Eukaryotic cilia and flagella are found in plants, animals and portozoa. They play major roles in human health and reproduction, their proper functioning being central to the fertility of spermatozoa and clearing of the respiratory pathways. This proposal is aimed toward the identification of components of eukaryotic flagellar and ciliary axonemes involved in their coordinated bending. In particular, I wish to determine what components are involved in the modulation of beat frequency and calcium-induced changes in the beat form. One possibility is that the dynein ATPases, which power the motility play a role. The question is but which ones and what portions of this large multiprotein complex(es) are involved? My plan is to use transient state kinetic analyses to determine rates of various steps in the dyneintubulin cycle, in conjunction with detailed motility analyses of intact organelles. Previously, these techniques have been used on different biological materials which made direct comparison of rates difficult to interpret. I plan to focus my efforts on Chlamydomonas in which considerable biochemical, ultrastructural and motility information is available. Furthermore, numerous mutants are available in Chlamydomonas, including those with defects in beat frequency and those with defects in calcium-dependent change in waveform. I will also attempt transient kinetic analysis on whole axonemes which will allow direct comparison of kinetic rates with motility parameters. Two-dimensional electrophoresis of the dynein complexes from mutants will further aid in identifying proteins interacting at different steps in the dynein and dynein-tubulin ATPase cycle. The long term goal is to identify kinetic parameters associated with molecular components involved in various parameters of motion of the intact organelle. Identification of kinetic steps involved in beat frequency modulation and change in beat form will illuminate interactions involved in this complex organelle to produce coordinated bending movements.
