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Temperature is a physiological factor that affects neuronal growth and synaptic homeostasis at the invertebrate neuromuscular junctions (NMJs); however, whether temperature stress could also regulate the structure and function of the vertebrate NMJs remains unclear. In this study, we useXenopus laevisprimary cultures as a vertebrate model system for investigating the involvement of heat shock protein 90 (HSP90) family of stress proteins in NMJ development. First, cold temperature treatment or HSP90 inhibition attenuates the formation of aneural acetylcholine receptor (AChR) clusters, but increases their stability after they are formed, in cultured muscles. HSP90 inhibition specifically affects the stability of aneural AChR clusters and their associated intracellular scaffolding protein rapsyn, instead of causing a global change in cell metabolism and protein expression inXenopusmuscle cultures. Upon synaptogenic stimulation, a specific HSP90 family member, glucose-regulated protein 94 (Grp94), modulates the phosphorylation and dynamic turnover of actin depolymerizing factor (ADF)/cofilin at aneural AChR clusters, leading to the recruitment of AChR molecules from aneural clusters to the assembly of agrin-induced postsynaptic specializations. Finally, postsynaptic Grp94 knock-down significantly inhibits nerve-induced AChR clustering and postsynaptic activity in nerve-muscle co-cultures as demonstrated by live-cell imaging and electrophysiological recording, respectively. Collectively, this study suggests that temperature-dependent alteration in Grp94 expression and activity inhibits the assembly of postsynaptic specializations through modulating ADF/cofilin phosphorylation and activity at aneural AChR clusters, which prevents AChR molecules from being recruited to the postsynaptic sites via actin-dependent vesicular trafficking, at developing vertebrate NMJs.