Segregation and Positioning of Basal Bodies

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Basal bodies and centrioles are microtubule organelles with both longitudinal and nine-fold rotational asymmetry. In addition to their role in ciliary assembly, basal bodies are important for the rotational positioning of motile cilia to properly orient the direction of ciliary beat. Little is known about the molecular components that establish or recognize the asymmetry to assemble appendages onto basal bodies at specific locations. Using the model organism Chlamydomonas reinhardtii, a unicellular, biflagellate green alga, the PI has isolated a series of mutants with a variable flagellar number (vfl) and with defects in rotational positioning of basal bodies, in orientation of probasal bodies with respect to mature basal bodies, and in segregation of basal bodies during cell division. Two VFL genes have been cloned. This new project continues the PI's study of the vfl mutants. The specific aims of the project are to 1) Clone and characterize the VFL4, VFL5, and VFL6 genes and localize the VFL5 and VFL6 gene products using light and electron microscopy; 2) Identify additional genes involved in basal body segregation and positioning using genetic approaches including insertional mutagenesis and chemical mutagenesis; 3) Examine interactions among vfl genes and identify interacting proteins using genetic approaches and yeast two-hybrid technology. The experimental approach will identify evolutionarily conserved molecules that specify rotational asymmetry in basal bodies as well as molecular components of fibers that bind to specific sites on the walls of basal bodies. The study will advance the long-term goal of understanding the molecular mechanisms that underlie the structure of basal bodies, the attachment of appendages to specific sites on the wall of the basal body, and the initiation of basal body duplication at a specified rotational position. The project will provide research training at three levels: undergraduate, graduate and postdoctoral.