Microtubule binding kinetics of membrane-bound kinesin-1 predicts high motor copy numbers on intracellular cargo
Microtubule binding kinetics of membrane-bound kinesin-1 predicts high motor copy numbers on intracellular cargo
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DOI：
10.1073/pnas.1916204116
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发表时间：
2019-12-26
影响因子：
11.1
通讯作者：
Hancock, William O.
中科院分区：
文献类型：
--
作者：
Jiang, Rui;Vandal, Steven;Hancock, William O.
囊泡沿着微管的双向运输对于细胞的存活和功能是必需的，特别是在神经元中。当多个马达连接到囊泡时，囊泡在解离之前行进的距离由结合马达的分离和未结合马达的连接之间的竞争决定。马达分离速率常数（k（off））可以通过单分子实验测量，但马达再附着速率常数（k（on））通常是未知的，因为它们涉及通过双层的扩散、马达系链长度的几何考虑以及马达的固有微管结合速率。为了了解与流体脂质双层结合的马达的附着动力学，我们在二维（2D）系统中定量了荧光标记的驱动蛋白-1马达的微管累积速率，其中马达与支持的脂质双层连接。从一阶累积率在不同的电机密度，我们推断出一个k（关闭），匹配单分子测量和测量的2D k（上）膜结合驱动蛋白-1电机结合到微管。这个k（on）与驱动蛋白-1在附着到微管时能够达到大约20个微管蛋白亚基是一致的。通过将胆固醇降低膜扩散率，我们证明，这k（上）是不受限制的电机扩散速率，而是由内在的电机结合率。对于胞内囊泡运输，这个2D k（on）预测100 nm直径囊泡的远程运输需要35个驱动蛋白-1马达，这表明不同马达类别和马达聚集之间的协同作用可能在远程囊泡运输中发挥重要作用。
Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rate constants (k(off)) can be measured via single-molecule experiments, but motor reattachment rate constants (k(on)) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand the attachment dynamics of motors bound to fluid lipid bilayers, we quantified the microtubule accumulation rate of fluorescently labeled kinesin-1 motors in a 2-dimensional (2D) system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a k(off )that matched single-molecule measurements and measured a 2D k(on) for membrane-bound kinesin-1 motors binding to the microtubule. This k(on) is consistent with kinesin-1 being able to reach roughly 20 tubulin subunits when attaching to a microtubule. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this k(on) is not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this 2D k(on) predicts that long-range transport of 100-nm-diameter vesicles requires 35 kinesin-1 motors, suggesting that teamwork between different motor classes and motor clustering may play significant roles in long-range vesicle transport.