| This thesis examines the actions of erbB receptor tyrosine kinases and their ligand neuregulin at the mammalian neuromuscular junction (NMJ). I determined the localization of the erbB receptors and additional synaptic proteins at the neuromuscular junction. I examined how these proteins distributed in myotube culture under different pharmacological conditions. Finally, I characterized a novel role for neuregulin: the regulation of acetylcholine receptor (AChR) distribution.; The neuregulin/erbB receptor and agrin/MuSK pathways are critical for communication between the nerve, muscle, and Schwann cell that establishes the precise topological arrangement at the NMJ. We determined that erbB2 and erbB4 are enriched in the secondary folds on the post-synaptic muscle membrane, along with proteins involved in erbB signaling: Erbin, p35, and caveolin-3. In contrast, erbB3 was concentrated at pre-synaptic Schwann cells. This distribution argues that erbB2/erbB4 hetero-dimers are the post-synaptic neuregulin receptors. Neuregulin localized to the axon terminal, secondary folds, and Schwann cells. MuSK co-distributed with AChRs in the post-synaptic primary gutter. Agrin was present at the axon terminal and/or in the basal lamina of the primary gutter. The differential distributions of neuregulin/erbB receptors and agrin/MuSK argue against neuregulin and erbB receptors being localized via direct interactions with either agrin or MuSK. Sarcoglycan complex proteins were differentially distributed in the post-synaptic membrane.; Next, I studied the distribution of AChRs, erbB receptors, caveolin and proteins of the dystroglycan complex in cultured myotubes. There was no enrichment of erbB receptors at agrin-induced AChR clusters. Although AChRs in untreated myotubes appeared partially associated with caveolin-3, there was no enrichment of caveolin-3 at agrin-induced AChR clusters. α and β-sarcoglycan were enriched at AChR clusters while γ and δ-sarcoglycans were not.; Finally, I examined the ability of neuregulin to regulate AChR distribution. I investigated neuregulin's ability to act as a factor, which mediates synaptic competition by destabilizing AChR clusters. I showed neuregulin reduces the number of AChR clusters in myotubes and increases their rate of disassembly. Cluster assembly and disassembly both involved AChR internalization into caveolae. Time-lapse microscopy of individual AChRs on living myotubes demonstrated that neuregulin increases the rate at which individual AChR clusters disassemble. This thesis was performed in the laboratory of Dr. Jonathan Cohen. |
