Tucker, P., 2011. A genetic dissection of actin regulation in Drosophila hemocytes. Thesis (Doctor of Philosophy (PhD)). University of Bath.
Cell migration is essential for embryonic development, it occurs in adult organisms during processes like wound healing and its misregulation contributes to pathological conditions such as metastasis. Despite this, most studies of cell migration have been undertaken in vitro. Ena/VASP proteins, believed to be actin anti-capping proteins, have been studied extensively in fibroblasts in vitro, and using Drosophila macrophages (hemocytes) within the developing embryo, the role of the Drosophila homologue of Mena, Ena, is investigated in vivo. Consistent with data from fibroblasts in vitro, Ena localised to regions of actin dynamics within migratory hemocytes, where this protein stimulated lamellipodial dynamics and positively regulated filopodial number and length. However, whilst overexpression of Ena/VASP proteins in fibroblasts reduced migration speeds, Ena overexpression in hemocytes dramatically increased migration speeds in three different assays. This positive regulation of migration speed closely resembled the increased motility of breast cancer cells that overexpress Mena and evidence presented here, suggests that this key difference may be explained by spatial constraints that are imposed upon cells within three dimensional environments. Indeed, such constraints prevented ruffling, a more detrimental form of retraction, in hemocytes in vivo. Furthermore, fibroblasts overexpressing Mena in vitro form membrane ruffles more frequently. Therefore Ena/VASP proteins drive migration by enhancing lamellipodial protrusion, but in certain environments these protrusions are lost as ruffles slowing migration. The method by which Ena regulates lamellipodial protrusion and migration speeds was then investigated: Ena increased Fascin-mediated actin bundling and the number of Fascin rich-actin bundles that coalesced. Analysis of individual actin bundles revealed that coalescence increased protrusion rate and that both protrusion rate and coalescence, increased cell migration speeds. This suggests that Ena facilitates an increase in cell migration by promoting the coalescence of Fascin bundles, and positions Ena as a key regulator of migration speeds in vivo.
|Item Type ||Thesis (Doctor of Philosophy (PhD))|
|Uncontrolled Keywords||cell migration,actin,ena,hemocytes,drosophila|
|Departments||Faculty of Science > Biology & Biochemistry|
|Publisher Statement||UnivBath_PhD_2012_P_K_Tucker.pdf: © The Author|
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