9728207 Asai Intracellular motility -- the ability to move subcellular structures from place to place within a cell -- is a critical fundamental property of organisms. Microtubules and their motors represent one of the two major mechanisms of intracellular motility in eukaryotes. While microtubule motors are implicated in many functions, it is not clear to what extent intracellular motility is regulated by utilizing a specific motor (i.e., isoform) for a specific task. In the case of cilia or flagella, there are approximately 12 separate dynein isoforms which each generate specific forces at specific sites within the complex flagellar structure that together produce the characteristic flagellar beating. Most eukaryotes also express multiple cytoplasmic dyneins. In this case, however, a key problem is to determine the contributions of each dynein isoform in a multi-dynein environment. This project will test the hypothesis that cytoplasmic dyneins are functionally specialized. If cytoplasmic dyneins are specialized, then it is anticipated that their intracellular locations are distinct, the elimination of a single isoform will give rise to a specific phenotype, and the biochemical and motility properties of the individual isoforms are different. The project focuses on two cytoplasmic dynein isoforms, DHC1a and DHC1b, which are expressed in all cells with multiple dyneins. The ciliated protozoan Tetrahymena, in which targeted gene replacement can be combined with large-scale protein biochemistry and immunocytochemistry, is an exceptional system in which to study dynein isoforms. Tetrahymena expresses more than 15 dynein heavy chains, including DHC1a and DHC1b. Dr. Asai and his colleagues have isolated Tetrahymena transformants in which the DHC1a and 1b genes have been separately knocked out. This provides the opportunity to determine the in vivo activities of an individual dynein in the context of other dyneins, then to isolate that dynein isoform and evaluate it in vitro. Th e examination of the knockout lines demonstrates that the two cytoplasmic dyneins are responsible for separate sets of tasks in vivo: DHC1a is required for micronuclear segregation in vegetatively growing cells, and is also required for phagocytosis; DNC1b participates in the normal organization of the cortical microtubule cytoskeleton and in the distribution of intracellular membranous organelles. Thus, the preliminary results support the hypothesis that the cytoplasmic dyneins are functionally specialized. The present project builds on these preliminary results and aims at defining mechanisms for specialization. Three questions will guide the work. (1) What are the in vivo tasks performed by DHC1a and 1b? the phenotypes of the dynein knockouts will be evaluated in a series of experiments that focus on cellular organization, secretion, ciliogenesis, and nuclear migrations during conjugation. (2) Where are dyneins 1a and 1b in the cell, and can one isoform substitute for the loss of the other? (3) How do dyneins 1a and 1b compare in terms of accessory proteins and motor activities in vitro? Utilizing the targeted gene replacement technology, tagged dynein genes will be constructed in order to affinity-purify individual dyneins and then examine the isolated dyneins. It is expected that functional specialization in vivo will be based on specific properties detected in vitro. With these three questions serving as a framework, the following specific aims will be pursued: 1, to assemble the tools for the project (characterizing the 1a and 1b genes and making isoform-specific peptide antibodies); 2, to determine the in vivo functions of the 1a and 1b dyneins by evaluating dynein knockouts, focusing on cellular architecture, secretion, nuclear movements, and ciliogenesis; 3, to compare the expression of 1a and 1b in wildtype and in dynein knockouts; 4, to affinity-purify individual dyneins; and 5, if time permits, to characterize the isolated dyneins in terms of associated proteins and in vitro motilities.
