Waller, Andrew Philip (2010)
Ph.D. thesis, University of Birmingham.
Cells depend on the actin cytoskeleton and the structures it forms for a wide range of processes. IRSp53 acts downstream of cytoskeleton master regulators Rac and Cdc42 in the formation of lamellipodia and filopodia, respectively. IRSp53 interacts with many other effectors in the formation of these structures, via various protein interaction domains. IRSp53 also contains an IMD (IRSp53/MIM homology domain), which is able to induce formation of filopodia-like structures when overexpressed in cells. Early reports suggested that the IMD exerts its effect by bundling actin filaments. However, the structure of the IMD revealed that it is related to BAR domains, which can induce membrane curvature by binding to membranes. It is still unsure whether IMD activity is dependent on actin or membrane interactions, or both. Data are presented here showing that actin destabilizing drug cytochalasin D prevents extension of IMD protrusions, suggesting a role for actin in this process. Fluorescence recovery after photobleaching experiments suggest that a large proportion of IMD is stably associated with the protrusions. Finally, in vitro lipid binding experiments suggest that IMD binds to membranes by mainly electrostatic interactions. These findings suggest that IMD activity may depend on interactions with both actin filaments and membranes.
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