Research Abstract


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

2015年10月6日 Nature Communications 6 : 8531 doi: 10.1038/ncomms9531



Noriko Takahashi, Wakako Sawada, Jun Noguchi, Satoshi Watanabe, Hasan Ucar, Akiko Hayashi-Takagi, Sho Yagishita, Mitsuyo Ohno, Hiroshi Tokumaru & Haruo Kasai

Corresponding Author

河西 春郎
東京大学大学院医学系研究科 附属疾患生命工学センター 構造生理学部門

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.