Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells

Noriko Takahashi, Wakako Sawada, Jun Noguchi, Satoshi Watanabe, Hasan Ucar, Akiko Hayashi-Takagi, Sho Yagishita, Mitsuyo Ohno, Hiroshi Tokumaru and Haruo Kasai ()
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Noriko Takahashi: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Wakako Sawada: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Jun Noguchi: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Satoshi Watanabe: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Hasan Ucar: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Akiko Hayashi-Takagi: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Sho Yagishita: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Mitsuyo Ohno: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku
Hiroshi Tokumaru: Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University
Haruo Kasai: Faculty of Medicine, Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, University of Tokyo, Bunkyo-ku

Nature Communications, 2015, vol. 6, issue 1, 1-15

Abstract: Abstract It remains unclear how readiness for Ca2+-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.

Date: 2015
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DOI: 10.1038/ncomms9531

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