REGULATION OF CYTOPLASMIC DYNEIN-BASED VESICLE TRANSPORT

基于微管的膜转运是一个复杂的过程， 机械化学酶动力蛋白和驱动蛋白以及可溶性激活剂 proteins.体外细胞器能动性的测定允许 对三种不同 鸡胚脑中动力蛋白驱动细胞器运输的激活剂。的 激活剂可能会控制体内的运动功能。的目标 提出的研究是阐明两种活化剂， 150/5 OkD和C3，调节运动活动，并启动研究 调节体内的运动活动。以下问题将是 处理。 1. 150/5 OkD激活剂如何调节细胞质动力蛋白活性？ 将进一步纯化150/50 kD激活剂，并将抗体纯化。 提出...激活剂对动力蛋白ATP酶活性的影响将在 测定抗激活剂抗体将用于免疫定位 活化剂在培养细胞和活细胞的显微注射研究中的应用。 2.膜会影响酶的活性吗？的膜结合性质 将测量动力蛋白，并确定膜是否刺激 动力蛋白ATP酶 3.胞质动力蛋白活性受磷酸化调节吗？所述C3 激活剂具有蛋白激酶活性;如果细胞质 动力蛋白是磷酸化的底物。动力蛋白磷酸化模式 将被分析以确定它们是否与活动模式相关。 4.细胞周期中细胞质动力蛋白的活性是否受到调节？膜 已知运输在有丝分裂期间被抑制;此时， 动力蛋白可能参与有丝分裂纺锤体的运动。将 确定是否“有丝分裂”动力蛋白可以作为一个细胞器马达，以及如何 动力蛋白活性在细胞周期中受到调节。这些研究可能 提供了动力蛋白在有丝分裂中的作用的见解。

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.