The goal of this project is to understand the role of the brain microtubule associated proteins (MAPs) in axonal transport, neuronal differentiation, cell division and other aspects of cell behavior. During the preceding project period MAP 1C was found to be a retrograde force producing ATPase and to represent a cytoplasmic form of the ciliary and flagellar ATPase dynein. An additional ATPase, dynamin, was also identified, with properties indicating a role in sliding between microtubules. The proposed work deals with the cellular function of dynein and dynamin and with the identification of functional domains in the fibrous MAPs MAP 1A, MAP 1B, and MAP 2. The cytoplasmic dynein and its many component subunits will be examined for co-localization with membranous organelles and kinetochores by immunocytochemistry. A dynein receptor in organelle membranes will be sought, and modification of the mechanochemical and membrane-binding activities of dynein by phosphorylation and fatty acylation will be investigated. Exogenous labelled dynein will be introduced into cultured cells and squid axoplasm to evaluate the behavior of the enzyme in vivo. The structure of dynamin will be investigated by high resolution electron microscopy. cDNA's will be cloned encoding the 100 kD dynamin polypeptide, and will be sequenced to obtain the predicted amino acid sequence of the polypeptide. The dynamin activator will be purified. The location of the dynamin ATPase site will be identified from the predicted amino acid sequence for the 100 kD polypeptide and by photoaffinity labelling of the dynamin holoenzyme. The direction of force production by dynamin will be determined by microtubule gliding using proteolytic fragments or bacterially expressed segments of the 100 kD polypeptide. The orientation of microtubules within dynamin-induced bundles will be determined from the polarity of the constituent microtubules. Together with immunocytochemical evidence on the distribution of dynamin, a model for the function of the protein in the cell will be constructed. The RII-binding domain of MAP 2 will be further defined by RII binding to truncated portions of MAP 2 expressed in E. coli, and the ability of RII dimers to cross-link microtubules will be evaluated. Efforts to clone and sequence cDNAs encoding the MAP 1 light chains will continue in order to determine whether these polypeptides contain regions of homology with the microtubule binding domains of other MAPs. This work is of direct relevance to the control of abnormal cell division and a variety of neurodegenerative diseases.
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