Cilia and flagella are present in a wide variety of eukaryotic cells. Their disfunction is correlated with respiratory ailments or male sterility. This proposal has the following two long-term objectives: analyze the molecular and structural features of those axonemal components that are essential for the generation of axonemal movement and second, identify the molecular mechanisms of regulation that lead to the formation of specific bending patterns of cilia and flagella. Both objectives will be approached with the analysis of the inner dynein arms, because in vivo these structures are necessary and sufficient to generate both ciliary and flagellar types of movement in the absence of outer dynein arms. The organism used is the unicellular green alga Chlamydomonas reinhardtii. This organism allows for the analysis of the axoneme by a combination of approaches including genetics, electron microscopy and biochemical procedures. The comparison of Chlamydomonas wild-type axonemes with mutant axonemes lacking the outer dynein arms or part of the inner arms forms the basis of many experimental procedures outlined in this proposal. The comparison of inner dynein arms prepared from motile flagella with those from nonmotile flagella and analysis of defective inner arms in mutant axonemes, will be adopted to explain the function of the posttranslational modification of inner arm heavy chains. On this basis the long-term objective will be approached with the following specific aims: 1. complete the characterization of the structure and molecular composition of all forms of inner dynein arms present in Chlamydomonas axonemes; 2. develop a detailed analysis of the structures and the modifications of inner arm heavy chains and determine whether the modification of heavy chains is part of mechanism controlling axonemal motility; 3. characterize the axonemal components whose function is correlated with the restoration of flagellar activity of radial spoke-defective mutants; 4. determine whether axonemal actin and caltractin directly are involved in those changes of inner dynein arm activity that occur when Chlamydomonas axonemes pass from the ciliary to the flagellar type of motion.
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