The long-range goal of this project is to determine the mechanism by which the molecular motor dynein is regulated to produce the complex waveforms characteristic of beating eukaryotic cilia and flagella. The proposed research is specifically aimed at understanding how changes in calcium modulate the size and shape of ciliary and flagellar bends to control motility. Several studies have indicated that the flagellar central apparatus is a key component of a signal transduction pathway that regulates dynein activity to modulate waveform. The objectives of this proposal are founded on the hypothesis that the central apparatus locally controls a calcium sensor to regulate dynein activity, and that calmodulin and an axonemal calmodulin dependent kinase mediate the calcium signal. The proposed experiments are designed to test this hypothesis and to identify axoneme components involved in the calmodulin-mediated signal transduction pathway.
The Specific Aims are: 1) to identify calmodulin-binding proteins associated with the central apparatus; 2) to characterize calmodulin dependent kinases associated with the axoneme; and 3) to determine if the activities of particular dynein subforms attached to specific subsets of doublet microtubules are preferentially modulated by changes in calcium. The unicellular green alga, Chlamydomonas reinhardtii, is the organism of choice for these studies as it is the only system that offers motility mutants, virtually unlimited material for biochemical approaches, and unique in vitro functional assays. Many of the genes encoding flagellar proteins in Chlamydomonas show high sequence similarity with genes in the human genome and EST databases. Therefore, the information obtained in Chlamydomonas will be directly applicable to higher eukaryotes and may provide insight into defects that result in primary cilia dyskinesia including Kartegener's syndrome. Studies of dynein regulation and control of flagellar waveform will also impact upon our understanding of certain developmental processes. For example, the nodal cilia in developing embryos have been implicated in the production of a morphogen gradient responsible for generating left-right asymmetry. This result explains the observation that about 50% of patients with immotile-cilia syndrome also have situs inversus. Interestingly, nodal cilia utilized during development do not assemble a central apparatus and beat with a different waveform than cilia of epithelial cells found in the same organism. This observation further illustrates the importance of controlling ciliary and flagellar waveforms appropriate for particular cell types.
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