Abstract
Endocytic protein machinery is essential in several neuronal processes such as synaptic growth, cargo trafficking, synaptic vesicle recycling and exosome formation. In order to study these processes in vivo, it is necessary to tease apart protein-protein interactions and their domain-specific contributions to cell function. The focus of my thesis was the Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) domain protein, Nervous Wreck (Nwk), which binds to membranes and induces membrane curvature, and its vast array of binding partners. Nwk has also been shown to act as a negative regulator of growth upstream of the BMP signaling pathway. Previous work with Dap160/Intersectin, a Nwk binding partner, has shown that two SH3 (Src Homology 3) domains in Dap160, SH3CD, specifically interact with Nwk SH3b domain, and contribute to relief of autoinhibition in combination with WASp. The DapCD fragment of this protein was overexpressed and using immunohistochemical \r techniques, its effect on Nwk and the cell phenotype was investigated. Moreover, using tissue-specific tagging of endogenous proteins (TSTEP), five plasmid constructs were designed for Dap160, Nwk and Dynamin to uncouple these protein interactions in vivo. The present study shows that the DapCD fragment might facilitate localization of Nwk at the NMJ, and enhance its suppression of growth at the NMJ, providing a functional significance of domain interactions between Nwk and Dap160. Future studies, especially using the T-STEP fly lines, can provide more insight into other specific protein interactions within the endocytic machinery and their contribution to cell function at the Drosophila NMJ.