REGULATION OF CYTOPLASMIC DYNEIN-BASED VESICLE TRANSPORT

基于细胞质动力蛋白的囊泡运输的调节

• 批准号: 3303775
• 负责人: TRINA A SCHROER
• 金额: $ 8.67万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-29 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303775
• 关键词: adenosinetriphosphatase; affinity chromatography; autoradiography; binding proteins; cell motility; cell population study; chick embryo; dynein ATPase; enzyme mechanism; gel filtration chromatography; high performance liquid chromatography; immunoprecipitation; membrane fusion; membrane permeability; microinjections; monoclonal antibody; phosphorylation; protein structure function; protoplasm motility; scintillation counter; stoichiometry; vesicle /vacuole

Microtubule-based membrane transport is a complicated process that involves the mechanochemical enzymes dynein and kinesin as well as soluble activator proteins. Assays of organelle motility in vitro have allowed the identification and preliminary characterization of three distinct activators of dynein-driven organelle transport in chick embryo brain. The activators are likely to govern motor function in vivo. The goal of the proposed research is to elucidate the mechanism(s) by which two activators, 150/5OkD and C3, regulate motor activity and to initiate studies of regulation of motor activity in vivo. The following questions will be addressed. 1. How is cytoplasmic dynein activity regulated by the 150/5OkD activator? The 150/5OkD activator will be purified further and antibodies will be raised. The effects of the activator on dynein ATPase activity will be determined. Anti-activator antibodies will be used to immunolocalize the activator in cultured cells and in microinjection studies of live cells. 2. Do membranes affect enzyme activity? The membrane binding properties of dynein will be measured and it will be determined if membranes stimulate dynein ATPase. 3. Is cytoplasmic dynein activity regulated by phosphorylation? The C3 activator has protein kinase activity; it will be determined if cytoplasmic dynein is a substrate for phosphorylation. Dynein phosphorylation patterns will be analyzed to determine if they correlate with patterns of activity. 4. Is cytoplasmic dynein activity regulated during the cell cycle? Membrane traffic is known to be inhibited during mitosis; at this time, cytoplasmic dynein may be involved in the mitotic spindle movements. It will be determined whether "mitotic" dynein can serve as an organelle motor and how dynein activity is regulated during the cell cycle. These studies may provide insight into the role of dynein in mitosis.

基于微管的膜运输是一个复杂的过程，涉及 机械化学酶动力蛋白和驱动蛋白以及可溶性激活剂 蛋白质。体外细胞器运动的测定使得 三个不同的识别和初步表征 鸡胚大脑中动力蛋白驱动的细胞器运输的激活剂。这 激活剂可能控制体内运动功能。的目标 拟议的研究是为了阐明两种激活剂的机制， 150/5OkD 和 C3，调节运动活动并启动以下研究 体内运动活动的调节。将提出以下问题 已解决。 1. 150/5OkD 激活剂如何调节细胞质动力蛋白活性？ 150/5OkD 激活剂将被进一步纯化，抗体将被 提出。激活剂对动力蛋白 ATP 酶活性的影响为 决定。抗激活剂抗体将用于免疫定位 培养细胞和活细胞显微注射研究中的激活剂。 2. 膜会影响酶活性吗？膜结合特性 将测量动力蛋白并确定膜是否刺激 动力蛋白 ATP 酶。 3. 细胞质动力蛋白活性是否受磷酸化调节？ C3 激活剂具有蛋白激酶活性；将确定是否是细胞质 动力蛋白是磷酸化的底物。动力蛋白磷酸化模式 将进行分析以确定它们是否与活动模式相关。 4. 细胞质动力蛋白活性在细胞周期中受到调节吗？膜 已知有丝分裂期间交通受到抑制；此时，细胞质 动力蛋白可能参与有丝分裂纺锤体的运动。这将是 确定“有丝分裂”动力蛋白是否可以充当细胞器马达以及如何发挥作用 动力蛋白活性在细胞周期中受到调节。这些研究可能 深入了解动力蛋白在有丝分裂中的作用。