title: Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells.
abstract: It remains unclear how readiness for Ca(2+)-dependent exocytosis depends on varying degrees of SNARE complex assembly. Here we directly investigate the SNARE assembly using two-photon fluorescence lifetime imaging (FLIM) of Förster resonance energy transfer (FRET) between three pairs of neuronal SNAREs in presynaptic boutons and pancreatic β cells in the islets of Langerhans. These FRET probes functionally rescue their endogenous counterparts, supporting ultrafast exocytosis. We show that trans-SNARE complexes accumulated in the active zone, and estimate the number of complexes associated with each docked vesicle. In contrast, SNAREs were unassembled in resting state, and assembled only shortly prior to insulin exocytosis, which proceeds slowly. We thus demonstrate that distinct states of fusion readiness are associated with SNARE complex formation. Our FRET/FLIM approaches enable optical imaging of fusion readiness in both live and chemically fixed tissues.
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Takahashi
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Wakako
authors.initials: W
authors.lastname: Sawada
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Noguchi
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Hasan
authors.initials: H
authors.lastname: Ucar
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Hayashi-Takagi
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Yagishita
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Mitsuyo
authors.initials: M
authors.lastname: Ohno
authors.affiliation: Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, Kagawa 769-2193, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumaru
authors.affiliation: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Haruo
authors.initials: H
authors.lastname: Kasai
mesh_terms: D000818:Animals; D005089:Exocytosis; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D050417:Insulin-Secreting Cells / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D061848:Optical Imaging / Q000379:methods*; D017729:Presynaptic Terminals / Q000737:chemistry* / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D050600:SNARE Proteins