentiation index was determined by dividing the total number of nuclei in eMyHC⁺ cells by the total number of nuclei counted (Jansen and Pavlath, 2006). The fusion index was determined by dividing the total number of nuclei in myotubes by the total number of nuclei counted (Jansen and Pavlath, 2006). The average myonuclear number was determined by dividing the total number of nuclei in myotubes (≥ 2 nuclei) by the total number of myotubes counted (Jansen and Pavlath, 2006). A total of 2500-5400 nuclei were analyzed per genotype or condition for each timepoint. To quantify myotube diameter using ImageJ, a range of 3-30 measurements were taken perpendicular to the axis of the myotube at equal distances along the axis of the myotube, depending on the length of the myotube. To quantify myotube length using ImageJ, a line was drawn along the entire myotube and only myotubes visible in their entirety were analyzed. Three to four independent isolates were analyzed per genotype or condition.

Endocytosis assays

Myoblasts were differentiated into myocytes for 18 hrs in DM and switched to serum-free media lacking transferrin (DMEM, 10 mM Hepes pH 7.4, 0.2% BSA) at 37°C for 3 hours prior to the assay. Cells were cooled to 4°C to inhibit endocytic internalization, followed by incubation with Alexa-594 conjugated transferrin (Invitrogen) in the presence or absence of Dynasore (Abcam), to allow both transferrin and Dynasore to equilibrate at the cell surface. Subsequently, in the continuous presence or absence of Dynasore, cells were either incubated at 37°C for 35 min to allow transferrin internalization, or kept at 4°C to prevent it. All cells were then returned to 4°C in the presence or absence of an acidic buffer (0.5 M NaCl, 15 mM MES (2-(N-Morpholino) ethanesulfonic acid hydrate (Sigma-Aldrich) pH 4.5 in PBS), to remove cell surface transferrin. Finally, while still at 4°C, all cells were fixed with 4% paraformaldehyde, and stained with DAPI. Intensities were measured with MetaMorph software version 6.1 (Molecular Devices, Sunnyvale, CA) as integrated pixel intensity, and the ratio (internalized/total) was plotted for some experiments. A total of 20-90 cells were analyzed per genotype or condition. One to three independent isolates were analyzed per genotype or condition.

Cell migration assays

Cell migration experiments were performed as previously described (Jansen and Pavlath, 2006). Myoblasts or myocytes (18 hrs in DM) were visualized using an Axiovert 200M microscope with a 0.3 NA 10X Plan-Neofluar objective (Carl Zeiss MicroImaging, Inc.), and images were recorded using a camera (QImaging) and OpenLab 5.5.2 (Improvision) software every 5 min for 3 hrs using time-lapse microscopy. Using ImageJ, the migratory paths of 60 individual cells were analyzed per genotype and mean cell velocities were calculated. Three to four independent isolates were analyzed per genotype.

Phalloidin staining

Differentiated muscle cells at 18 hrs 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 Life Sciences) in PBS with 1% BSA for 20 min. Nuclei were counterstained with DAPI. The percentage of cells with lamellipodia was quantified. A total of 130-160 cells were analyzed per genotype for each timepoint. Three independent isolates were analyzed per genotype.

Immunostaining

Differentiated muscle cells at 18 hrs in DM were fixed with 3.7% formaldehyde and incubated in blocking buffer (5% donkey serum, 0.1% or 0.25% Triton X-100, 0.5% or 1% BSA in PBS), followed by an overnight incubation at 4°C 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 streptavidin (PerkinElmer) and the tyramide signal amplification red reagent (Tyramide Signal Amplifcation (TSA) kit, Perkin Elmer). Nuclei were counterstained with DAPI.

Retroviral infection

Mouse Bin3 cDNA (Open Biosystems) with 3xHA tags at the C-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. WT and Bin3 KO myoblasts were subjected to 2-4 rounds of retroviral infection (Abbott et al., 1998) using either the HA-Bin3 vector or the control vector. Infected cells were subsequently grown in GM under puromycin selection at 0.75 μg/ml for a minimum of 48 hours. The infection efficiency was >95% based on cell survival in the presence of 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 protease inhibitors (Mini Complete; Roche) and centrifuged at 21,000 × g for 15 min. The pellet was then discarded and the supernatant was subjected to SDS-PAGE and immunoblotting. An equal amount of protein from each sample was loaded onto 10% or 12% SDS-polyacrylamide gels and transferred to either a 0.2 μm or a 0.45 μm nitrocellulose membrane (Bio-Rad Laboratories). After blocking non-specific binding, the membranes were incubated with the appropriate primary antibodies. Primary antibodies used were as follows: Bin3 (3A4; Ramalingam et. al, 2008), Cdc42 (Santa Cruz), EF1α (Millipore), eMyHC (F1.652; Developmental Studies Hybridoma Bank), HA (Covance), Hsp90 (Santa Cruz), Myogenin (F5D; Developmental Studies Hybridoma Bank), Rac1 (BD Biosciences) and Tubulin (Sigma-Aldrich), followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated IgG (Jackson ImmunoResearch) secondary antibodies.

To perform Rac1 and Cdc42 activity assays, myocytes were lysed in supplied magnesium lysis buffer (MLB; Millipore) containing protease/phosphatase inhibitors (Sigma), and centrifuged at 14,000 × g for 10 min. The pellet was then discarded and the supernatant was incubated for 30 minutes with 10 mM EDTA. The reaction was stopped by the addition of 60 mM MgCl₂. An equal amount of protein from each sample was incubated with 20 μl PAK1-PBD beads (Millipore) for 1.5 hours at 4°C. The beads were then washed, spun quickly and the supernatants were subjected to SDS-PAGE and immunoblotting. The relative amounts of active Rac1 and Cdc42 were determined by densitometric analysis. Two to three independent isolates were analyzed per genotype or condition.

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 μg) was reverse transcribed using random primers (Invitrogen) and M-MLV reverse transcriptase (Invitrogen). mRNA levels were determined by real-time PCR using the SYBR Select Master Mix (Invitrogen), the StepOnePlus Real-Time PCR System and StepOne Software version 2.2.2 (Applied Biosystems, Life Technologies). All reactions were run in duplicate. Primers for mouse Amphiphysin (PPM30542A), Bin1 (PPM25097A), Bin2 (PPM66366A) and Bin3 (PPM26566A) were obtained from SABiosciences. Three independent isolates were analyzed. Mouse gastrocnemius muscle or brain was used as a positive control.

Image acquisition

For the analysis of myofiber branching in vivo, the differentiation and fusion assays in vitro, as well as the Bin3 immunostaining in differentiated cells, images were obtained using an Axiovert 200M microscope (Carl Zeiss MicroImaging) with a 0.3 NA 10X Plan-Neofluar objective (Carl Zeiss MicroImaging) and were recorded using a camera (QImaging) and OpenLab 5.5.2 (Improvision) software. For all other experiments, images were obtained using an Axioplan microscope (Carl Zeiss MicroImaging) with either a 0.3 NA 10X Plan-Neofluar objective (Carl Zeiss MicroImaging) or with a 0.8 NA 25X Plan-Neofluar objective (Carl Zeiss MicroImaging) and were recorded with a camera (Carl Zeiss MicroImaging) and Scion Image 1.63 (Scion Corporation) software. All images were assembled using Adobe Photoshop CS5.1 for Macintosh (Adobe) and equally processed for size, color levels, brightness, and contrast.

Statistical analysis

Statistical analysis to determine significance between two groups was performed using a Student’s t test. One-way or two-way ANOVA with Bonferroni’s posttest was used for comparisons between multiple groups as appropriate. All statistical analyses were performed using GraphPad Prism 5.0 for Macintosh (GraphPad Software). Differences were considered to be statistically significant at P<0.05.

Highlights.

• Bin3 controls myofiber size in vitro and in vivo

• Bin3 promotes muscle cell migration and lamellipodia formation

• Bin3-dependent pathway is a key regulator of Rac1 and Cdc42 activity in muscle cells

Abbreviations List

BAR

Bin-Amphiphysin-Rvs

Bin

Bridging integrator

CSA

Cross-sectional area

DM

Differentiation media

eMyHC

Embryonic myosin heavy chain

F-actin

Filamentous actin

GAP

GTPase-activating protein

GEF

Guanine nucleotide exchange factor

GM

Growth media

GRAF1

GTPase regulator associated with focal adhesion kinase-1

KO

Knockout

PAK

p21-activated protein kinase

PBD

p21 binding domain

RV

Retrovirus

WT

Wild-type