The long term objective of this research is to understand the mechanism of ciliary and eukaryotic flagellar motility. The significance of the work derives from the ubiquity of cilia and flagella and related intracellular-microtubule dependent cell movements. Our focus is on the subunit composition, structure, and function of the force generating ATPase dynein. The specific goals of this work include: (1) Study dynein structure of quick-frozen, deep etched reactivated sperm flagella by the deep-etch, rotary shadow method. We will analyze systematic changes in dynein structure along axonemal bends by these new electron microscopic procedures. These results will add to our understanding of the hypothetical step-wise mechanism by which dynein generates force and complement data on dynein's ATPase kinetics. (2) Study of protein domains in isolated dynein 1 and in situ outer dynein arms by electron microscopic localization of monoclonal antibodies to polypeptide subunits of dynein 1. In related, complementary work we will study the specificity of binding of isolated dynein 1 subunits to doublet microtubules and study the structure of proteolytically cleaved dynein 1. Together, these approaches will help in determining structural domains in dynein. (3) Analysis of patterns of microtubule sliding in MgATP2-, Ca++ induced quiescent, demembranated sperm and patterns of microtubule sliding in elastase treated axonemes. These results will aid in the understanding of regulation of dynein activity such that microtubule sliding is converted to bending waves characteristic of flagella. All of these results will add to our general knowledge of microtubule function and knowledge of energy transduction mechanisms. Additionally, it is recognized that certain human respiratory and fertility related ailments and development abnormalities result from ciliary and flagellar immotility due to dynein arm defects. Fundamental knowledge of dynein function may permit more precise description of the molecular basis of such dynein related diseases.
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