Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.
Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.
复制标题
DOI：
10.1038/nature25481
复制
发表时间：
2018-02-08
期刊：
影响因子：
64.8
通讯作者：
Chapman ER
中科院分区：
文献类型：
--
作者：
Bao H;Das D;Courtney NA;Jiang Y;Briguglio JS;Lou X;Roston D;Cui Q;Chanda B;Chapman ER
融合孔是胞吐过程中形成的第一个至关重要的中间体，但关于决定这些瞬时结构的大小和动力学特性的机制知之甚少。在这里，我们减少了神经元中可用的SNAREs的数量，并观察到提示融合孔改变的递质释放的变化。为了解决这个问题，我们采用了重组融合实验，使用纳米片将孔隙捕获在初始开放状态。光学测量表明，增加SNARE复合物的数量可以提高单个孔隙的释放速率，并使较大的货物能够逃逸。为了确定这是否是由于新生孔径与稳定性的变化，我们开发了一种基于纳米盘和平面脂质双分子层电生理学的新方法，该方法在单事件水平上提供μ秒的时间分辨率。值得注意的是，这两个参数都受到SNARE拷贝数的影响。将每个纳米片的v-SNAREs数量从3个增加到5个，导致孔隙大小增加了2倍，孔隙关闭率降低了3个数量级以上。此外，trans-SNARE配对是高度动态的：在添加v-SNARE片段后，闪烁的新生孔隙关闭，这表明在胞外作用的这个阶段不会形成完全组装的、稳定的SNARE复合物。最后，SNARE复合物底部的缺失，模仿肉毒杆菌神经毒素a的作用，极大地降低了融合孔的稳定性。总之，跨snare复合物是动态的，用于驱动融合的SNAREs的数量决定了单个孔隙的基本性质。
The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known regarding the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs in neurons and observed changes in transmitter release suggestive of alterations in fusion pores. To address this, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores, and enabled the escape of larger cargos. To determine whether this was due to changes in nascent pore size versus stability, we developed a novel approach, based on nanodiscs and planar lipid bilayer electrophysiology, that affords μsec time resolution at the single event level. Remarkably, both parameters were affected by SNARE copy number. Increasing the number of v-SNAREs per nanodisc from three to five caused a two-fold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, trans-SNARE pairing was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable, SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, that mimics the action of botulinum neurotoxin A, dramatically reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determine fundamental properties of individual pores.
登录
查看更多内容
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
16.8
作者：
Bhalla, A;Chicka, MC;Chapman, ER
通讯作者：
Chapman, ER
影响因子：
64.8
作者：
MUELLER, P;RUDIN, DO;WESCOTT, WC
通讯作者：
WESCOTT, WC
影响因子：
3.7
作者：
Bao H;Duong F
通讯作者：
Duong F
影响因子：
5.5
作者：
Guvench, Olgun;Hatcher, Elizabeth;Venable, Richard M.;Pastor, Richard W.;MacKerell, Alexander D., Jr.
通讯作者：
MacKerell, Alexander D., Jr.