Direct Observation of Dynein Motility Using Biophysics

描述(申请人提供)：细胞质动力蛋白是细胞内沿微管行走的主要负端定向运动蛋白，但其复杂的结构使动力蛋白难以纯化和体外研究。这项研究的目的是提纯动力蛋白，并用荧光团或珠子标记发动机，以检测它们的失速力、步长和可加工性。动力蛋白包裹的珠子将被光学镊子抓住，以检测步数和作用力。当单个荧光动力蛋白沿着微管行走时，可以使用全内反射荧光显微镜和1纳米精度的荧光成像来确定步长和处理能力，从而准确地定位单个荧光动力蛋白。这项工作不仅将揭示动力蛋白运动蛋白的内部工作原理，还将进一步告知我们与运动神经元疾病相关的动力蛋白故障。

DESCRIPTION (provided by applicant): Cytoplasmic dynein is the main minus-end directed motor protein to walk along microtubules inside cells, but the complex structure has made dynein difficult to purify and study in vitro. This study aims to purify dynein and label the motors with fluorophores or beads to probe their stalling force, step size, and processivity. Dynein coated beads will be grabbed by optical tweezers to detect steps and forces. Single fluorescent dyneins can be accurately located as they walk along microtubules using total internal reflection fluorescence microscopy and fluorescence imaging with one nanometer accuracy to determine the step size and processivity. This work will not only reveal the inner workings of the dynein motor protein, but will also further inform us of dynein malfunction related to motor neuron diseases.