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.
Dai
Z
Z
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Luo
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X
Xie
H
H
Peng
H B
HB
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002478:Cells, Cultured; D051356:Cortactin; D047630:Depsipeptides; D015870:Gene Expression / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000032:analysis / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008840:Microfilament Proteins / Q000032:analysis / Q000235:genetics; D008863:Microspheres; D009132:Muscles / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D009469:Neuromuscular Junction / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014981:Xenopus
D000199:Actins; D000970:Antineoplastic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D051356:Cortactin; D047630:Depsipeptides; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D010456:Peptides, Cyclic; D011108:Polymers; D011950:Receptors, Cholinergic; D013844:Thiazoles; D053778:Thiazolidines; C057531:jasplakinolide; D049452:Green Fluorescent Proteins; C037067:latrunculin A