PROJECT SUMMARY/ABSTRACT During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE complexes catalyze fusion, the structure and composition of fusion pores remain unknown. This is the central question in the field of membrane fusion, as the mechanism of fusion cannot be solved until the structure of the first key intermediate in this pathway, the fusion pore, has been elucidated. The main objective of this proposal is to gain new insights into fusion pore composition, structure, and dynamics, using both reconstitution and cell-based approaches. A major limitation in the biochemical study of fusion pores in cells concerns their low abundance and ephemeral nature. For example, in neuroendocrine cells, the duration of the initial open state of the fusion pore is of the order of msec; the pore then either closes (kiss-and-run exocytosis), or dilates to yield full fusion. To overcome this limitation, we have begun to study fusion pore structure, in vitro, by exploiting the rigid framework of nanodiscs. SNARE-bearing proteoliposomes dock and fuse with nanodiscs that harbor cognate SNAREs. Since nanodiscs are bounded by membrane scaffolding proteins, the pores cannot dilate, and hence can be studied biochemically. Using this system, we have begun to interrogate the properties of reconstituted fusion pores. Our preliminary data indicate that, contrary to the common view that fusion pores are purely lipidic, they are in fact hybrid structures, composed of both lipids and proteins. We will use a simulation approach to derive a new model for fusion pore structure, and conduct cryo-electron microscopy studies to visualize this structure. We will also use a variety of cargos of varying diameter, in conjunction with optical sensors that report their release during fusion, to determine the size of the pore, and to determine whether pore diameter is `plastic' and varies with the number of SNARE proteins. We will also probe for interactions between cargo and SNARE transmembrane domains by exploiting electrostatic interactions between them. The nanodisc system will be adapted to single molecule studies, to monitor pore opening and closing of individual pores in real-time, and to directly assess the impact of regulatory factors on pore stability. These in vitro experiments will be complimented by our ongoing direct measurements of fusion pores in chromaffin cells, using carbon fiber amperometry, by comparing the effects of SNARE mutations in these two systems. We will draw parallels between these systems so that we can arrive at unified, physiologically relevant models for pores. Finally, we will also design novel optical probes, based on pH sensitive dyes conjugated to quantum dots, to study fusion pores in cultured neurons. These latter studies will address the highly controversial topic of kiss-and-run exocytosis versus full fusion. Together, the work described here will provide unparalleled comparisons between in vitro and cell based observations, and will reveal new insights in the first crucial intermediate in the exocytotic pathway: the enigmatic fusion pore.