How membrane proteins are targeted to dense-core and synaptic vesicles - Project Summary/Abstract Neurons and neuroendocrine cells release neurotransmitters, neuromodulators, and hormones by calcium- triggered exocytosis of dense-core and synaptic vesicles. Synaptic and dense-core vesicles share the same core membrane proteins that mediate exocytosis, but they arise from different parts of the secretory pathway and otherwise have different proteomes. There is an enormous literature on how these proteins govern membrane fusion, but how they initially reach their different target vesicles is largely a mystery. What determines whether a membrane protein goes to dense-core vesicles, synaptic vesicles, or both, and how do they get there? In this F32 proposal, I will investigate two key questions on this topic in rodent neurons and neuroendocrine cells. In Aim 1, I will determine what sequence(s) within a membrane protein are recognized to target it to dense core vs synaptic vesicles. The synaptotagmin family of calcium sensors will be test cargoes, since despite being structurally very similar, some isoforms go to both dense-core and synaptic vesicles, while others only go to dense-core vesicles. To identify what parts of the protein dictate targeting, I will create chimeras between synaptotagmin isoforms that traffic to different vesicles and see where they localize. In Aim 2, I will investigate what route through the secretory pathway membrane proteins take to reach dense-core vs. synaptic vesicles. There is consensus that synaptic vesicles are created and loaded with cargo via endocytosis from the plasma membrane and sorting at endosomes. By contrast, it is completely unknown how membrane proteins traffic to dense-core vesicles. I will test whether membrane proteins similarly transit through the plasma membrane en route to dense-core vesicles, or if they take a different pathway, using a cutting-edge HaloTag surface labeling strategy. This research will address basic but unanswered questions about how essential proteins traffic to neurosecretory vesicles. Aberrant sorting of proteins to dense-core and synaptic vesicles contributes to various neurological and metabolic disorders, ranging from psychiatric disorders to neurodegeneration to type 2 diabetes. The discoveries resulting from this work will serve as a foundation to understand how the physiology of protein trafficking goes awry in disease. Supported by the NINDS NRSA F32, this research will be part of a mentored research training plan with my sponsor, Dr. Edwin Chapman, in the Department or Neuroscience at University of Wisconsin-Madison. The Chapman lab, and UW-Madison as a whole, is an ideal place to execute my proposed research training plan, with state-of-the-art equipment and other leaders in my field to serve as collaborators and mentors. On the path to becoming a successful research professor, my training plan includes developing my light microscopy and biochemistry skills, growing in my knowledge of secretory organelle biogenesis and protein trafficking in neurons, and through career development acquiring all the tools I need to excel as future faculty. This will form a path to independence with my own lab studying the biogenesis of neurosecretory vesicles.
