Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors
Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors
复制标题
DOI：
10.1073/pnas.1201390110
复制
发表时间：
2013-01-08
影响因子：
11.1
通讯作者：
Kojima, Hiroaki
中科院分区：
文献类型：
--
作者：
Furuta, Ken'ya;Furuta, Akane;Kojima, Hiroaki
细胞内运输被认为是由与货物结合的运动蛋白组实现的。然而，由于在控制电机数量和组成方面的实验困难，团队内部的协调仍然知之甚少。在这里，我们开发了一个实验系统，该系统将DNA支架上确定数量的马达与确定的间距连接在一起。通过该系统，我们在体外连接了两种不同类型的运动蛋白马达的多个分子，即进程性运动蛋白1或非进程性运动蛋白Ncd（运动蛋白14）。两种类型的运动蛋白均随运动数的增加而显著增加加工能力。值得注意的是，尽管单个Ncd电机的加工能力较差，但两个Ncd电机的耦合可以沿着微管（mt）进行超过1 μ m的加工运动。随着电机间距的减小，这种改进进一步增强。力测量结果表明，当2 ~ 4个Ncd电机一起工作时，Ncd组产生的力是可加性的，比单个电机产生的力大得多。相反，多个运动蛋白-1的作用力对运动数量的依赖性较弱。数值模拟和单分子解结合测量表明，Ncd施加的力的加性依赖于快速的MT结合动力学和单个Ncd马达的大阻力。这些特性将使小组Ncd电机能够交联mt，同时通过形成簇快速调节其力。因此，我们的实验系统可以为从下到上研究运动蛋白的集体行为提供一个平台。
Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 mu m along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to cross-link MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.