The long-term goal of the project is to determine the mechanism and regulation of ciliary and flagellar motility. The focus is on the regulation of dynein by phosphorylation. Much is know about general role of dyneins in powering microtubule sliding, however, little is known about localized control of dynein activity required for control of bending. The work proposed takes advantage of genetics and functional studies using Chiamydomonas, and focuses on one flagellar dynein, inner arm dynein Ii and its regulatory intermediate chain IC 138, which plays a central role in control of flagellar waveform. The work also focuses on a network of kinases, including PKA and CKI, built into the axoneme for control of motility.
The specific aims are: Determine how phosphorylation of ICl38 regulates dynein motor activity, using several in vitro motility assays to define the mechanochemistry and control of Ii. Identify regulatory domains in IC 138, taking advantage of Chiamydomonas mutant strains bop5 and mia2, defective in IC 138 or the phosphorylation of IC 138. Define the axonemal machinery that anchors PKA and CKI in position to control of IC 138 phosphorylation and control of dynein-driven microtubule sliding. The focus is on the axonemal A-kinase anchoring protein (AKAP); radial spoke protein 3 (RSP3). The experiments address central questions of the physiology of ciliary and flagellar dynein, important in humans for normal embryonic development, male and female reproduction and epithelial physiology. Moreover, the results may reveal an asymmetry in organization of axonemal kinases that is fundamental to localized control of dynein activity and, ultimately, control of axonemal bend formation. The results also address a general model for the regulation of the dynein motors, responsible for several vital cell functions involving directed cytoplasmic transport and organelle assembly, and the role of kinases, and kinase anchoring proteins including AKAPs, for control of movement.
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