Fusion pore structure and dynamics - Project Summary/Abstract Membrane fusion is a fundamental process in eukaryotic cells, yet the precise molecular mechanism(s) by which proteins catalyze the merger of lipid bilayers has yet to be elucidated. We address the nanomechanics of membrane fusion reactions by focusing on the fusion of synaptic vesicles (SV) with the presynaptic plasma membrane in neurons. This fusion event results in the release of neurotransmitters and thus underlies neuronal function. In synapses, interactions between v- and t-SNAREs initiate the first step in the fusion reaction: the formation of the fusion pore. These are short-lived, channel-like structures that represent the first aqueous connection between the lumen of secretory vesicles and the extracellular space. To understand the biophysics of the fusion reaction, it is imperative to address the dynamics of SNARE complexes and the fusion pores that they form. In project 1, we leverage a novel system, innovated via our current R35 (that ends soon), that enabled the first µsec measurements of single, recombinant, reconstituted fusion pores. In this system, a fusion pore forms between a v-SNARE-bearing nanodisc (ND) and a black lipid membrane (BLM) that harbors t-SNAREs (ND-BLM system). The rigid membrane scaffold around the ND limits pore dilation and thus holds pores in a nascent state. Importantly, pores are interrogated electrophysiologically, and the high sampling rate revealed dynamics that had not been previously observed. This system enables us to quantitatively measure opening and closing rates, and to estimate pore size via unitary pore currents. We will continue this line of study, to: address the regulation of fusion pores by accessory proteins, relate structural transitions in SNAREs with kinetic transitions in fusion pores, create progressively larger NDs to quantitatively study the pore dilation step, determine the molecular basis for the cation selectivity of pores, and address the activation energy for pore opening. In project 2, we will determine the structure of fusion pores formed between v- and t-SNARE- bearing NDs, via cryo-EM. For this work, we utilize DNA nanostructures to obtain the required sample homogeneity needed for single-particle averaging. These DNA nanostructures will also be used to dictate the organization of SNARE proteins, so that we can infer the structure of the fusion pore via functional assays. Finally, in project 3, we extend our studies of fusion pores to study the modes of SV release in neurons. We will develop robust, rigorous, optical approaches to “size” the SV fusion pore using dyes with different radii. This work will directly address the controversy of whether SVs always undergo full fusion collapse (FFC), or if they sometimes undergo kiss-and-run (K&R) exocytosis in which the fusion pore closes prior to dilation and merger. K&R exocytosis, through a non-dilating fusion pore, would hinder glutamate efflux; this would have a major impact on the post synaptic response to dramatically impact information flow in the nervous system. In summary, our goals are to understand the dynamics, and determine the structure, of the SV fusion pore, and directly address the FFC versus K&R controversy.
