Supplementary MaterialsFIGURE S1: Image of spontaneous clusters of AChR that form in C2C12 myotubes in the absence of agrin and laminin. which create gaps between AChR-rich areas. In cultured myotubes, the inhibition of podosome formation leads to modified distribution of AChR receptors in postsynaptic clusters (Proszynski et al., 2009). However, the function of podosomes in NMJ development has not been elucidated. Apart from Niraparib R-enantiomer podosomes, the actin cytoskeleton is definitely important for the formation and maintenance of postsynaptic AChR assemblies. AChR are anchored to F-actin (Mitsui et al., 2000) and actin dynamics drives AChR trafficking and clustering (Dai et al., 2000; Lee et al., 2009). Specifically, the rules of actin cytoskeleton by Rho family GTPases appears to be involved in postsynaptic AChR clustering (Luo et al., 2002; Weston et al., 2003; Shi et al., 2010). The mechanisms of recruitment and rules of Rho GTPases in the NMJ are poorly recognized. The dystrophin-glycoprotein complex (DGC) is a major muscle mass receptor for extracellular laminins and an important component of the postsynaptic NMJ machinery (Ervasti and Campbell, 1991; Nishimune et al., 2008; Gawor and Prszyski, 2018). The core of the DGC complex consist of dystrophin, syntrophin, -dystroglycan, -dystroglycan, the sarcoglycan complex, sarcospan, and -dystrobrevin (Nakamori and Takahashi, 2011; Aittaleb et al., 2017; Belhasan and Akaaboune, 2020). The dysfunction of the DGC core components prospects to myopathies in humans, including Duchenne muscular dystrophy, a disease characterized Niraparib R-enantiomer by progressive damage and impaired regeneration of skeletal muscle tissue (Campbell, 1995). DGC core parts can recruit additional, peripherally associated proteins. For instance the cytoplasmic protein -dystrobrevin 1 (aDB1) is definitely believed to be an adaptor for recruitment of various signaling molecules (Oh et al., 2012; Gingras et al., 2016; Gawor and Niraparib R-enantiomer Prszyski, 2018). The loss of aDB1 in mice results in irregular NMJ morphology and impaired Rabbit polyclonal to PIWIL2 maturation of the postsynaptic apparatus (Grady et al., 2003, 2000, 1999). In humans, aDB1 mutations cause congenital heart disease with remaining ventricular non-compaction (Ichida et al., 2001). The function of aDB1 is dependent at least in part on its phosphorylation by tyrosine kinases (Grady et al., 2003; Schmidt et al., 2011; Gingras et al., 2016). To identify the mechanisms of the rules of NMJ maturation by aDB1, we have previously searched for proteins that interact with aDB1 inside a phosphorylation-dependent manner using a protein pull-down assay followed by mass spectrometry (Gingras et al., 2016). One of the proteins that we therefore identified as an aDB1 interactor was Arhgef5. Arhgef5 is definitely a guanidine nucleotide exchange element (GEF) for the small GTPases from your Rho family and is involved in the rules of actin dynamics (Xie et al., 2005). Interestingly, Arhgef5, which also interacts with another aDB1-binding protein -catulin (Lyssand et al., 2010; Gingras et al., 2016) was shown to be pivotal for the Src-dependent formation of podosomes (Kuroiwa et al., 2011). We consequently hypothesized that Arhgef5 may cooperate with aDB1 and -catulin to regulate the maturation and stability of the NMJ postsynaptic machinery by altering the dynamics of the actin cytoskeleton via Rho-family GTPases. Here, we display that Arhgef5 localizes in the NMJ and concentrates in the postsynaptic machinery. Loss of Arhgef5 in mouse skeletal muscle tissue results in NMJ defects characterized by increased fragmentation of the postsynaptic apparatus, an effect that may be attributed to the irregular function of the GTPases RhoA and Cdc42. Results Arhgef5 Binds to Phosphorylated aDB1s Arhgef5 was originally recognized in our unbiased mass spectrometry-based display for interaction partners of the phosphorylated form of aDB1. Arhgef5 was one of the top proteins from myotube components that specifically bind the aDB1-derived phosphopeptide TQPEDGNpY ENESVRQ (Y713-P; related to phosphorylated tyrosine 713 of aDB1) but not to its unphosphorylated control peptide TQPEDGNY ENESVRQ (Y713) (Number 1A). Arhgef5 has a standard domain structure of Rho GEFs: it contains a Dbl homology (DH), a pleckstrin homology (PH), and a Src homology 3 (SH3) website (Number 1B). Additionally, it contains an N-terminal website that has several proline-rich motifs (Kuroiwa et al., 2011). In humans, in addition to the full-length protein, a shorter isoform called Niraparib R-enantiomer TIM lacking the N-terminal website is indicated, but this isoform has not been Niraparib R-enantiomer recognized in mice. Using western blot, we confirmed the C-terminal website of Arhgef5 binds the Y713-P, but not the Y713 peptide (Number 1C). We also individually showed that full-length Arhgef5 binds to full-length.