Fusion pores in endocrine and synaptic exocytosis

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PROJECT SUMMARY/ABSTRACT Neurons and endocrine cells release signaling molecules through Ca2+‐triggered exocytosis. Ca2+ enters a nerve terminal or endocrine cell, binds to a Ca2+ sensor protein, and triggers the fusion of vesicle and plasma membranes to expel neurotransmitters and hormones. To investigate the mechanisms of exocytosis our research focuses on fusion pores and Ca2+. Ca2+ triggers the opening and evolution of the fusion pore; the fusion pore is an aqueous passage between the vesicle interior and cell exterior. All secreted molecules pass through a fusion pore, which is strategically situated to exert finely tuned control over secretion. We use biophysical techniques to probe fusion pores at the single‐pore level, track their transitions, and monitor their responses to biological signals. Studies of the fusion pore have given us valuable insights into the roles of specific proteins in the control of exocytosis. We showed that SNARE protein transmembrane domains alter flux through initial fusion pores in both endocrine and synaptic exocytosis. We have made important advances in understanding the nascent fusion pores of endocrine exocytosis, but progress has been slow in understanding endocrine fusion pore expansion, and how fusion pores impact synaptic transmission. Innovations from this laboratory have created opportunities to take on these new challenges. Project 1. We have developed a new method for analyzing amperometric recordings to probe the dynamics of late‐stage endocrine fusion pores. This method tracks fusion pore permeability as vesicles lose catecholamine, and led to the novel findings that a fusion pore sequentially expands, contracts, and settles into a metastable state. We will use measurements of late‐ stage fusion pores to address long‐standing questions about the biological control of secretion. We will probe late‐ stage fusion pores for control by lipid bilayer elasticity, Ca2+, synaptotagmins, and synaptophysin/dynamin. Project 2. To study synaptic fusion pores we developed a co‐culture system with neurons and HEK293 cells expressing 4 postsynaptic proteins, neuroligin 1, GluA2, stargazin, and PSD95. These HEK cells serve as sensors of synaptic release, yielding miniature synaptic current data of exceptional quality in which fusion pore contributions are more clearly resolved. In parallel with Project 1, we will use HEK cell‐neuron co‐cultures to determine how synaptic fusion pores are controlled by bilayer elasticity, Ca2+, synaptotagmins, and synaptophysin/dynamin. The results on endocrine and synaptic fusion pores will be synthesized into a comprehensive framework for regulated secretion. We will then adapt this co‐culture system to the study of synaptic kiss‐and‐run and presynaptic contributions to synaptic plasticity. Project 3. We will adapt HEK cell synaptic sensors to the study of synaptic release from neurons derived from human stem cells. Collaborators have been recruited to provide neurons, which we will use to evaluate synaptic release and fusion pores in Down syndrome, fragile X mental retardation, aging, Parkinson's disease, and tuberous sclerosis complex. This work will provide insight into the molecular mechanisms of exocytosis, illuminate its molecular control, and show us how synaptic release goes awry in diseases.