9604137 Gavin Technical: Basal body-associated actin and myosin could constitute a contractile system that might have a role in ciliary motion and theefore have significance beyond motility in ciliated protozoa. Ciliated basal bodies with associated fibrillar complexes are present in many vertebrate tissues, such as, tracheal epithelium and ciliary motion, that are an integral part of the physiology of these tissues. The head domain of an unconventional myosin heavy chain gene (TETMYO-1) was previously cloned and sequenced. Preliminary immunofluorescence microscopy results indicated that the myosin encoded by the TETMYO-1 gene is localized in the basal bodies of the Tetrahymena cytoskeleton. The objectives of this one-year project are to determine the ultrastructural localization of the myosin encoded by TETMYO-1 and to prepare a construct for a genomic knockout of this gene in Tetramymena. Specific antibodies will be prepared from 156-nucleotide fragment of the gene that will be cloned intoa vector and expressed in E. Coli. The expressed protein will be ourified and used as an immunogen to elicit polyclonal antibodies. The antibodieq will be used for immunogold localization of the TETMYO-1 in Tetrahymena cells. The ultrastructural localization will focus on cortical structures, the macronucleus, and the cytoplasm for evidence of TETMYO-1 localization. Particular attention will be focused on basal body-associated microfilaments in order to determine whether the mysosin colocalizes with actin filaments that have been shown to form a cage-like structure around each cytoskeletal basal body in Tetrahymena. To prepare for a genomic knockout of TETMYO-1 function in Tetrahymena, a disruption construct will be devleoped. A cassette that contains an antibiotic resistance coding sequence flanked by promoter and a terminator sequence will be ligated to a cloned TETMYO-1 fragment. Non Technical: The beating of cilia provides the motive force for the movement of many unicellular organisms and to mo ve fluids over the surface of some animal organs. A remarkable feature of the motion of cilia in both unicellular organisms and in vertebrate tissues is that the beating and direction of the cilia is coordinated with that of both adjacent and distal cilia. How this coordination is achieved is an important question in cell biology that is essential to understand physiological processes of unicellular organisms and the function of some vertebrate organs. This project will investigate the function of a contractile protein that may have an essential role in coordinating the direction of cilia beating in the unicellular organism Tetrahymena. Elucidating the function of this protein will provide important insights into how coordinated movement occurs in some cells and tissues.
