The neuromuscular junction is a complex structure resulting from the interaction of an innervating neuron and skeletal muscle fiber. The neuron releases a molecule called agrin which acts via a muscle specific tyrosine kinase, MuSK, to initiate the localized differentiation and specialization of the muscle membrane at the synapse. A defining characteristic of the postsynaptic aspect neuromuscular junction is the concentration of acetylcholine receptors (AChRs) at the crests of junctional folds. Also clustered to these sites is the dystrophin associated protein (DAP) complex, a collection of proteins previously associated with muscular dystrophy and synapse formation. A central component of this complex, dystroglycan, had previously been shown to bind to agrin with high affinity. Inhibition studies that blocked agrin binding to dystroglycan indicated that dystroglycan might be required for synapse formation. In addition, several properties of dystroglycan, including its ability to co-cluster with the AChR and to bind both the neural and muscle forms of agrin, made it an obvious candidate as the putative co-receptor for MuSK. Based on these results we have attempted to define the role of dystroglycan in synaptogenesis. / We studied nerve muscle co-cultures derived from Xenopus embryos and found that dystroglycan is present at almost all neural agrin and AChR clusters. This holds true even in developing synapses as well as in extrasynaptic clusters of AChRs. AChR and dystroglycan aggregates can be induced in vitro, by treating myotubes with agrin and/or exogenous laminin. The AChR clusters formed by laminin application were larger and more dense then those formed by agrin treatments and could be inhibited by the addition of anti-laminin antiserum or laminin fragments that do not self-polymerize. In addition, laminin, unlike agrin, was found to induce AChR and dystroglycan clustering independent of MuSK activation. The introduction of an antisense dystroglycan construct into the C2C12 muscle cell line and the resulting reduction in expressed dystroglycan on myotubes also had little effect on phosphorylation but instead reduced the number of agrin induced AChR clusters on myotubes. These finding suggested that dystroglycan is not the co-receptor for MuSK but rather functions at a point in AChR clustering downstream of agrin-MuSK signaling. When similar experiments were conducted on dystroglycan null myotubes we found that these myotubes respond to agrin in a manner similar to wild-type myotubes but that the AChR clusters formed on null myotubes were two to three times larger, half as dense and significantly less stable. Synapses in chimeric mice with dystroglycan deficient muscle were similarly affected. In culture and in vivo the absence of dystroglycan also resulted in the disruption of laminin, perlecan and acetylcholinesterase localization to AChR clusters but did not affect rapsyn or agrin localization. Finally, I present unpublished observations that suggest that the close association of dystroglycan with rapsyn and AChRs may not result from a direct interaction between dystroglycan and rapsyn. From these results we propose that dystroglycan is a key element of a "trap" requir
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.36958 |
| Date | January 2000 |
| Creators | Jacobson, Christian B. |
| Contributors | Carbonetto, Salvator (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001808752, proquestno: NQ70049, Theses scanned by UMI/ProQuest. |
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