ABSTRACT Many vital cellular processes such as neuronal communication, insulin secretion and immune responses rely upon highly regulated fusion events between cargo containing vesicles and target membranes. All these processes require the assembly between vesicular (v-) SNARE and target (t-) SNARE proteins into a single SNARE complex, which brings the bilayers into close proximity and triggers their fusion. Sec1/Munc18 (SM) family proteins and calcium sensor bind to and modulate the function of specific sets of SNARE proteins in different cell types in many different ways not yet well understood. The goal of this project is to shed light into the mechanisms by which SM proteins and calcium sensors control granule exocytosis by focusing on the exocytosis of lytic granules (LGs) in cytotoxic t-cells as a model system. Insights into the molecular machinery that drives LG exocytosis emerged from genetic analyses of Familial Hemophagocytic Lymphohistiocytosis (FHL) patients in which LG exocytosis is impaired. With this completely novel approach we will exploit the slower kinetics, well-defined steps during LG exocytosis, and the higher energetic barrier imposed by the atypical lipid-anchored SNARE (STX11) involved in this fusion event, to dissect how SM proteins and calcium sensors control SNARE machinery during exocytosis. Using an in vitro “flipped” cell-cell fusion assay developed in our lab, we found that lipid-anchored STX11 mainly supports hemifusion. Strikingly, addition of Munc18-2 –a SM isoform of immune cells- promotes the transition from hemifusion to complete fusion suggesting that SM proteins has a direct role on membrane merging. We hypothesize that physiologically lipid- anchored STX11 mediates incomplete merging of LGs with the PM and requires an extra set of regulatory proteins – Munc18-2 and calcium sensors – for fusion pore opening and content release. To address these questions we will test: 1)- whether interactions of Munc18-2 with STX11 alone or with SNARE complexes stabilize and/or facilitate SNARE complex formation and drive membrane fusion. This will be done through a detailed protein:protein interaction analyses, functional “flipped” cell-cell fusion assays, and optical tweezers to assess the strength of forces on single SNAREs complex; 2)- how Munc18-2 controls STX11-mediated LG granule fusion at physiologically relevant immunological synapses of cultured cells and whether Munc13-4 and/or Synaptotagmin-7 confer calcium sensitivity to this process. To do this we will use TIRF-microsocpy in genetically-modified human CTLs, cell lines or FHL patient-derived cells that either lack these proteins or express functional mutant constructs. Answering these specific questions will provide insights into what SM functions are universal from those that dictate cell-type specific differences, how lipid-anchored SNAREs mediate fusion in general, how cells control a potentially dangerous exocytic event and may guide approaches to intervene ...