Mechanism of transport of secretory vesicles in regulated secretory pathway

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DESCRIPTION (provided by applicant): Obliteration of regulated secretion of hormones, neuropeptides, and neurotrophins leads to disorders in children, such as diabetes, obesity, and deficits in neural development, memory, as well as learning. Thus, elucidation of the mechanism for secretory vesicle transport in the regulated secretory pathway (RSP) will facilitate development of new approaches to treat such diseases. The candidate found that post-Golgi transport of newly synthesized BDNF/ACTH secretory vesicles from the cell body of neurons and endocrine cells to the release site at axonal/dendritic terminals or plasma membrane requires interaction among vesicular carboxypeptidase E (CPE) cytoplasmic tail, snapin, and microtubule motors. It was also found that snapin interacted with actins, which are known to be involved along with myosin V in the transport of peptidergic secretory vesicles in an activity-dependent manner, in the terminal stages of secretion. The candidate hypothesizes that snapin-CPE tail interacting proteins govern the anterograde transport of RSP vesicles along neurites, and are also involved in capturing these vesicles from the microtubule transport platform onto the actin network at the terminals upon stimulation. Thus, the primary research goal is to identify, in endocrine cells and neurons, CPE-snapin-interacting proteins (a) that regulate the anterograde transport of RSP vesicles along processes/neurites in the regulated secretory pathway; and (b) that tether/capture these vesicles at the terminals upon stimulation and moves them to the release site for activity-dependent secretion of their cargo. The research proposed in Specific Aim 1 is designed to identify proteins necessary for post-Golgi transport of ACTH and BDNF vesicles along microtubules. Proteins that interact with snapin-CPE-motor will be purified by chromatography and analyzed for their function using microtubule pulldown and live cell imaging assays. In Specific Aim 2, he will purify and identify CPE-snapin associated proteins involved in tethering peptidergic vesicles to the actin network at the plasma membrane for activity-dependent secretion. CPE-snapin-interacting proteins will be assayed for enhancement of tethering of ACTH vesicles to actin with stimulation using live cell imaging. The proposed studies will uncover the molecular mechanism for regulation of ACTH/BDNF vesicle transport in (neuro)endocrine systems and potential treatments of diseases associated with the disruption of the mechanism.