Mechanobiological regulation of the assembly and topological maturation of postsynaptic apparatus at developing neuromuscular synapses

The mechanical properties of the extracellular matrix (ECM) environment can trigger different signaling cascades that affect various cellular processes. Cell-ECM interaction is primarily mediated by heterodimeric transmembrane receptors, integrins, that physically connect to the cytoskeletal elements via different focal adhesion molecules to generate downstream signaling in controlling cell proliferation, differentiation, migration, and adhesion. The vertebrate neuromuscular junction (NMJ) is a tetrapartite chemical synapse composed of a spinal motor neuron, a skeletal muscle fiber, a terminal Schwann cell, and the ECM. The ECM environment of the basal lamina can influence all other players at the NMJs to regulate synapse formation and maintenance. In our recently completed GRF project (project number: 17100718), we showed that focal ECM remodeling is detected at perforations of aneural and synaptic acetylcholine receptor (AChR) clusters at NMJs, which is regulated by the vesicular trafficking and surface insertion of membrane-type 1 matrix metalloproteinase to the sites of podosome-like structures (PLSs). To build upon the conclusions of these studies, this continuation project (n.b. resubmission application of 17105324 in GRF 2024/25) aims to further examine the mechanobiological regulation of the assembly and topological maturation of the postsynaptic apparatus at developing NMJs. Advanced live-cell imaging and molecular manipulation will be performed to answer a series of cell biology questions regarding the molecular basis of Ca2+ mobilization and signaling that regulates the development of vertebrate NMJs in vitro and in vivo. Specifically, we will test the hypothesis that submembrane Ca2+ elevation is triggered by ECM stiffness-induced activation of the mechanosensitive transient receptor potential canonical type 1 (TRPC1) channel, which in turn promotes calpain-mediated proteolysis of PLS components that directly regulate integrin activation and PLS turnover during the assembly and topological remodeling of postsynaptic apparatus at NMJs. On the other hand, we will
also investigate the divergent roles of agrin-induced Src activation in kindlin-2-mediated integrin activation and TRPC1-caveolin-3 interaction at developing NMJs. Given that dysregulated Ca2+ signaling is implicated in many eurodegenerative and cognitive disorders, results of this project could potentially contribute to new insights into the detailed molecular mechanisms that underlie the physiological and pathophysiological regulation of synaptic development and maintenance.