# Exocytosis of Lytic granules

> **NIH NIH R01** · THOMAS JEFFERSON UNIVERSITY · 2021 · $306,150

## Abstract

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 ...

## Key facts

- **NIH application ID:** 10116419
- **Project number:** 5R01GM123020-06
- **Recipient organization:** THOMAS JEFFERSON UNIVERSITY
- **Principal Investigator:** Claudio Guillermo Giraudo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $306,150
- **Award type:** 5
- **Project period:** 2017-06-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10116419

## Citation

> US National Institutes of Health, RePORTER application 10116419, Exocytosis of Lytic granules (5R01GM123020-06). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10116419. Licensed CC0.

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