MOLECULAR CHARACTERIZATION OF A CYTOPLASMIC DYNEIN

细胞质动力蛋白的分子表征

• 批准号: 2519022
• 负责人: MICHAEL P KOONCE
• 金额: $ 11万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2519022
• 关键词: Dictyostelium; SDS polyacrylamide gel electrophoresis; affinity chromatography; binding proteins; cell biology; centromere; cytoplasm; density gradient ultracentrifugation; dynein ATPase; electron microscopy; gene expression; in situ hybridization; laboratory rabbit; molecular cloning; protein sequence; protein structure function

Cytoplasmic dynein is a high molecular weight, microtubule-based mechanochemical ATPase that is thought to provide a motive force for a number of intracellular motilities. These include membrane-bound organelle transport, endocytotic trafficking, and intracellular organization of structures such as the golgi. Cytoplasmic dynein also localizes to the mitotic spindle and to the kinetochore region of condensed chromosomes where it may play active roles in spindle assembly, position, and chromosome movement during cell division. While in vitro studies and localization data have been suggestive, there is very little direct functional data on what cytoplasmic dynein does in cells. This application describes the initial stages of an in depth analysis of cytoplasmic dynein function. We are proposing here to pursue four distinct but overlapping lines of investigation: (1) a molecular genetic approach to delete or modify the dynein heavy chain sequence in situ (in Dictyostelium) to ask how important these gene products are to cell viability or cytoplasmic organization; (2) an indepth fine structure analysis to correlate the molecular sequence with the visible structural domains of this large polypeptide; (3) a detailed biochemical identification of functional domains such as microtubule-, kinetochore-, and organelle-binding sites; and (4), identification and molecular cloning of polypeptides that interact with the heavy chain, such as regulatory molecules, or polypeptides located on organelles or kinetochores. These four complementary approaches are designed to take maximal advantage of Dictyostelium as an experimental system. The overall goal of this project is to provide understanding of a molecule that potentially participates in a variety of essential cellular functions.

细胞质动力蛋白是一种高分子量，基于微管 机械化学ATPase被认为为A提供了动力 细胞内运动的数量。这些包括膜结合的细胞器 运输，内吞贩运和细胞内组织 高尔基体等结构。细胞质动力蛋白也定位于 有丝分裂主轴和冷凝染色体的动力学区域 它可以在主轴组件，位置和 细胞分裂期间的染色体运动。在体外研究和 本地化数据已经暗示，很少有直接的 有关细胞质动力蛋白在细胞中的功能数据。 该应用程序描述了深入分析的初始阶段 细胞质动力蛋白功能。我们在这里提议追求四个独特的 但是研究的重叠线：（1）分子遗传方法 删除或修改原位动力蛋白重链序列（在 dictyostelium）询问这些基因产物对细胞的重要性 生存能力或细胞质组织； （2）深入的精细结构 分析以将分子序列与可见的结构相关联 这个大多肽的域； （3）详细的生化 鉴定功能域，例如微管，动脉底座 - 和细胞器结合位置； （4），识别和分子克隆 与重链相互作用的多肽（例如调节） 分子或位于细胞器或动力学上的多肽。 这四种互补方法旨在具有最大优势 作为实验系统的Dictyostelium。总体目标 项目是为了提供对潜在的分子的理解 参与各种必需的细胞功能。