Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicular trafficking, 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 and function as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetic study and may be purified in large amount for biochemistry. Major questions remain about how dynein motor function is controlled and the mechanisms by which the enzyme is attached to the appropriate cargo. This application proposes study of four distinct light chains (LCs) that we have recently identified within the dynein particle. LC4 is a Ca2+-binding EF-hand protein associated with the gamma heavy chain that we hypothesize is involved in Ca2+-mediated control of motor function. The proposed experiments will test this directly both in vitro and in vivo using mutant versions of LC4 exhibiting different Ca affinities. The second project will analyze the LcI protein which is associated with the motor domain of the gamma heavy chain. This LC interacts directly in situ with a second protein (p45) that is thereby targeted to the motor unit. We propose to identify p45 and determine where LC1 binds on the heavy chain. Subsequent experiments will focus on defining the role of this novel system in dynein function. We also will examine the generic roles played by two essential classes of LC (Tctex1/Tctex2 and LC7/roadblock) that are located at the base of the dynein particle and are found in both cytoplasmic and flagellar isozymes. we will express altered versions of LC2 (Tctex2) in the odal2 null mutant background which is unable to assemble an outer arm to determine the function of this LC class. Dominant negative mutations for the LC7/roadblock proteins have been identified in Drosophila. Therefore, we will overexpress mutant forms of this protein in vivo and subsequently subject the strains to detailed biochemical/cell biological analysis. Thus, this project will provide detailed information on the assemble, regulation and activity of this complex molecular motor.
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