Proteomics of a neurotransmitter recycling domain in glia of the visual system

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DESCRIPTION (provided by applicant): Glia cells perform numerous specialized tasks that are required for the continued working of the nervous system. Two of the most prominent examples are the role of oligodendrocytes in the formation of myelin sheets and the contribution of astrocytes to the homeostasis of neurotransmitters. For the execution of such functions different glia cells elaborate many unique cellular compartments including myelin sheets or the 'astrocyte feet' onto endothelial cells along blood vessels. Despite the importance of these and other distinctive glial compartments, the understanding of their formation and function is still limited due to our incomplete knowledge of their components. Achieving this goal has been hampered by the difficulty of determining the proteins present in such complex glia compartments, which are not well recapitulated in cultured cells in vitro. Here, we propose the development of a novel proteomics tool for a systematic analysis of such glia compartments in vivo. The method is based on the locally restricted biotin-labeling of proteins present in differen compartments and their subsequent isolation and identification by mass spectroscopy. As a proof-of-principle model we are focusing on capitate projections, which are deep invaginations of glial cells into the nerve endings of photoreceptors in the Drosophila visual system. We have recently demonstrated a direct role of this glia compartment in neurotransmitter recycling. Using this model system we will in Aim 1 optimize the biotin-labeling of proteins present in this compartment in vivo and their subsequent identification by mass spectroscopy. Because for any new proteomics approach it is important to critically validate the identified candidate proteins, i Aim 2 we will focus on systemically testing the role of identified proteins in neurotransmitter recycling and in the formation of capitate projections. This analysis of the candidates and the evaluation of their roles in the function of capitate projections will then aid us in the further optimization of this proteomics approach towards understanding the composition and function of different glia compartments.