Defining the Substrates and Interactome of Focal Adhesion Kinase that Regulate Exocytic Vesicle Fusion

PROJECT SUMMARY Developing neurons achieve a complex, elongated morphology, involving substantial increases in plasma membrane surface area that far exceed the accompanying growth in cytoplasmic volume. We have previously shown that during exocytosis, the fusion of secretory vesicles provides sufficient membrane material for developmental plasma membrane expansion. Similar to the synapse, the synaptic SNARE proteins, VAMP2, syntaxin1, and SNAP25 are present in developing neurons and mediate vesicle fusion. At the synapse, several synaptic vesicles are tethered to the presynaptic plasma membrane, representing the readily releasable pool. Their synchronous fusion and release of neurotransmitter is evoked by a Ca++ spike. In contrast, in developing neurons, our studies have demonstrated that fusion is not synchronous, and single vesicle fusion events occur in what has previously been considered a constitutive manner. However, we recently reported a surprising requirement for the activity of the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in promoting vesicle fusion, in particularly during the transition between SNARE complex formation and vesicle fusion. The goal of this proposal is to investigate how FAK scaffolding function and/or FAK kinase activity regulates SNARE complexes and exocytosis. Our work utilizes neuronal cultures, multiple modes of light microscopy to visualize single vesicle fusion events at high spatial and temporal resolution, and quantitative mass spectrometry. Completion of this work will provide fundamental information about regulation of exocytosis during neuronal development.