The regulated assembly and disassembly of microtubules is essential for cell division, cell motility, and for the cell-cycle dependent growth of cilia and flagella. Proteins associated with the primary assembly and disassembly site (the plus end) of a microtubule are likely to be important regulators of its growth. Two proteinaceous structures associated with the plus ends of microtubules are kinetochores, bound to centromeric regions of chromosomes, and caps that link the ends of microtubules to membranes in cilia and flagella. The caps are essential for ciliary and flagellar microtubule assembly and are bound to the site of microtubule assembly and disassembly as cilia and flagella grow and shorten during the cell cycle. Caps also link the microtubule ends to the membrane. In respiratory and reproductive tract cilia, caps are linked through the membrane to the ciliary crown, a bristle-like structure that contacts and transports mucus across the epithelial surface. The assembly of the caps and their interactions with microtubules appear to be essential for the proper growth and functioning of cilia and flagella. These properties of caps are similar to those exhibited by chromosomal kinetochores, which capture microtubules and link chromosomes to the mitotic apparatus. This linkage stabilizes microtubules and may regulate their disassembly as chromosomes are pulled toward the poles during mitosis. The lack of proper chromosome movement during mitosis leads to aneuploidy and to a variety of age-related human developmental disorders, including Down's syndrome. One protein common to mammalian kinetochores and ciliary caps has been identified, which suggests that these structures bound to the microtubule plus ends may have similar properties. The goals of this study are to characterize the function of capping structures in cilia and flagella, using a multidisciplinary approach. Biochemical studies are designed to identify and isolate cap proteins in Tetrahymena cilia and to study their interactions with microtubules in vitro. Genetic analysis of cap function in vivo will be done with Chlamydomonas reinhardtii bald mutants and a new class of stumpy mutants that cannot assemble caps. Mutants are generated using DNA-transformation and insertional mutagenesis, which facilitates the identification of the mutant genes using wild type DNA libraries and verification of gene function by the transformation of the rescued genes back into the mutants. While the mutants are expected to be defective in cap formation or function, these mutants also should reveal other parts of the assembly process, including signal transduction mechanisms that regulate flagellar microtubule growth and disassembly. The sequences of the rescued genes and antibodies produced against their protein products also will be used to examine mammalian ciliated cells, including those in testis, trachea, oviduct, and retinal cells for functional or structural homologies.
