School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14850, USA.
Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):14249-54. doi: 10.1073/pnas.1306699110. Epub 2013 Aug 12.
The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.
SNARE 复合物由三种蛋白质组成：突触融合蛋白 2、突触融合蛋白和突触相关蛋白 25（SNAP25），它被认为在驱动膜融合和胞吐作用时执行大的构象变化。然而，SNARE 复合物的变化与融合孔打开之间的关系仍然未知。我们在这里报告了一种直接测量方法，将 SNARE 复合物的变化与毫秒时间尺度上的囊泡融合联系起来。在单个嗜铬细胞中，我们通过全内反射荧光共振能量转移（FRET）显微镜跟踪 SNAP25 的构象变化，同时使用微制造电化学探测器阵列同时记录单个囊泡中儿茶酚胺的胞吐释放。局部快速和瞬态 FRET 变化恰好发生在单个囊泡释放儿茶酚胺的地方。为了克服收集足够信号强度所需的成像帧的低时间分辨率，开发了一种名为事件相关显微镜的方法，该方法表明 FRET 变化是突然的，并且在胞吐融合孔打开之前发生了约 90 毫秒。FRET 变化与融合孔的打开在时间上相关，而不是与融合孔的扩张相关。
