Antagonistic actions of agrin on the clustering of acetylcholine receptors

Abstract

This dissertation explores the mechanisms underlying synapse formation and maintenance. Skeletal muscle cells in culture were used as a model system in which to study how the distribution of synaptic molecules is regulated. On cultured myotubes, acetylcholine receptors (AChRs) aggregate into "clusters" either spontaneously or in response to various exogenous factors such as agrin. I report here that a soluble fragment of agrin caused the disruption of preexisting AChR clusters. The disruption occurred quickly after agrin addition (2-3 h) and was more discernible with large clusters (>100 μm²). Various experiments performed to examine the mechanism underlying the disruption of AChR clusters revealed the following. First, the dose-dependence for the disruption of large clusters was similar to that for the induction of small clusters (<50 μm²). Second, a non-neuronal isoform of agrin did not cause the disruption of clusters. Third, heparin, which inhibits agrin-induced clustering, did not inhibit agrin-induced disruption of clusters. Fourth, agrin was unable to disrupt clusters that had been previously induced by laminin-1. A full-length, insoluble form of neuronal agrin was tested for its ability to disrupt AChR clusters at a distance from the site of application. This focally applied agrin caused the disruption of clusters that had arisen spontaneously or had been induced by soluble agrin, and it prevented the induction of clusters by either soluble agrin or laminin-1. The disruptive effect was more marked at distances <50 μm and for clusters >50 μm². Two broad classes of mechanism are proposed to explain how agrin disrupts clusters. Previous studies have led to the hypothesis that agrin binding to chondroitin sulfate chains on α-dystroglycan is required for AChR clustering. As evidence against this hypothesis, we observed that protease-free chondroitinase did not inhibit AChR clustering and that the presence of CS did not always coincide with the presence of α-DG as judged from gel electrophoresis. Future experiments to further explore the effects of agrin on the distribution of synaptic molecules are proposed, and the functional implications of the actions of agrin are discussed in the context of development.