PILOT STUDY--CONTROL OF HEPATOCYTE CYTOPLASMIC DYNEIN

基于微管的运动在泡状细胞中起着重要的作用， 肝细胞运输。 这一过程的关键动力是 肝细胞质动力蛋白。 动力蛋白的激活可能是 在特定囊泡的运输开始或在 控制运输速度。 事实上，我们不知道 细胞质动力蛋白在任何细胞中都变得活跃。 使用体外分析， 这个项目将探索动力蛋白的分子基础是否 激活可以被证明涉及cAMP依赖性磷酸化 细胞质动力蛋白重链或轻链。 该提案是仿照 在我们目前对轴丝动力蛋白激活的理解之后， 29kDa调节轻链的cAMP依赖性磷酸化。 细胞质动力蛋白将从培养的大鼠肝细胞获得， 在生物化学上，在结构上由负染色电子表征 显微镜和功能通过体外微管易位测定。 条件将探讨细胞质动力蛋白可能是 在透化肝细胞中以cAMP依赖性方式磷酸化，或 在细胞匀浆中。 目前尚不清楚是否有任何细胞质 微管运动受第二信使依赖性调节 磷酸化 如果能找到这样的条件， 将制备动力蛋白用于与非磷酸化对照进行比较。 磷酸化可增加微管转位率。 如果这 如果是这样，该项目将扩展到定义这种分子基础， 增加，更具体地说，以探索是否轻链 与轴丝动力蛋白相对应， 以及它的cAMP依赖性磷酸化是否控制了易位 率和速度的水泡贩运。

Microtubule-based motility plays an important role in vesicular trafficking in hepatocyte. A critical motor for this process is hepatocyte cytoplasmic dynein. Activation of dynein could be a critical event in the initiation of transport of specific vesicles or in the control of the speed of transport. Virtually nothing is known about how cytoplasmic dynein becomes active in any cell. Using in vitro assays, this project will explore whether the molecular basis of dynein activation can be demonstrated to involve cAMP-dependent phosphorylation of cytoplasmic dynein heavy or light chains. The proposal is modelled after our current understanding of the activation of axonemal dynein by cAMP-dependent phosphorylation of a 29kDa regulatory light chain. Cytoplasmic dynein will be obtained from cultured rat hepatocyte and characterized biochemically, structurally by negative stain electron microscopy and functionally by in vitro microtubule translocation assays. Conditions will be explored whereby cytoplasmic dynein may be phosphorylated in a cAMP-dependent manner in permeabilized hepatocyte or in cell homogenates. It is not yet known whether any cytoplasmic microtubule motor is regulated by 2nd messenger dependent phosphorylation. If such conditions can be found, the phosphorylated dynein will be prepared for comparison to non-phosphorylated controls. Phosphorylation could increase microtubule translocation rate. If this is so, the project will be extended to define the molecular basis of this increase, more specifically to explore whether a light chain corresponding to that of axonemal dynein is present in cytoplasmic dynein and whether its cAMP-dependent phosphorylation controls translocation rate and the speed of vesicular trafficking.