TRIM67 as a novel regulator of exocytosis in developing neurons

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PROJECT SUMMARY/ABSTRACT The expansion of the plasma membrane is a critical, continuous process in the developing neuron. The addition of membrane material primarily occurs through the secretory system, in which secretory vesicles are transported to and fuse with the plasma membrane. These vesicles undergo SNARE-complex mediated fusion, in which a v-SNARE on the vesicle and t-SNAREs on the plasma membrane interact and fuse the vesicular membrane and plasma membrane together, opening a fusion pore and expelling vesicular cargo. Presumably, membrane addition occurs primarily through the classic mechanism of exocytosis, full-vesicle fusion (FVF), in which the fusion pore dilates and the vesicle collapses into the plasma membrane. During kiss-and-run (KNR) fusion, however, the fusion pore closes and the vesicle remains intact. The high temporal and spatial resolution of imaging needed to capture these events has been a roadblock in differentiating between these two mechanisms of fusion. Using my newly developed image analysis software, which automatically detects and characterizes exocytic events, I have been able to discern between two modes of exocytosis in developing neurons, which are consistent with FVF and KNR. Furthermore, the E3 ubiquitin ligase, TRIM67, biases the mode of exocytosis away from KNR and toward FVF. Our lab has previously shown that the guidance cue netrin-1 increases the frequency of exocytosis in mouse cortical neurons in vitro. Netrin-1 and its receptor DCC also regulate axon guidance, with axons turning toward higher concentrations of netrin-1. The asymmetrical addition of plasma membrane is hypothesized to underlie this turning and guidance of growing axons, which is expected to primarily occur through FVF. Preliminary findings suggest that TRIM67 is necessary for a netrin-1- depedendent increase in the frequency of exocytosis. This proposal will test the hypotheses that TRIM67 biases the mode of fusion toward FVF and that TRIM67 is involved in axon turning in response to netrin-1 through its ability to promote FVF. Our first aim will be to confirm that the two modes of exocytosis revealed by the automated image analysis are genuine FVF and KNR events and to identify the domains of TRIM67 necessary for biasing exocytosis toward FVF and the domains necessary for the netrin-1 response. Our second aim will determine how a gradient of netrin affects the spatial occurrence of exocytosis in the growth cone, to identify whether this exocytic response is disrupted in Trim67-/- growth cones and to identify axon guidance defects in Trim67-/- neurons using a novel microfluidic chamber.