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Identification and analysis of novel proteins required for Wingless secretion

Final Report Summary - WINGLESS SECRETION (Identification and analysis of novel proteins required for Wingless secretion.)

Intercellular communication is essential for embryonic development and homeostasis of multicellular organisms. Signalling proteins, such as Wnts, spread within tissues to coordinate cell growth, differentiation and survival. Wnts, including Wingless (the main Drosophila Wnt) are lipoglycoproteins that tightly associate with cellular membranes, yet they can act some distance from their site of production. Several genes have been identified to be essential for Wingless secretion. One of them is evenness-interrupted (evi), also called wntless or sprinter. Evi is a multipass transmembrane protein that directly binds Wg and is thought to escort Wg from the Golgi to the plasma membrane. We and others have shown that the Retromer complex, which transports cargo from endosomes to the Golgi, is also essential for Wg secretion. According to a current model, Evi is endocytosed and recycled to the Golgi by Retromer. In the absence of Retromer, Evi is trafficked to the lysosome and degraded, and Wg fails to reach the plasma membrane. The main objective of this project has been to determine how membrane-associated Wingless can act over a distance. Our working hypothesis is that distinct cofactors or organelles are required to ensure that Wg is released from secreting cells an allowed to spread in the extracellular space and reach other cells. Specifically, our goals are:
1. To define the pathway of Wingless secretion and site of export.
2. To identify novel proteins required for Wingless secretion and release.

To determine the pathway of Wingless secretion we have used BAC technology and recombineering to create a tagged form of Wingless that is expressed at endogenous levels to use for immuno-EM analysis. This is has been generated and we have obtained the first EM images of tagged Wingless. This approach will continue to be pursued in collaboration with Mark Marsh LMCB (London), a collaboration that was initiated as a result of the grant. We have also developed the tools that allow us to trigger a pulse of Wingless secretion to determine the route taken by Wingless during secretion at high temporal and spatial resolution.

To identify novel proteins required for Wingless secretion and release we have taken a genetic and a biochemical approach. The genetic approach made use of RNAi lines generated by the VDRC (Vienna) and identified several candidate genes that could contribute to Wingless secretion. These will be cross-referenced with the result of the following biochemical approach. We have initiated a collaboration with Dr. Solange Monier and Dr. Roland LeBorgne at the University of Rennes to analyse the state of Wingless by Drosophila cultured cells. Again, this collaboration would not have been possible without the grant. We have found that some Wingless is present on exosome, small membranous vesicles that are produced by a variety of cells, particularly cancer cells. We have begun to analyse the composition of these exosomes by mass spectrometry and have designed functional assays to determine the role of these components both in the spread of Wingless in vivo and in the formation of exosomes in cell culture. In parallel, we have created genetic tools to study the role of two proteins that have been suggested to participate in exosome function in mammalian cells (rab27 and rab35).

On the whole this grant has allowed the main researcher to acquire new skills in cell biology and biochemistry. It has also triggered two important collaborations. Progress has been slower than expected but important tools have been generated during the course of the grant and these will be capitalised upon in the next few years. Therefore, it is still our hope that this work will lead to a greater understanding of the mechanisms that control Wnt signalling. As this signalling pathway is often deregulated in cancer, the result of our investigation could have health benefits in the long term.

A general view of the project can be seen on http://www.nim