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在体外纯化和研究。本研究旨在纯化动力蛋白，并用荧光团或荧光珠标记马达，以探测其失速力、步长和处理能力。涂有动力蛋白的珠子将被光学镊子抓住，以检测步骤和力。单个荧光动力蛋白可以精确定位，因为他们沿着微管行走使用全内反射荧光显微镜和荧光成像与一纳米精度，以确定步长和处理能力。这项工作不仅将揭示动力蛋白运动蛋白的内部工作原理，还将进一步揭示与运动神经元疾病相关的动力蛋白功能障碍。

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.