Biochemical and single-molecule characterization of the human cytoplasmic dynein motor complex

人细胞质动力蛋白是一种重要的分子马达，参与多种细胞功能，如沿微管运输货物、细胞分裂和细胞迁移等。动力蛋白及其辅因子与包括无脑畸形和佩里综合征在内的神经系统疾病有关，并参与病毒转运和癌细胞分裂等病理过程。开发治疗这些疾病的药物，以及在治疗其他疾病时操纵动力蛋白的功能，都需要对人类运动有详细的了解。然而，在体外，人类动力蛋白的生化和生物物理性质仍未得到表征。调控动力蛋白多种细胞功能的机制与其单分子特性还没有充分的相关性，但很可能是酵母和其他真核动力蛋白研究结果不明确的原因。在这个研究项目中，我将从人类细胞培养物中纯化动力蛋白，并通过蛋白质印迹和质谱仪分析运动复合体的生化组成。使用单分子荧光和光学捕获显微镜，然后我将仔细检查动力蛋白的动力和力产生特性。结合生化分析，这些单分子研究可能揭示体内动力蛋白的潜在调节点，如可变的磷酸化状态或亚单位组成。除了澄清动力蛋白领域的争议外，对人类动力蛋白的深入表征可能对动力蛋白依赖的信号转导途径以及癌症和细胞生物学具有跨学科的影响。

Human cytoplasmic dynein is an essential molecular motor involved in multiple cellular functions such as cargo transport along microtubules, cell division and cell migration. Dynein and its cofactors are implicated in neurological diseases including lissencephaly and Perry syndrome, and contribute to pathologic processes such as viral transport and cancer cell division. Developing drugs for these conditions, and manipulating dynein function in the treatment of other diseases, requires a detailed knowledge of the human motor. However, the biochemical and biophysical properties of human dynein remain uncharacterized in vitro. Mechanisms regulating dyneins multiple cellular functions have not been correlated sufficiently with its single-molecule characteristics, yet are likely responsible for ambiguous results on yeast and other eukaryotic dyneins. In this research project, I will purify dynein from human cell cultures and analyze the biochemical composition of the motor complex by Western blot and mass-spectrometry. Using single-molecule fluorescence and optical trapping microscopy, I will then scrutinize dyneins motive and force-generating properties. In combination with the biochemical analysis, these single-molecule studies may reveal potential points of dynein regulation in vivo, such as variable phosphorylation states or subunit composition. In addition to clarifying controversies in the dynein field, an in-depth characterization of human dynein is likely to have cross-disciplinary implications for dynein-dependent signal transduction pathways, as well as cancer and cell biology.