Septins are a family of 14 cytoskeletal proteins that dynamically form 
hetero-oligomers and organize membrane microdomains for protein complexes. The 
previously reported interactions with SNARE proteins suggested the involvement 
of septins in exocytosis. However, the contradictory results of up- or 
down-regulation of septin-5 in various cells and mouse models or septin-4 in 
mice suggested either an inhibitory or a stimulatory role for these septins in 
exocytosis. The involvement of the ubiquitously expressed septin-2 or general 
septin polymerization in exocytosis has not been explored to date. Here, by 
nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in 
mouse brain, we identified not only SNARE proteins but also Munc-18-1 
(stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) 
(disassembles SNARE complexes after each membrane fusion event), and the 
chaperones Hsc70 and synucleins (maintain functional conformation of SNARE 
proteins after complex disassembly). Importantly, α-soluble NSF attachment 
protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE 
complex, did not interact with septin-2, indicating that septins undergo 
reorganization during each exocytosis cycle. Partial depletion of septin-2 by 
siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive 
and stimulated exocytosis of secreted and transmembrane proteins in various cell 
types. Forchlorfenuron impaired the interaction between SNAP-25 and its 
chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and 
inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor 
neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic 
reorganization of septin oligomers in exocytosis.