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Please use this identifier to cite or link to this item: http://hdl.handle.net/1783.1/7186
Title: The assembly of the postsynaptic cytoskeletal scaffold in the development of the neuromuscular junction
Authors: Gong, Zhuolin
Issue Date: 2010
Abstract: During the development of the vertebrate neuromuscular junction (NMJ), muscle acetylcholine receptors (AChRs) are concentrated in the postsynaptic membrane in response to the activation of the muscle-specific tyrosine kinase MuSK by nerve-secreted agrin. The aggregation of muscle-surface AChRs is tightly regulated by intracellular tyrosine kinase signaling and occurs in an actin polymerization-dependent manner. Although intracellular signaling through many synaptic proteins has been intensively studied, the regulation of the assembly of the cytoskeletal scaffold at the developing NMJ remains poorly understood. Our group previously found that cortactin, a substrate of src-family tyrosine kinases and an actin regulator, was localized at AChR clusters in muscle cells. In this study, the role of cortactin in NMJ formation was therefore investigated, and agrin was found to enhance the tyrosine phosphorylation of cortactin at three src-target sites. The functional involvement of cortactin in actin-dependent AChR clustering was assayed in cells expressing exogenous cortactin constructs. The tyrosine-phosphorylation of cortactin was found to be important for AChR aggregaiton, since the mutation of the three src-target tyrosines or the depletion of endogenous cortactin in muscle inhibited the clustering of AChRs induced by synaptogenic stimuli. The expression of mutant cortactin in muscle cells also increased the mobile fraction of AChRs, suggesting that AChRs were poorly trapped by the cytoskeleton in these cells. This is consistent with a role of cortactin in promoting actin polymerization and the enhancement of this function by cortactin’s phosphorylation during AChR cluster assembly. Phosphorylation by src enables cortactin to interact with other actin regulators through the adapter protein Nck to potently stimulate polymerization. Because Nck is a key effector of cortactin, its role was examined in actin-dependent AChR clustering. Deletion of Nck’s SH2 domain, which binds to cortactin, suppressed agrin-stimulated AChR aggregation, and the down-regulation of Nck inhibited AChR clustering to a similar extent as the depletion of cortactin in muscle cells. The depletion of both Nck and cortactin did not enhance the inhibition of AChR clustering produced by reducing cortactin’s expression alone, suggesting that Nck functions in concert with cortactin to potentiate F-actin scaffold assembly for AChR aggregation. The postsynaptic F-actin scaffold allows AChRs to be anchored and concentrated by a diffusion-mediated trapping mechanism. The AChR-associated protein, rapsyn, is the best candidate linker of AChRs to the cytoskeleton, and rapsyn’s function was further studied here. Over-expression of rapsyn in muscle cells significantly decreased the fraction of mobile, non-clustered AChRs and led to the generation of AChR micro-clusters. Such rapsyn-induced AChR clustering was dependent of tyrosine kinase signaling and F-actin scaffold assembly, since the disruption of either interfered with the receptor clustering process. These findings suggest that rapsyn mediates tyrosine kinase cascade- and actin polymerization-dependent AChRs cluster formation. Collectively, my findings suggest that the assembly of a cytoskeletal scaffold by cortactin, Nck and other regulators of actin polymerization, the protein rapsyn, and the local, postsynaptic elevation of tyrosine phosphorylation, all work together to trap mobile AChRs and generate high-density AChR clusters at the NMJ.
Description: Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2010
xiv, 115 p. : ill. (some col.) ; 30 cm
HKUST Call Number: Thesis BIOL 2010 Gong
URI: http://hdl.handle.net/1783.1/7186
Appears in Collections:BIOL Doctoral Theses

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