Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells.
Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells.
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DOI：
10.1038/ncomms9531
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发表时间：
2015-10-06
影响因子：
16.6
通讯作者：
Kasai H
中科院分区：
文献类型：
--
作者：
Takahashi N;Sawada W;Noguchi J;Watanabe S;Ucar H;Hayashi-Takagi A;Yagishita S;Ohno M;Tokumaru H;Kasai H
目前还不清楚如何准备Ca 2+依赖性胞吐依赖于不同程度的陷阱复合物组装。在这里，我们直接调查SNARE组装使用Förster共振能量转移（FRET）的双光子荧光寿命成像（FLIM）在突触前终末和胰岛β细胞的胰岛中的三对神经元SNARE之间。这些FRET探针在功能上拯救其内源性对应物，支持超快胞吐作用。我们发现，反式陷阱复合物积累在活性区，并估计与每个停靠囊泡的复合物的数量。相比之下，SNARE在静息状态下未组装，并且仅在胰岛素胞吐之前不久组装，其进展缓慢。因此，我们表明，不同的融合准备状态与陷阱复杂的形成。我们的FRET/FLIM方法能够在活体和化学固定组织中进行融合准备的光学成像。 突触囊泡在其快速释放之前保持在可融合状态，这被认为取决于反式SNARE复合物的形成。Takahashi等人使用FLIM/FRET直接对这种启动状态进行成像，并证明了神经元和β细胞之间基础SNARE组织的差异。
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. Synaptic vesicles are held in a fusion-competent state prior to their rapid release, which is thought to depend upon formation of trans-SNARE complexes. Takahashi et al. directly image this primed state using FLIM/FRET, and demonstrate differences in basal SNARE organization between neurons and β cells.