title: Septin dynamics are essential for exocytosis.
abstract: 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.
authors.affiliation: From the Departments of Physiology and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073.
authors.forename: Elmira
authors.initials: E
authors.lastname: Tokhtaeva
authors.affiliation: The Neuropsychiatric Institute-Semel Institute, Pasarow Mass Spectrometry Laboratory, UCLA, Los Angeles, California 90024.
authors.forename: Joe
authors.initials: J
authors.lastname: Capri
authors.affiliation: Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073, Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Marcus
authors.affiliation: The Neuropsychiatric Institute-Semel Institute, Pasarow Mass Spectrometry Laboratory, UCLA, Los Angeles, California 90024.
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center of the Russian Academy of Sciences, Kazan 420111, Russia, Kazan Federal University, Kazan 420008, Russia.
authors.forename: Venera
authors.initials: V
authors.lastname: Khuzakhmetova
authors.affiliation: Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center of the Russian Academy of Sciences, Kazan 420111, Russia, Kazan Federal University, Kazan 420008, Russia.
authors.forename: Ellya
authors.initials: E
authors.lastname: Bukharaeva
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, and.
authors.forename: Nimrod
authors.initials: N
authors.lastname: Deiss-Yehiely
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, and.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Dada
authors.affiliation: From the Departments of Physiology and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073.
authors.forename: George
authors.initials: G
authors.lastname: Sachs
authors.affiliation: Department of Pathology, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109.
authors.forename: Ester
authors.initials: E
authors.lastname: Fernandez-Salas
authors.affiliation: From the Departments of Physiology and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073, olgav@ucla.edu.
authors.forename: Olga
authors.initials: O
authors.lastname: Vagin
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004285:Dogs; D005089:Exocytosis; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D016716:PC12 Cells; D010671:Phenylurea Compounds / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D051381:Rats; D058112:Septins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism
chemical_list: D010671:Phenylurea Compounds; D020543:Proteome; D011725:Pyridines; D050825:Synaptosomal-Associated Protein 25; C106863:Sept2 protein, mouse; D058112:Septins; C116093:N-(2-chloro-4-pyridyl)-N'-phenylurea
keywords: Cell Biology; Cytoskeleton; Exocytosis; Membrane Fusion; Membrane Trafficking; Protein Secretion; Septins