REGULATION OF CYTOPLASMIC DYNEIN-BASED VESICLE TRANSPORT

基于微管的膜转运是一个复杂的过程，涉及 机械力化学酶动力蛋白和动蛋白以及可溶性激活剂 蛋白质。细胞器运动的体外分析已经允许 三个不同菌株的鉴定及初步鉴定 动力蛋白驱动的鸡胚脑细胞器运输激活物。这个 激活剂很可能控制体内的运动功能。的目标是 拟议的研究是为了阐明(S)两种激活剂， 150/5OkD和C3，调节运动活动，并启动对 体内运动活动的调节。以下问题将是 地址。 1.150/5OkD激活剂对细胞质动力蛋白活性的调节 150/5OkD激活剂将进一步纯化，抗体将被 养大的。激活剂对动力蛋白ATPase活性的影响将是 下定决心。抗激活剂抗体将用于免疫定位 在培养细胞和活细胞的显微注射研究中使用激活剂。 2.膜会影响酶的活性吗？膜结合性能的研究 将测量动力蛋白，并将确定膜是否刺激 动力蛋白ATPase。 3.细胞质动力蛋白活性是否受磷酸化的调节？C3 激活剂具有蛋白激酶活性；它将被确定是否胞质 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.细胞质动力蛋白活性在细胞周期中是否受到调节？膜 已知在有丝分裂期间交通受到抑制；此时，细胞质 动力蛋白可能参与了有丝分裂纺锤体的运动。会是 确定了“有丝分裂”动力蛋白是否可以作为细胞器马达以及如何发挥作用。 动力蛋白的活性在细胞周期中受到调节。这些研究可能 洞察动力蛋白在有丝分裂中的作用。

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.