Defining the regulation of exocytosis in developing neurons

About This Grant

ABSTRACT During brain development, neurons undergo dramatic growth and branching to establish the intricate morphology required for neural network connectivity and function. This cellular expansion involves a substantial and rapid increase in neuronal surface area and therefore requires extensive insertion of material into the plasma membrane. Defects in neuronal morphogenesis can result in improper synaptic connectivity, neurodevelopmental disorders, and psychiatric syndromes. Insertion of membrane material is facilitated by SNARE-mediated exocytosis. Two SNARE proteins, Vesicle-associated membrane protein (VAMP) 2 and VAMP7, are enriched in the embryonic brain and are associated with distinct vesicle populations even prior to synapse formation. While knockout studies in mice have shown that both VAMP2 and VAMP7-mediated vesicle fusion are required for proper neuronal morphogenesis, the specific mechanisms regulating the trafficking and fusion of these vesicles during development is not known. My preliminary data reveal that VAMP2 and VAMP7 mediate non-synaptic exocytic events that cluster in different areas of the developing neuron, suggesting the existence of distinct regulatory pathways governing their distribution and fusion. Additionally, my preliminary data indicate that VAMP2-mediated exocytosis is sensitive to Ca2+ chelation, whereas VAMP7-mediated exocytosis is not, mirroring the differential Ca2+ sensitivity of VAMP2 and VAMP7 vesicle pools observed at the synapse of mature neurons. This suggests a potential role for Ca2+ signaling in the regulation of VAMP2 and VAMP7- mediated exocytosis during neuronal morphogenesis. Endoplasmic reticulum (ER)-PM membrane contact sites, key regulators of Ca2+ signaling, may also be involved in this regulatory process. The goal of this proposal is to investigate the regulation of VAMP2 and VAMP7-mediated exocytosis during neuronal morphogenesis using hypothesis driven science coupled with unbiased proteomic analysis. I will utilize total internal reflection fluorescence (TIRF) microscopy and advanced image analysis techniques to quantitatively define the interplay between SNARE-mediated exocytosis, Ca2+ signaling, and ER-PM membrane contact sites during neurite outgrowth in stage 2 developing neurons. Using a proximity biotinylation approach I will define the interactome of VAMP2 and VAMP7 during neuronal morphogenesis, providing insights into the molecular machinery regulating their trafficking and fusion. These findings will advance our understanding of the complex regulation of exocytic membrane trafficking during neuronal development. Successful completion of this proposed research will provide advanced training in live cell microscopy, the development of advanced image analysis pipelines, proteomics, and bioinformatics.