Extracellular matrix protein-induced assembly of podosome-like structures for aneural acetylcholine receptor cluster remodeling

Abstract

The presence of high acetylcholine receptor (AChR) density is a prominent feature of neuromuscular junctions (NMJs). Previous studies have shown that integrin beta-1 is localized at the cortex domain of podosome-like structures at aneural AChR clusters induced by extracellular matrix (ECM) proteins, suggesting the involvement of integrin-mediated signaling pathway in the formation and/or remodeling of aneural AChR clusters. However, the causal relationship among different ECM proteins, podosome-like structures, and aneural AChR cluster remodeling is not fully understood. In this study, we first showed that the formation of aneural AChR clusters is spatially induced in muscle cells cultured on micro-patterns of various extracellular matrix (ECM) proteins by utilizing multiphoton-crosslinking fabrication technique. Additionally, several typical core and cortex markers of podosome-like structures are detected in the perforated regions of the aneural AChR clusters induced by those ECM proteins. Next, we used total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) to further understand the functional involvement of podosome-like structures at developing NMJs. Live cell time-lapse imaging of microtubule plus-end protein EB1 demonstrated that microtubules are targeted to and captured at the cortex domains of perforated aneural AChR clusters, suggesting that the podosome-like structures may direct the local surface delivery of new AChR molecules to the aneural AChR clusters via microtubule-based vesicle transport. Further work aimed to investigate the molecular mechanisms involved in ECM protein-induced assembly of podosome-like structures through integrin-mediated signaling pathways to facilitate the formation and/or remodeling of aneural AChR clusters at developing NMJs.