The broad long-term objectives of this project are (i) to determine the structural, chemical and enzymatic properties of outer-arm dynein, including its interaction with tubulin, and to relate these properties to the role of the arms in producing flagellar and ciliary motility, (ii) to compare the properties of the other isoforms of axonemal dynein to those of outer-arm dynein and to relate the differences to their respective roles in axonemal beating, (iii) to study the differences between cytoplasmic and axonemal dyneins and to relate them to their roles in cell motility. In the up-coming five-year project period much of the effort will be concentrated on using a combination of protein chemistry and molecular biology to follow up the various leads that have been opened by our recent sequencing of the Beta heavy chain of outer arm dynein from sea urchin embryos. Photoaffinity labeling will be used to identify the several candidate nucleotide-binding sites revealed in the amino acid sequence. Localized regions of conserved amino acid sequence that span the hydrolytic ATP-binding site will be used to amplify and sequence the corresponding 400 base pair regions of other dynein isoforms from sea urchin embryos and testis, as well as from selected tissues of Drosophila and mouse. By grouping the isoforms into families according to their degree of homology to each other and to the Beta heavy chain and then correlating the families with their tissue of origin, it should be possible to distinguish axonemal and cytoplasmic dyneins, and to identify the functional equivalents of the various axonemal dynein isoforms in different species. As this grouping into families proceeds, the sequence of selected isoforms will be extended in order to look for regions of conserved sequence that are further removed form the hydrolytic ATP-binding site and that show homology to the microtubule-binding sites of the other microtubule-associated proteins whose sequence is known. Selected conserved regions of the Beta chain will be expressed in E coli and examined in order to determine whether the expressed protein shows ATP-sensitive or ATP-insensitive microtubule-binding properties. The cellular localization of the various dynein isoforms will be examined with poly-clonal antibodies to expressed peptides representing unique regions of their sequence. As the project progresses, attempts will be made to express functional domains of the Beta chain that retain its capability to translocate microtubules over glass. The results of this study are expected to clarify the fundamental mechanisms by which dynein produces transport along microtubules in mitosis and in nerve axons, and so to contribute to understanding the many problems associated with improper chromosomal segregation and the lack of nerve regeneration after injury.
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