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Contribution of planar cell polarity to dorsal closure in drosophila

Contribution of planar cell polarity to dorsal closure in drosophila

Objective

Dorsal closure is a well studied morphogenetic process of Drosophilae embryo genesis during which the lateral epidennis moves dorsally, driven in part by dynamic acting protrusions of a specialised population of epidermal cells, the leading edge (LE) cells. Recently, a molecular map of the epidermal cells has revealed that LE cells are polarised in the plane of the epithelium. This polarisation reflects planar cell polarity (PCP) and genetic studies have suggested an involvement of PCP genes in the organisation and dynamics of the cytoskeleton in the LE cells during dorsal closure. Here I propose to study this involvement, focussing on the requirements and interactions between the PCP genes frizzled, dishevelled, flamingo and strabismus and their products. I shall do this by using studies of intracellular protein localisation to refine the molecular map of these proteins in LE cells and analyse their functional relationships through genetic analysis. I will also probe the molecular basis of interactions between these proteins by studying the contribution of their different domains to the generation of asymmetry and to their likely mutually dependent localisation. Finally, in an attempt to identify new components involved in the PCP process I will analyse complexes contributing to polarisation of LE cells using proteomic approaches.
The combination of these experiments will provide a mechanical explanation for the establishment of polarity in the LE cells during dorsal closure that is likely to be relevant to other situations involving PCP.In addition it should provide insights into how PCP is transformed into acting dynamics. The study of intracellular proteins localisation in LE cells will be achieved by confocal imaging, either on fixed embryos stained with antibodies, or on live embryos expressing fluorescent fusion proteins under the Ga14/UAS.system. The imaging of live embryos will also include time-laps movies of their development. I will use molecular cloning to generate -GFP or -CFP fusion proteins with PCP products, which will be expressed under the control of UAS sites.
Those constructs will be either injected to generate transgenic fly lines or used for transient expression assays in the embryo. A LE cells specific Ga14 driver fly line will also be generated. Finally, proteomic approaches will enable the analysis of complexes contributing to polarisation of LE cells. In addition, the overall project will involve classical genetic techniques. This project will allow me to learn and get familiar with the confocal imaging, transient expression assays and proteomic techniques while I will use my experience in genetic, generation of transgenic lines and molecular cloning. It will allow me to introduce the proteomic techniques, available at the University of Cambridge, in the laboratory.

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Coordinator

University of Cambridge

Address

Downing Street
Cb2 3eh Cambridge

United Kingdom

Administrative Contact

Stephen KELLEHER (Mr)

Project information

Grant agreement ID: HPMF-CT-2002-01937

  • Start date

    1 September 2002

  • End date

    31 August 2004

Funded under:

FP5-HUMAN POTENTIAL

  • Overall budget:

    € 114 072

  • EU contribution

    € 114 072

Coordinated by:

University of Cambridge

United Kingdom