PILOT STUDY--CONTROL OF HEPATOCYTE CYTOPLASMIC DYNEIN

微管运动在囊泡发育中的重要作用 肝细胞的运输。这一过程的一个关键发动机是 肝细胞胞浆动力蛋白。动力蛋白的激活可能是一个关键的 在启动特定囊泡的运输过程中或在 对运输速度的控制。实际上，人们对此一无所知。 细胞质动力蛋白在任何细胞中都变得活跃。使用体外测试， 该项目将探索动力蛋白的分子基础 可以证明激活涉及cAMP依赖的磷酸化。 胞质动力蛋白重链或轻链。该提案是模仿性的 在我们目前对轴丝动力蛋白激活的理解之后 CAMP依赖的29 kDa调节轻链的磷酸化。 细胞质动力蛋白将从培养的大鼠肝细胞和 以负染色电子为特征的生化、结构特征 显微镜检查和体外微管移位功能检测。 将探索细胞质动力蛋白可能被 在通透性肝细胞中以cAMP依赖的方式磷酸化 在细胞匀浆中。目前尚不清楚是否有细胞质 微管运动受第二信使依赖的调节 磷酸化。如果能找到这样的条件，磷酸化的 将制备动力蛋白，以与非磷酸化对照进行比较。 磷酸化可增加微管移位率。如果这个 如果是这样，该项目将扩展到定义这一分子基础 增加，更具体地说是探索轻链是否 与轴膜动力蛋白相对应的是胞浆动力蛋白 以及其依赖cAMP的磷酸化是否控制易位 囊泡运输的速率和速度。

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.