The 9+2 axoneme is a microtubule-based extraordinary nanomachine that powers the oscillatory beating of motile cilia and flagella. The tightly controlled locomotion is crucial for the normal function of vital organs. Defects in this nanomachine caused common congenital disorders and severe chronic respiratory tract infections. A crucial device in this intricate biological machine is the radial spoke (RS) complex that is postulated as a mechanochemical transducer controlling the motor-driven sliding of the microtubules. Intriguingly, the molecular motifs resembling the domain for docking cAMP-dependent protein kinase A (PKA) to A-kinase anchoring protein (AKAP) are present in four constitutive components in the RS. However, they appear to tether crucial mechanisms related to calcium sensing, mechanic transduction and nucleotide metabolism but independent to PKA. Molecules analogous to these RSPs are involved in distinct cellular reactions in diverse organisms and yet their roles remain largely unknown. Hence, the ubiquity and versatility of these docking motifs are far beyond recognized currently. This proposal seeks to test the hypothesis that a single protein previously shown to be the base of the RS and predicted to be a spoke AKAP actually serve as a structural scaffold to anchor the four different non-PKA regulatory moieties that respectively confer the rigidity to the entire RS complex for the precise and repetitive mechanic transduction during oscillatory beating or modulate the RS upon the signaling of calcium and nucleotide derivatives to alter the beating. The strategy for testing this hypothesis is to mutate the putative binding sites in this scaffold protein in the flagellar model systems, Chlamydomonas. The in vivo experiments are essential for testing the interactions involving the four similar docking motifs. The defective RS complex in the mutants can be unequivocally defined by motility, protein biochemistry and electron microscopy. The reagents necessary for the experiments have been generated from a previous systematic proteomic project of the RS. The preliminary data of first-generation mutant strains strongly support the hypothesis and confirmed the experimental approach. The carefully designed, hypothesis-driven projects will offer excellent training, intellectually and experimentally, for both graduate and undergraduate students. These results will 1) shed crucial insight on the control mechanism of the axonemal nanomachine, the long standing central question in the field;2) provide a solid foundation for designing new strategies for treating diseases related to immotile cilia;3) reveal a novel mechanism in integration of chemical signaling with mechanic transduction that is gaining appreciation rapidly and 4) demonstrate the differential recognitions of the similar docking motifs for a wide variety of cellular reactions.
The proposed experiment will elucidate how a mechanism, once known for only anchoring a crucial protein kinase, is adopted to integrate mechanic regulation, chemical signaling and nucleotide metabolism for local control of flagellar beating and like many other cellular reactions that are vital for human health.
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