Grp94 Regulates the Recruitment of Aneural AChR Clusters for the Assembly of Postsynaptic Specializations by Modulating ADF/Cofilin Activity and Turnover

Abstract

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 use Xenopus laevis primary 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 in Xenopus muscle 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 nervemuscle 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.

Nerve-independent formation of membrane infoldings at topologically complex postsynaptic apparatus by caveolin-3

“…6G and movie S1). Vesicular trafficking of AChRs has been implicated in agrin-induced AChR transcytosis for the assembly of nascent postsynaptic specializations at developing Xenopus NMJs in cultures ( 11 , 46 , 47 ) and activity-dependent AChR recycling for the modulation of receptor density and synaptic efficacy at mature NMJs in living mice ( 48 , 49 ). We therefore hypothesize that membrane infoldings associated with AChR clusters may serve as an endo/exocytic hub for AChR vesicular trafficking in nerve-muscle co-cultures (Fig.…”