MICROTUBULE-BASED MOTOR PROTEINS AND ORGANELLE TRANSPORT

基于微管的运动蛋白和细胞器运输

• 批准号: 2179356
• 负责人: RONALD D VALE
• 金额: $ 24.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179356
• 关键词: Cephalopoda; Dictyostelium; adenosine triphosphate; alternatives to animals in research; binding proteins; biological transport; cell motility; chemical binding; chemotaxis; cytoplasm; dynein ATPase; enzyme activity; gel filtration chromatography; human tissue; immunocytochemistry; kinesin; microscopy; microtubules; northern blottings; organelles; protein transport; protoplasm motility; southern blotting; western blottings

Microtubules and force-producing enzymes termed "motor proteins" power chromosome segregation during cell division, ciliary beating, and the rapid transport of membranous organelles in the endocytic and secretory pathways. Deciphering the molecular basis of microtubule-based motility is therefore germane to understanding many intracellular processes that are needed for the growth, survival and development of eukaryotic cells. Furthermore, mutations in kinesin and dynein motors often produce defects in ciliary movement, meiosis, mitosis and axonal transport in many organisms; similar mutations may underlie certain human diseases as well. The long-term objectives of this laboratory are to elucidate the biological roles and biophysical properties of kinesin and dynein motors. One aim of this grant is to understand chemomechanical transduction, the process by which motors move along microtubules. In vitro motility assays and high resolution microscopy capable of detecting motion with nanometer precision on a millisecond time scale will be used to measure the discrete mechanical actions of individual motor proteins. By relating such mechanical events to chemical intermediates in the ATP hydrolysis cycle using ATP analogues, a general mechanism for the force- generating process may be defined. A second aim of this grant is to identify the proteins that are needed for initiating and regulating organelle transport along microtubules. Reconstituted organelle transport assays developed in this laboratory will be used in combination with classical biochemical techniques to purify proteins that are needed in conjunction with kinesin and dynein to move organelles in a specific direction. The long range goal of this effort is to derive a completely defined organelle transport system using purified components, so that the transport machinery can be dissected at a molecular level. Our third objective is to define the biological roles of kinesins in living cells by genetic approaches as well as by immunocytochemical localization of the motors. Several kinesin genes in Dictyostelium discoideum, a unicellular motile slime mold, will be disrupted by homologous recombination, and the phenotype of the mutant cell will be examined. Through such studies, biological functions can be assigned to specific kinesin motors. The roles of two human conventional kinesin heavy chains, one of which is ubiquitously expressed and the other of which is brain specific, will be examined by determining their respective cellular and subcellular distributions and by disrupting their function by antibody microinjection or anti-sense oligonucleotide treatment in cell culture systems.

微管和产生力的酶称为“运动蛋白”功率 细胞分裂期间的染色体隔离，睫毛跳动和 在内吞和分泌中快速运输膜细胞器 途径。 基于微管运动的分子基础解密 因此，与理解许多细胞内过程有关 需要真核细胞的生长，生存和发展。 此外，驱动蛋白和动力蛋白电动机的突变经常产生缺陷 在睫状运动，减数分裂，有丝分裂和轴突运输中 有机体；类似的突变也可能是某些人类疾病的基础。 该实验室的长期目标是阐明 驱动蛋白和动力蛋白电机的生物学作用和生物物理特性。 这笔赠款的目的之一是了解化学力学转导， 电动机沿微管移动的过程。 体外运动 测定和高分辨率显微镜能够检测运动 毫秒时间尺度上的纳米精度将用于测量 单个运动蛋白的离散机械作用。 经过 将此类机械事件与ATP中的化学中间体有关 使用ATP类似物的水解循环，ATP类似物是力的一般机制 可以定义生成过程。 这笔赠款的第二个目的是 确定启动和调节所需的蛋白质 细胞器沿微管运输。 重构的细胞器 该实验室开发的运输分析将组合使用 使用经典的生化技术来纯化所需的蛋白质 与驱动蛋白和动力蛋白结合使用，以特定的方式移动细胞器 方向。 这项工作的远距离目标是完全得出 使用纯化的组件定义的细胞器传输系统，以便 可以在分子水平上解剖运输机械。 我们的第三个 目的是定义驱动蛋白在活细胞中的生物学作用 通过遗传方法以及免疫细胞化学定位 电动机。 迪斯特尔迪斯特尔的几个驱动基因，一个 单细胞运动粘液模具，将被同源 重组和突变细胞的表型将被检查。 通过此类研究，可以将生物学功能分配给特定 动力机电机。 两个人类传统动力蛋白重的角色 链条，其中一条无处不在，另一个是 大脑特异性将通过确定各自的细胞来检查 和亚细胞分布，并通过 细胞中的抗体显微注射或抗义寡核苷酸处理 培养系统。