MOLECULAR CHARACTERIZATION OF A CYTOPLASMIC DYNEIN

细胞质动力蛋白的分子表征

• 批准号: 2190120
• 负责人: MICHAEL P KOONCE
• 金额: $ 9.43万
• 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/2190120
• 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的，以微管为基础的 机械化学ATP酶，被认为是提供动力， 细胞内运动的数量。这些包括膜结合细胞器 转运，内吞运输和细胞内组织 像高尔基体这样的结构。细胞质动力蛋白也定位于 有丝分裂纺锤体和浓缩染色体的动粒区 在那里它可以在主轴装配、定位和 细胞分裂时染色体的运动。在体外研究中， 本地化数据已经暗示，很少有直接的 细胞质动力蛋白在细胞中的功能数据。 本申请描述了对以下内容进行深入分析的初始阶段： 细胞质动力蛋白功能我们在此建议， 但重叠的研究路线：（1）分子遗传学方法 为了原位删除或修饰动力蛋白重链序列（在 Dictyosteopathy）来询问这些基因产物对细胞有多重要 生存力或细胞质组织;（2）深入的精细结构 分析以将分子序列与可见结构相关联， 结构域的这种大多肽;（3）详细的生化 鉴定功能结构域如微管-，动粒-， 和细胞器结合位点;（4）鉴定和分子克隆 与重链相互作用的多肽，如调节性多肽， 分子或位于细胞器或动粒上的多肽。 这四种互补方法旨在最大限度地利用优势 作为实验系统。这个项目的总体目标是 该项目旨在提供对一种分子的理解， 参与多种重要的细胞功能。