title: Dilation of fusion pores by crowding of SNARE proteins.
abstract: Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion).
authors.affiliation: Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, United States.|Nanobiology Institute, Yale University, West Haven, United States.
authors.forename: Zhenyong
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Nanobiology Institute, Yale University, West Haven, United States.|Department of Cell Biology, School of Medicine, Yale University, New Haven, United States.
authors.forename: Oscar D
authors.initials: OD
authors.lastname: Bello
authors.affiliation: Department of Physics, Columbia University, New York, United States.
authors.forename: Sathish
authors.identifier: 0000-0001-8545-8886
authors.initials: S
authors.lastname: Thiyagarajan
authors.affiliation: Nanobiology Institute, Yale University, West Haven, United States.|Department of Cell Biology, School of Medicine, Yale University, New Haven, United States.
authors.forename: Sarah Marie
authors.initials: SM
authors.lastname: Auclair
authors.affiliation: Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, United States.|Nanobiology Institute, Yale University, West Haven, United States.
authors.forename: Wensi
authors.initials: W
authors.lastname: Vennekate
authors.affiliation: Nanobiology Institute, Yale University, West Haven, United States.|Department of Cell Biology, School of Medicine, Yale University, New Haven, United States.
authors.forename: Shyam S
authors.identifier: 0000-0001-6148-3251
authors.initials: SS
authors.lastname: Krishnakumar
authors.affiliation: Department of Chemical Engineering, Columbia University, New York, United States.
authors.forename: Ben
authors.initials: B
authors.lastname: O'Shaughnessy
authors.affiliation: Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, United States.|Nanobiology Institute, Yale University, West Haven, United States.|Molecular Biophysics and Biochemistry, Yale University, New Haven, United States.|Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique, Paris, France.
authors.forename: Erdem
authors.identifier: 0000-0002-5934-8728
authors.initials: E
authors.lastname: Karatekin
mesh_terms: D005089:Exocytosis; D006367:HeLa Cells; D006728:Hormones / Q000378:metabolism; D006801:Humans; D018377:Neurotransmitter Agents / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism*
chemical_list: D006728:Hormones; D018377:Neurotransmitter Agents; D050600:SNARE Proteins
keywords: E. coli; SNARE proteins; biophysics; cell biology; exocytosis; fusion pore; membrane fusion; protein crowding; structural biology