Takahashi Noriko, Sawada Wakako, Noguchi Jun, Watanabe Satoshi, Ucar Hasan, Hayashi-Takagi Akiko, Yagishita Sho, Ohno Mitsuyo, Tokumaru Hiroshi, Kasai Haruo
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.
Nat Commun. 2015 Oct 6;6:8531. doi: 10.1038/ncomms9531.
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.
目前尚不清楚钙离子依赖性胞吐作用的准备状态如何取决于SNARE复合体组装的不同程度。在此，我们利用Förster共振能量转移（FRET）的双光子荧光寿命成像（FLIM），直接研究突触前终扣和胰岛中胰腺β细胞内三对神经元SNARE之间的SNARE组装。这些FRET探针在功能上拯救了它们的内源性对应物，支持超快速胞吐作用。我们发现反式SNARE复合体聚集在活性区，并估计了与每个停靠囊泡相关的复合体数量。相比之下，SNARE在静息状态下未组装，仅在胰岛素胞吐作用之前不久组装，而胰岛素胞吐作用进行得较慢。因此，我们证明了融合准备的不同状态与SNARE复合体形成有关。我们的FRET/FLIM方法能够对活组织和化学固定组织中的融合准备状态进行光学成像。
