The long-term goal of this proposal is to elucidate the control mechanism of motile cilia and flagella, and the focus, founded on new data, is on the radial spoke structure and calcium control of the dynein-driven motility. The broad significance of this work is best illustrated by the congenital syndrome, primary cilia dyskinesia. Noted symptoms include situs inversus, infertility, severe chronic infection of respiratory tract and hydrocephaly. Elucidating the control mechanism is essential for understanding the roles of these organelles in diverse cell types and for averting defective motility. The important questions include how the dynein motor activity is coordinated and how calcium and cyclic nueleotides modulate the dynein-driven motility. Independent lines of evidence indicate that radial spoke play a vital role in control of dynein motors and based on structural analysis and informative Chlamydomonas mutants, the radial spokes operate as mechano-chemical transducers to control dynein via a network of kinases, phosphatases and calcium sensors. Among the key molecules are two constitutive spoke proteins, RSP2 and calmodulin, that are essential for motility. Calmodulin, the prototypical calcium sensor located in spoke, is involved in calcium-induced motility changes but the mechanism is not known. RSP2, a recently cloned phosphoprotein, contains two calmodulin-binding motifs and binds calmodulin in a calcium-dependent manner. Most intriguing, RSP2 and isolated spokes display kinase activity. The simplest hypothesis is that RSP2/calmodulin complex mediates calcium control of motility by changing the physical and enzymatic properties of the radial spokes.
Three aims are designed to test this hypothesis. [1] Assess mutant constructs of recombinant RSP2, defective in calmodulin-binding and phosphotransfering domain in a RSP2 mutant (pf24). The mutant constructs are expected to rescue spoke assembly but fail to rescue calcium control of motility. [2] Measure the effect of calcium on kinase activity of isolated radial spokes and phosphorylation of RSP2. Predictably, spoke kinase activity is calcium sensitive. [3] Define radial spoke structure using new electron microscopic approaches, and define the location and molecular interactions of calmodulin in the spoke. Predictably calcium binding will change spoke structure. These experiments directly test the hypothesis and address the fundamental mechanism of control of ciliary and flagellar motility. The results will also have broad impact on how dynein-driven motility is controlled and how kinases and calcium sensors are anchored in the microtubule cytoskeleton.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CDF-4 (02))
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Gindhart, Joseph G
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Marquette University
Other Domestic Higher Education
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Yang, Chun; Yang, Pinfen (2006) The flagellar motility of Chlamydomonas pf25 mutant lacking an AKAP-binding protein is overtly sensitive to medium conditions. Mol Biol Cell 17:227-38
Yang, Chun; Compton, Mark M; Yang, Pinfen (2005) Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella. Mol Biol Cell 16:637-48

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