Molecular Analysis of Flagellar Dynein Function

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

DESCRIPTION (provided by applicant): Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicle trafficking, nuclear envelope breakdown, ciliary/flagellar motility and cell division. The 1.9 MDa outer dynein arm from flagella of Chlamydomonas offers an excellent model system in which to study dynein structure, function and regulation as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetics and can be purified in large amounts for biochemical analysis. Dynein is a member of the AAA+ family of ATPases, however, the mechanisms by which ATP hydrolysis is converted to mechanical movement and how that motor activity is regulated at the molecular level remain almost completely unknown. This application proposes four specific areas of investigation. 1) We will use site-directed mutagenesis and in vitro biochemistry to address which domains bind and/or hydrolyze nucleotide. We will also test whether inter-domain interactions regulate ATP/ADP binding and whether the microtubule-binding stalk plays an active role in transmitting ATP-driven conformational change. 2) We will use electron microscopy and mutagenesis to test our model for subdomain assignments within the dynein motor unit. Further, we will insert a glycine linker between AAA1 and the N-terminal domain to test whether the power stroke occurs between these two segments as has been recently proposed. 3) We will examine whether thioredoxin light chains and the redox-sensitive docking complex protein (DCS) are involved in regulating dynein motor function in response to alterations in cellular redox poise. This will involve analysis of mutant strains expressing altered versions of DC3 and mutagenesis (Cys to Ser) of the thioredoxin redox-active sites combined with both in vitro and in vivo analysis of motor function. 4) We will use site-directed mutagenesis to test the hypothesis (based on our NMR structural studies) that the C-terminal domain of the LC1 protein controls gamma heavy chain ATPase in a manner similar to that observed with the GAPs that activate Ras/Rho GTPases. Finally, we will also examine whether the y heavy chain-associated Ca2+-binding LC4 protein acts as the Ca2+ sensor responsible for modulating ATP-dependent dyneinmicrotubule interactions. This project will provide detailed information on the fundamental motor mechanism of dynein and enable us to define the molecular pathways by which motor function is controlled.