The long-term goals of my research are to understand the cellular and molecular properties of cilia/flagella that underlie their functions. My laboratory uses the biflagellated green alga Chlamydomonas reinhardtii as a model system to study cilium-generated signaling and ciliary/flagellar shortening. During the Chlamydomonas mating reaction, adhesion receptors (agglutinins) on the flagella of minus gametes bind to their cognate agglutinins on the flagella of plus gametes, thereby activating a cilium-generated signaling pathway in both cells that activates the gametes for cell-cell fusion to form a zygote. Immediately after zygote formation (and also during experimentally-imposed stress), Chlamydomonas cells shorten and completely resorb their flagella. We propose to use Chlamydomonas to dissect novel functions and mechanisms of regulation of the intraflagellar transport (IFT) machinery. Because almost every mammalian cell possesses a primary cilium that is used for signal transduction and also must be resorbed during cell cycle entry, our studies will provide novel insights into fundamental cellular mechanisms essential for human development and homeostasis. In previous studies of flagellar adhesion-induced signaling, we had shown that a regulatory protein, a flagellar protein tyrosine kinase (PTK), was activated early in the pathway. Moreover, using mutant gametes conditionally defective in IFT, we presented evidence that IFT is required for signal transduction in an intact cilium/flagellum. In the current funding period we discovered that a second regulatory protein in the pathway, a cGMP-dependent protein kinase, becomes associated with large assemblies within flagella whose formation requires IFT. To our surprise, the large, newly formed assemblies also contain IFT particles. Here, in our studies on signaling, we propose experiments to test the model that, in addition to its roles in flagellar assembly and disassembly, the IFT machinery participates directly in cilium-generated signaling and links membrane receptor interactions to gamete activation. Our previous studies on flagellar shortening showed that an aurora-like protein kinase (CALK) was essential for regulated shortening. In the current funding period, we made the surprising discovery that IFT trafficking within flagella and cargo loading onto IFT particles in the cell body are regulated during shortening. Moreover, we found that a protein that disassembles microtubules, a depolymerizing kinesin, in the cell body is phosphorylated and transported into flagella as microtubule disassembly is triggered during shortening. Our specific aims are to dissect the function of the intraflagellar transport machinery in flagellar adhesion-induced signaling, investigate the molecules that couple flagellar adhesion to gamete activation, and study the cellular and molecular mechanisms of flagellar shortening.
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