Mechanisms of Lytic Granules Exocytosis

Membrane fusion is a fundamental biological process, and imbalances in its regulation give rise to important diseases, such as diabetes, brain disorders and immunodeficiency. All these processes rely upon the assembly between vesicular SNARE and target SNARE proteins into a single SNARE complex, which brings the bilayers into close proximity and triggers their fusion. Munc13 and the Sec1/Munc18 (SM) family proteins 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 proposal is to study regulation of exocytosis from a completely novel angle by focusing on the exocytosis of lytic granules (LGs) in cytotoxic t-cells as a model system. We will exploit the slower kinetics of the LG exocytosis, well-defined fusion steps, higher energetic barrier imposed by the atypical lipid-anchored SNARE (STX11) involved in this fusion event, and the less complex domain- structure of Munc13-4 to dissect different functions of Munc13 and SM proteins. Using our unique liposome- liposome fusion assays developed in our lab, we established that lipid-anchored STX11-SNARE complex is unable to support fusion by itself since STX11 adopt a “closed” conformation involving its N-terminal domain, most likely folding back onto the SNARE domain. Preliminary data show that addition of Munc18-2 and Munc13-4 to the assay cooperatively promotes membrane fusion and that topological membrane constrains and DAG levels modulate Munc13-4 activity in a Ca2+-dependent manner. Thus, implying a direct role of both proteins on membrane merging as we recently predicted from studies of dominant-negative Munc18-2 mutants found in f-HLH patients. Based on these findings we hypothesize that physiologically lipid-anchored STX11 is constitutively autoinhibited and that a series of interactions with Munc18-2 and Munc13-4 induce conformational changes that relieve STX11 of this inhibition and promote membrane fusion and LG content release. To address these questions we will: 1)- establish the mechanism by which Munc13-4 and Munc18-2 control STX11-SNARE complex formation and drive membrane fusion through a detailed analyses of protein:protein/ protein:lipids interaction and functional analysis using liposome fusion assays; 2) Define structure-function requirements of Munc13-4/Munc18-2 and STX11-SNARE complexes by using cryo-EM, SPR and optical tweezers to assess the strength of forces on single SNAREs complex; 3) Establish the role of different structural domains of Munc13-4 during LG granule maturation and exocytosis at the synpase by using superresolution au-STED and TIRF microscopy in patient cells, genetically-modified CTLs. Answering these questions will provide insights into what functions of Munc13 and SM proteins are universal from those that dictate cell-type specific differences. Moreover, while exploiting a genetic disease to dissect a basic biological process, our proposal will also provide critical insights into how defects in exocytosis contribute to disease. Project Number: 1R01GM159406-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Claudio Giraudo | Institution: THOMAS JEFFERSON UNIVERSITY, PHILADELPHIA, PA | Award Amount: $567,411 | Activity Code: R01 | Study Section: Cell Structure and Function 1 Study Section[CSF1] View on NIH RePORTER: https://reporter.nih.gov/project-details/11315431