PILOT STUDY--CONTROL OF HEPATOCYTE CYTOPLASMIC DYNEIN

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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.