Molecular Mechanism of the Cytoplasmic Dynein-Dynactin Motor Complex

项目摘要/摘要 我们的长期目标是阐明细胞质动力蛋白-动力蛋白的分子机制 运动复合体，并确定人类动力蛋白相关疾病的分子基础。动力蛋白 是真核细胞中微管负端定向转运的主要载体。这个 这种重要的马达及其调节蛋白的功能和功能障碍在很大范围内起作用。 细胞功能和人类疾病的关系。尽管人们越来越努力地定义动力蛋白的功能 性质，但支配动力蛋白机械力化学的分子机制仍然很差 明白了。这一缺陷很大程度上源于动力蛋白的结构复杂性。动力蛋白属于 AAA+类的ATP水解酶，可以组装成环状结构， 因此，与其他两种细胞骨架马达相比，具有明显的结构特征 蛋白质家族，肌动蛋白和肌球蛋白。动力蛋白也非常大(~1.4丙二醛)和结构功能 直到最近，动力蛋白的研究一直受到功能性药物可用性的限制。 重组动力蛋白。增加了dynein的复杂性，dynein与多个附件关联 链和动力蛋白复合体，所有这些都是几乎所有细胞功能所必需的 动力蛋白。动力蛋白重链和动力蛋白最大亚基p150gled的突变 含有动力蛋白推测的微管结合结构域，导致毁灭性的神经 疾病。然而，对动力蛋白功能的机械知识--因此也就是它的功能障碍-- 与肌动蛋白和肌球蛋白相比是有限的，这构成了发展的主要障碍 有针对性的治疗。在这笔赠款中，我们试图通过结合 具有诱变和结构-功能研究的超灵敏单分子分析。我们会 利用酿酒酵母、昆虫和人类细胞表达系统生产稳定的 野生型和突变型两种多蛋白复合体。使用这些生化工具和 多色单分子荧光和光镊法，我们将破译 动力蛋白-动力蛋白复合体前进运动的分子机制 确定动力蛋白如何调节动力蛋白力量的产生。这些信息将提供深入的见解 进入细胞生理学并识别动力蛋白-动力蛋白复合体中的靶点以进行治疗 干预措施。

PROJECT SUMMARY/ABSTRACT Our long-term goal is to elucidate the molecular mechanism of the cytoplasmic dynein-dynactin motor complex, and to define the molecular bases of dynein-related diseases in humans. Dynein is the primary vehicle for microtubule minus-end-directed transport in eukaryotic cells. The function and dysfunction of this vital motor and its regulatory proteins contribute to a broad range of cellular functions and human diseases. Despite increasing efforts to define dynein’s functional properties, the molecular mechanisms that govern dynein’s mechanochemistry remain poorly understood. This deficiency largely stems from dynein’s structural complexity. Dynein belongs to the AAA+ class of ATP-hydrolyzing mechanoenzymes that assemble into ring-shaped structures, and therefore, possesses distinct structural features compared to the other two cytoskeletal motor protein families, kinesin and myosin. Dynein is also exceptionally large (~1.4 MDa) and structure function studies on dynein have been limited until recently by the availability of functional recombinant dynein. Adding to dynein’s complexity, dynein associates with multiple accessory chains and the dynactin complex, all of which are essential for nearly every cellular function of dynein. Mutations in the dynein heavy chain and dynactin's largest subunit, p150glued, which contains dynactin’s putative microtubule-binding domain, cause devastating neurological diseases. However, mechanistic knowledge of dynein’s function—and therefore its dysfunction— is limited compared to kinesin and myosin, which poses a major barrier for the development of targeted therapies. In this grant, we seek to overcome these limitations by combining ultrasensitive single-molecule assays with mutagenesis and structure-function studies. We will employ S. cerevisiae, insect and human cell-based expression systems to produce stable wildtype and mutant versions of both multiprotein complexes. Using these biochemical tools and multicolor single-molecule fluorescence and optical tweezers methods, we will decipher the molecular mechanism underlying the processive motion of the dynein-dynactin complex and determine how dynactin regulates dynein force generation. This information will provide insights into cellular physiology and identify targets within the dynein-dynactin complex for therapeutic interventions.