B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, 01069, Dresden, Germany.
Zernike Institute for Advanced Materials, University of Groningen, Groningen, AE, 9700, Netherlands.
Small. 2021 May;17(18):e2007388. doi: 10.1002/smll.202007388. Epub 2021 Mar 23.
Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, a highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods is introduced. Tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths, and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads are applied. Importantly, the 16 µm long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. The approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.
细胞骨架马达将化学能转化为机械功，驱动基本的细胞功能。光学捕获实验为这些分子马达在负载下的工作提供了至关重要的见解。然而，这种力谱实验的通量通常一次只能进行一次测量。在这里，引入了一种基于微流控的高通量方法，可以快速收集细胞骨架马达的力依赖性运动参数，与目前可用的方法相比，通量提高了两个数量级。通过 DNA 连接珠施加可调谐的流体动力来驱动步进驱动蛋白-1 马达，并利用大视场同时跟踪数百个单个驱动蛋白-1 分子在不同的阻力和辅助负载下的速度、运行长度和相互作用时间。重要的是，马达和珠子之间的 16 µm 长 DNA 系绳显著降低了将马达从微管拉开的施加力的垂直分量。该方法易于应用于其他分子系统，是对细胞骨架马达进行并行化单分子力研究的新方法。
