Title The function of p 120 Catenin in Filopodial Growth and SynapticVesicle Clustering in Neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve–muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve–muscle interactions, here we test p120ctn’s function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve–muscle interaction and NMJ assembly. INTRODUCTION During the development of the vertebrate neuromuscular junction (NMJ), initial contact between nerve and muscle facilitates signaling between the synaptic partners. This bidirectional signaling leads to the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane of muscle to a density of ∼10,000/μm2 (Fertuck and Salpeter, 1976) and to the accumulation of synaptic vesicles (SVs) and mitochondria within the nerve terminal (Hughes et al., 2006; Lee and Peng, 2006, 2008). In molecular terms, the best-understood part of this early neuromuscular signaling involves the activation of the muscle-specific tyrosine kinase by the nerve-derived factor agrin (Glass et al., 1996), which results in the clustering of AChRs by the protein rapsyn in an actin polymerization–dependent manner (Glass and Yancopoulos, 1997; Banks et al., 2003; Madhavan and Peng, 2005). On the presynaptic side, after nerve contact with muscle, active zones appear and SVs become concentrated in the incipient nerve terminals (Weldon and Cohen, 1979; Nakajima et al., 1980; Buchanan et al., 1989), but the control of presynaptic differentiation remains poorly described. Although previous work suggested that several fibroblast growth factors (FGFs), neurotrophins, and cell adhesion molecules expressed by muscle influence the transformation of motor axon growth cones into nerve terminals specialized for neurotransmitter release (Dai and Peng, 1995; Fitzsimonds and Poo, 1998; Fox and Umemori, 2006; Fox et al., 2007; Johnson-Venkatesh and Umemori, 2010), further studies are needed to understand the mechanistic underpinnings of presynaptic development at the NMJ. When growing motor axons reach muscle, synaptic differentiation is induced locally in nerve and muscle at the sites where they contact each other, which suggests that physical interaction between the synaptic partners facilitates NMJ assembly. Although the Monitoring Editor Paul Forscher Yale University Received: Jan 4, 2012 Revised: May 16, 2012 Accepted: May 21, 2012 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E12-01-0004) on May 30, 2012. *Present address: Department of Medicine, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China. †These authors contributed equally. Address correspondence to: H. Benjamin Peng (penghb@ust.hk). © 2012 Chen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: AChR, acetylcholine receptor; bFGF, basic fibroblast growth factor; NMJ, neuromuscular junction; p120ctn, p120 catenin; R-BTX, rhodamineconjugated α-bungarotoxin; SV, synaptic vesicle.