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© The Rockefeller University Press,
0021-9525/2000//1321 $5.00
The Journal of Cell Biology, Volume 150, Number 6,
, 2000 1321-1334
Original Article |
The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters
A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin–binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule–coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead–muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein–coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR 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 agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.
Key Words: acetylcholine receptor cluster • actin • neuromuscular junction • latrunculin • jasplakinolide
© 2000 The Rockefeller University Press
Abbreviations used in this paper: AChR, acetylcholine receptor; F-actin, filamentous actin; G-actin, globular actin; GFP, green fluorescent protein; HB-GAM, heparin-binding growth-associated molecule; Ltn A, latrunculin A; MSD, mean-square displacement; MuSK, muscle-specific kinase; NMJ, neuromuscular junction; OG-BTX, Oregon green–conjugated
-bungarotoxin; PY, phosphotyrosine; Rh-phalloidin, rhodamine-conjugated phalloidin; R-BTX, rhodamine-conjugated -bungarotoxin; RTK, receptor-tyrosine kinase.
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