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Title: Structure-function analysis of the role of amphiphysin in T-tubule organisation in Drosophila
Author: Deshpande, A. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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To investigate how amphiphysin contributes to T-tubule organisation in Drosophila, I initiated a structure-function analysis of the protein in this process. I assayed the ability of different domains and mutant forms of the protein to rescue the amphiphysin null mutant phenotype. When either an N-terminal fragment that includes the BAR domain, or the SH3 domain, was expressed in an amphiphysin mutant background, there was no obvious rescue of the T-tubule defect. However, expression of full-length protein lacking the first 40 amino acids resulted in some rescue of reticular structures. When full-length amphiphysin, carrying a mutated SH3 domain that does not interact with praline-containing motifs, was expressed in amphiphysin mutants, tubular structures similar to those of wild type were seen, but they failed to localise normally. When full-length amphiphysin, carrying point mutations in the BAR domain that inhibit its binding and tabulating activity, was expressed in amphiphysin mutants, there was an partial rescue of the phenotype. Although amphiphysin is known to dimerise, neither an N-terminal region containing the BAR domain, nor the SH3 domain, had any detectable dominant negative phenotype in a wild type background. I also examined muscle development in embryos, to detect whether amphiphysin expression occurred before or after T-tubule formation. I examined the time course of amphiphysin and Dig (another T-tubule marker) expression in developing muscles. Amphiphysin appeared first on the plasma membrane and as intracellular puncta, then on a reticular network, presumably T-tubules. Dig could be detected on T-tubules only subsequently to this. Therefore, amphiphysin expression in muscles precedes the earliest time at which T-tubules can be detected with any markers available to me.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available