Gennerich Arne, Reck-Peterson Samara L
Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA.
Methods Mol Biol. 2011;783:63-80. doi: 10.1007/978-1-61779-282-3_4.
Cytoplasmic dynein, which is the largest and arguably the most complex cytoskeletal motor protein, plays fundamental roles during cell division, nuclear positioning, and organelle and mRNA transport, by generating force and movement toward the minus ends of microtubules. Consequently, dynein is central to many physiological processes, and its dysfunction is implicated in human diseases. However, the molecular mechanism by which dynein produces force and movement remains poorly understood. Here, we describe the use of optical tweezers to probe the nanometer-scale motion and force generation of individual dynein molecules, and provide a hands-on protocol for how to purify cytoplasmic dynein from budding yeast in amounts sufficient for single-molecule studies.
胞质动力蛋白是最大且可能是最复杂的细胞骨架运动蛋白，通过产生朝向微管负端的力和运动，在细胞分裂、核定位以及细胞器和信使核糖核酸运输过程中发挥着重要作用。因此，动力蛋白对于许多生理过程至关重要，其功能障碍与人类疾病有关。然而，动力蛋白产生力和运动的分子机制仍知之甚少。在这里，我们描述了使用光镊来探测单个动力蛋白分子的纳米级运动和力的产生，并提供了一个实用方案，介绍如何从出芽酵母中纯化足够用于单分子研究的胞质动力蛋白。
