Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Cell Metab. 2010 Jul 7;12(1):19-29. doi: 10.1016/j.cmet.2010.05.013.
When cells release hormones and neurotransmitters through exocytosis, cytosolic Ca(2+) triggers the fusion of secretory vesicles with the plasma membrane. It is well known that this fusion requires assembly of a SNARE protein complex. However, the timing of SNARE assembly relative to vesicle fusion--essential for understanding exocytosis--has not been demonstrated. To investigate this timing, we constructed a probe that detects the assembly of two plasma membrane SNAREs, SNAP25 and syntaxin-1A, through fluorescence resonance energy transfer (FRET). With two-photon imaging, we simultaneously measured FRET signals and insulin exocytosis in beta cells from the pancreatic islet of Langerhans. In some regions of the cell, we found that the SNARE complex was preassembled, which enabled rapid exocytosis. In other regions, SNARE assembly followed Ca(2+) influx, and exocytosis was slower. Thus, SNARE proteins exist in multiple stable preparatory configurations, from which Ca(2+) may trigger exocytosis through distinct mechanisms and with distinct kinetics.
当细胞通过胞吐作用释放激素和神经递质时，胞质中的 Ca(2+) 触发分泌囊泡与质膜融合。众所周知，这种融合需要 SNARE 蛋白复合物的组装。然而，对于理解胞吐作用至关重要的 SNARE 组装相对于囊泡融合的时间尚未得到证明。为了研究这种时间安排，我们构建了一个探针，通过荧光共振能量转移（FRET）检测两个质膜 SNARE 蛋白 SNAP25 和 syntaxin-1A 的组装。通过双光子成像，我们同时测量了胰岛中胰腺 Langerhans 岛的β细胞中的 FRET 信号和胰岛素胞吐作用。在细胞的某些区域，我们发现 SNARE 复合物已经预先组装，这使得快速胞吐作用成为可能。在其他区域，SNARE 组装紧随 Ca(2+) 内流，胞吐作用较慢。因此，SNARE 蛋白存在多种稳定的预备构象，Ca(2+) 可能通过不同的机制和不同的动力学触发胞吐作用。
