The actin-driven movement and formation of acetylcholine receptor clusters

A new method was devised to visualize actin polymerization induced by posts ynaptic differentiation signals in cultured muscle cells. This entails mask ing myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing n ew actin assembly with fluorescent phalloidin. With this procedure, actin p olymerization associated with newly induced acetylcholine receptor (AChR) c lustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed, The beads induced local F-actin assembly that colocali zed with AChR clusters at bead-muscle contacts, whereas both the actin cyto skeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a p rotein that binds to active F-actin, the dynamic nature of the actin cytosk eleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced A ChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and argin-induced AChR clusters as well as phosphotyrosine accumulati on, as shown by their blockage by latrunculin A, a toxin that sequesters gl obular (G)-actin and prevents F-actin assembly. These results show that act in polymerization induced by synaptogenic signals is necessary for the move ment and formation of AChR clusters and implicate a role of F-actin as a po stsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.