Abstract：
Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein\'s molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein\'s multiple AAA+ ATPase domains regulate dynein\'s attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.
摘要：
细胞质动力蛋白，最大和最复杂的细胞骨架运动蛋白，为许多细胞内货物向微管（MT）的负端移动提供动力。尽管它在真核细胞中起着重要作用，动力蛋白的分子机制，其亚基和辅助蛋白的调节功能，人类疾病突变对动力蛋白力产生的影响仍不清楚。最近的工作结合诱变，单分子荧光，和基于光学镊子的力测量为动力蛋白的多个AAAATPase结构域如何调节动力蛋白对MT的附着提供了有价值的见解。这里,我们描述了力依赖性动力蛋白-MT脱离率测量的详细方案.我们为尾部截短的单头酿酒酵母动力蛋白的表达和纯化提供了更新和优化的方案，对于极性标记的MT聚合，以及将MT非共价连接到覆盖玻璃表面，以测量动力蛋白-MT分离力。
