Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores
Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores
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DOI：
10.1038/s41467-019-14072-7
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发表时间：
2020-01-13
影响因子：
16.6
通讯作者：
Chapman, Edwin R.
中科院分区：
文献类型：
--
作者：
Das, Debasis;Bao, Huan;Chapman, Edwin R.
在胞吐过程中，融合孔的打开在囊泡的管腔和细胞外空间之间建立了第一次水连接。可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARES)介导了这些动态结构的形成，其动力学转换受到突触辅助蛋白的严格调控。在这里，我们利用两种单分子方法，基于纳米盘的平面双层电生理学和单分子FRET，来研究SNARE复杂组装和快速(微毫秒)融合孔转变之间的关系，并确定辅助蛋白的作用。突触聚集素(Syt)1是突触囊泡胞吐的主要钙感受器，它驱动形成一种中间体：承诺的反式SNARE复合体，形成大而稳定的毛孔。一旦打开，这些毛孔只能通过ATPase，NSF的作用来关闭。时间分辨测量显示，NSF介导的孔道闭合通过复杂的“口吃”机制发生。因此，这个简化的系统揭示了熔融气孔的动态形成和溶解。
The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.