Regulatory role of the ubiquitin ligase Itch in endocytosis and synaptic vesicle recycling

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***Endocytosis is a fundamental cell process in which a cell absorbs a portion of its membrane as well as the proteins associated to it. This phenomenon plays a crucial role in the regulation of intracellular signalling by controlling the amount of cell-surface receptors. It is also closely linked to the maintenance of neuronal activity. When neurons are firing, excitatory neurotransmitters are released at the synapse by fusion of synaptic vesicles with the synaptic membrane. These vesicles are filled with neurotransmitters that are freed into the intercellular space upon fusion of the synaptic vesicles with the cell membrane. To ensure the maintenance of the membrane and of the synaptic vesicle pools, synaptic vesicles as well as their associated proteins must be recovered from the cell membrane by endocytosis. The molecular mechanisms controlling receptor internalization and synaptic vesicle recycling are very similar, and the regulatory proteins involved in both processes are the same. Endophilin is one of the proteins governing endocytosis and is essential for synaptic vesicle recycling. We discovered that endophilin is targeted by an enzyme called Itch that modifies endophilin by fusing it to ubiquitin, which is a signal for protein degradation. When we knocked down Itch in developing zebrafish embryos, the development was perturbed due to the misregulation of growth factor receptors, leading to an excess of their target genes and developmental defects; however, Endophilin is not the only target of Itch's activity, and it is not known how Itch really affects these receptors. Our research program is dedicated to the elucidation of regulatory mechanisms of synaptic vesicle recycling. To establish the contribution of Itch and Endophilin in this process, we propose to specifically interfere with the interaction between Itch and Endophilin, and thereby establish a potentially new regulatory mechanism of endocytosis and synaptic vesicle recycling. We will genetically alter somatic and neuronal cells to modify the gene coding the Itch protein in a way that it will no longer be able to recognize and modify endophilin, while leaving its ability to modify other substrates intact. We expect that blocking the interaction between Itch and endophilin will result in increased endophilin levels, perturbing receptor internalization and synaptic vesicle recycling. We will use different molecular and cell imaging techniques to measure the extent of these perturbations. These experiments will provide important fundamental knowledge on the fine-tuning of crucial processes in the maintenance of cell function and maybe provide new avenues in research for the treatment of cellular diseases such as cancer and neurodegeneration.**