Dynein is of fundamental importance in the processes of fertilization and early embryonic development as well as in intracellular molecular movements including cell division and nerve cell axonal transport. The various tasks which dynein mediates are produced by different dynein heavy chain isoforms. A key element in the understanding of dynein function and regulation is the elucidation of the mechanisms that specify the intracellular location of each dynein isoform. The goal of this project is to determine the molecular basis for the accumulation of specific isoforms in specific places in the cell. This analysis will be made in Paramecium, an experimental model system particularly well suited for the proposed studies. There are five specific aims: (1) clone and sequence the ATP-binding domains of the dynein heavy chains expressed in Paramecium in order to be able to describe as completely as possible the structures and functions of the dynein isoforms in a single organism; (2) measure the steady state mRNA levels of each of the heavy chain isoforms after deciliation in order to classify the isoforms into those that are ciliary and those that are cytoplasmic; (3) determine the intracellular location of individual dynein isoforms by using site-directed antibodies in order to establish directly the linkage between heavy chain sequence and isoform function; (4) sequence the complete tail domains and complete motor domains of the functionally divergent ciliary and cytoplasmic isoforms in order to identify conserved regions which may represent common functional domains, such as the microtubule binding site, and nonconserved regions which may represent sequences that specify the intracellular location of dynein; and (5) construct and express in Paramecium chimeric heavy chain genes in which the tail domains will be interchanged between ciliary and cytoplasmic isoforms in order to determine the roles of particular sequences in directing the intracellular location of the dyneins.
