Dynamic analysis of actin protrusion assembly and function during Drosophila dorsal closure

• Main Author: Woolner, Sarah
• Published: University College London (University of London) 2005
• Subjects: 571.9466
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422328

The coordinated migration and fusion of epithelial sheets is employed on numerous occasions to shape the developing embryo and to repair tissues as part of the wound healing response. The morphogenetic episode of dorsal closure, whereby a large hole in the dorsal epithelium of the Drosophila embryo is drawn closed, provides a powerful model for the study of such cell movements because of its amenability to live imaging and the outstanding genetic tractability of Drosophila. During dorsal closure, the leading edge epithelial cells surrounding the hole extend dynamic actin protrusions - filopodia and lamellipodia - which reach across the exposed amnioserosa layer and help to zip the epithelial edges together. In this thesis I have employed live imaging to investigate the assembly of these protrusions and to explore their roles during the closure process. I have carried out a detailed live analysis of wild type dorsal closure, which has provided a valuable staging framework with which to compare defective closure in mutant embryos. To begin to understand more about how actin protrusions are assembled by the epithelial leading edge, I have investigated the function of the small GTPase, Rac, by ectopic expression of constitutively active and dominant negative forms of Race1 and also using a triple Rac loss-of-function mutant. The phenotypes I see in Rac mutant cells, where assembly of actin protrusions is either increased or reduced from wild type levels, suggest three roles for the actin protrusions during dorsal closure. Two of these are sensory, enabling leading edge cells to "find" their correct partner on the opposing epithelium, and to "read" contact inhibition cues once the two edges have met. The third is to function in priming cell-cell adhesion events as the two epithelial surfaces fuse. To begin to assess how the filopodia may fulfil these functions I have focused on a subset of unconventional myosins, which in other systems have been found to localise to filopodial tips. I show that one such myosin, Myo10A, localises to the tips of filopodia in cultured Drosophila cells, whilst its knockdown by RNA interference leads to intriguing dorsal closure defects, including segmental mismatching along the midline seam.