The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

doi:10.1083/jcb.150.6.1321

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Published online 18 September 2000. doi:10.1083/jcb.150.6.1321

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© The Rockefeller University Press, 0021-9525/2000/9/1321/ $5.00
The Journal of Cell Biology, Volume 150, Number 6, September 18, 2000 1321-1334

Original Article

The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

Zhengshan Dai^a, Xiaoyan Luo^a, Hongbo Xie^a, and H. Benjamin Peng^a,b,c
^a Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599
^b Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina 27599
^c Curriculum in Neurobiology, University of North Carolina, Chapel Hill, North Carolina 27599

Correspondence to: H. Benjamin Peng, Department of Cell Biology and Anatomy, University of North Carolina, CB#7090, 108 Taylor Hall, Chapel Hill, NC 27599-7090. Tel:(919) 843-7185 Fax:(919) 966-1856

A new method was devised to visualize actin polymerization inducedby postsynaptic differentiation signals in cultured muscle cells.This entails masking myofibrillar filamentous (F)-actin withjasplakinolide, a cell-permeant F-actin–binding toxin,before synaptogenic stimulation, and then probing new actinassembly with fluorescent phalloidin. With this procedure, actinpolymerization associated with newly induced acetylcholine receptor(AChR) clustering by heparin-binding growth-associated molecule–coatedbeads and by agrin was observed. The beads induced local F-actinassembly that colocalized with AChR clusters at bead–musclecontacts, whereas both the actin cytoskeleton and AChR clustersinduced by bath agrin application were diffuse. By expressinga green fluorescent protein–coupled version of cortactin,a protein that binds to active F-actin, the dynamic nature ofthe actin cytoskeleton associated with new AChR clusters wasrevealed. In fact, the motive force generated by actin polymerizationpropelled the entire bead-induced AChR cluster with its attachedbead to move in the plane of the membrane. In addition, actinpolymerization is also necessary for the formation of both beadand agrin-induced AChR clusters as well as phosphotyrosine accumulation,as shown by their blockage by latrunculin A, a toxin that sequestersglobular (G)-actin and prevents F-actin assembly. These resultsshow that actin polymerization induced by synaptogenic signalsis necessary for the movement and formation of AChR clustersand implicate a role of F-actin as a postsynaptic scaffold forthe assembly of structural and signaling molecules in neuromuscularjunction formation.

Key Words: acetylcholine receptor cluster, actin, neuromuscular junction,latrunculin, jasplakinolide

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