PROJECT SUMMARY Mast cell-derived TNF (tumor necrosis factor) plays important roles in immune responses, but when in excess, causes inflammatory disorders including asthma and rheumatoid arthritis. Unlike other TNF producing cells, mast cells pre-store TNF in cytoplasmic granules, which can undergo rapid exocytosis/degranulation under specific conditions. Mast cells can also be activated to promote TNF transcription, and newly synthesized TNF is released over time via constitutive secretory carriers such as Golgi-derived vesicles. Both regulated and constitutive TNF secretion require specific interactions between vesicle/granule-anchored SNAREs (v-SNAREs) and SNAREs anchored to the plasma membrane (the target membrane; hence t-SNAREs), resulting in the formation of trans-SNARE complexes and concomitant membrane fusion. Exocytic trans-SNARE complexes in immune cells are typically composed of a VAMP (v-SNARE), a SNAP23 (t-SNARE) and a syntaxin (t-SNARE). However, the identities of the SNAREs that underscore TNF release from activated mast cells are largely unknown, which has hindered the understanding of the stimulus-specific regulation of TNF exocytosis in these cells. The overall objective of this proposal is to uncover the missing v-SNAREs required in regulated and constitutive TNF secretion from primary mast cells. New evidence from our lab has suggested that TNF is associated with exosomes within MVBs (multivesicular bodies; a type of late endosomes), which rely on VAMP7 or Ykt6 (a unique, multi-purpose v-SNARE) to fuse with the plasma membrane. Meanwhile, VAMP2 has recently emerged as the v-SNARE that mediates the exocytosis of Golgi-derived vesicles. Based on these fresh insights, we hypothesize that mast cells employ VAMP7/Ykt6 and VAMP2 to release prestored and newly synthesized TNF respectively in response to different environmental cues. To test this hypothesis, we will exploit primary mast cells derived from KO mice and their wildtype littermates to pursue two specific aims. Aim1 will assess the requirement of VAMP7 and Ykt6 in acute/regulated TNF exocytosis. Aim2 will assess the requirement of VAMP2 in constitutive TNF exocytosis. The completion of this study will delineate the catalytic machineries that drive TNF exocytosis under different environmental conditions, setting a stage for revealing stimulus-specific regulation of TNF release unique to mast cells.