Grp94 regulates AChR cluster formation at the neuromuscular junction by ADF/cofilin

Abstract

Aggregation of acetylcholine receptors (AChRs) on muscle membrane represents a crucial step in the development of vertebrate neuromuscular junction (NMJ). This process depends on both localized signals to trigger AChR clustering, and global signals to induce redistribution of AChRs on the muscle membrane. However, the mechanism underlying AChR redistribution remains largely unknown. Here, we describe a novel role of glucose-related protein 94 (Grp94), an endoplasmic reticulum-localized heat shock protein 90 (HSP90) family member, in AChR clustering and redistribution at developing NMJs. First, we found that Xenopus muscle cells treated with 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a HSP90 inhibitor, attenuated aneural AChR cluster formation and reduced localization of podosome-like structures (PLS) associated with aneural AChR clusters. Knockdown of Grp94 by morpholino in muscle cells mimicked the effects of pharmacological inhibition of HSP90 activity. In control nerve-muscle cocultures, we observed that both diffuse and aneurally clustered AChRs were recruited for the assembly of synaptic AChR clusters induced by agrin beads. More importantly, knockdown of Grp94 impaired the recruitment of AChRs from aneural to synaptic clusters through modulating the localization and turnover of actin-depolymerizing factor (ADF)/cofilin in muscle cells, resulting in an attenuation of agrin-induced AChR clusters formation. Using Xenopus nerve-muscle co-cultures, we further showed that either treatment of a specific Grp94 inhibitor PU-WS13 or knockdown of muscle Grp94 decreased both the intensity and percentage of nerve-induced AChR clusters. Finally, electrophysiological studies demonstrated that muscle Grp94 knockdown reduced the frequency and amplitude of spontaneous synaptic currents (SSCs) at the NMJ, demonstrating the requirement of postsynaptic Grp94 for synaptic functions. Taken together, Grp94 that regulates the recruitment of AChRs from aneural to synaptic clusters by modulating ADF/cofilin is a novel regulator for the assembly of synaptic structures and the maintenance of synaptic functions at developing NMJs.