In ciliated cells, basal bodies are contained within separate, filamentous cages connected to basal body microtubules. A recent study showed that actin is a component of the basal body cage complex in Tetrahymena and may be part of an actomyosin system associated with these organelles (Hoey and Gavin, J. Cell. Sci., 103, November 1992). There are three main objectives to this project. The first is to identify the myosin component of the putative actomyosin system in the Tetrahumena basal body cage complex; the second is to localize myosin within the complex; and the third is to determine whether basal body cage myosin mediates actin filament motility. An anti-oral apparatus anti-serum will be screened for cross-reactivity with myosins I and II on immunoblots. Antibodies that cross-react with myosin polypeptides will be affinity-purified and used to localize myosin-like components of the Tetrahymena basal body cage complex using immunogold labeling. To show actin filament interactions, in vitro assembled actin filaments will be mixed with purified basal body cage myosin and applied directly to a carbon-coated grid, negatively stained, and viewed by electron microscopy. %%% One of the most important forms of eukaryotic cellular motility is that mediated by axonemal beating: i.e., the highly regulated back- and-forth movement of slender, microtubule-containing cellular extensions (cilia or flagella). Depending on circumstances, such movements enable a cell to either swim in a regulated manner or to create local currents in its vicinity resulting in the movement of the immediate environment with respect to the cell. Specific cases of ciliary or flagellar motility include such diverse examples as: swimming of mammalian sperm toward the ovum, a process critical to mammalian reproduction; swimming of unicellular algae or protozoans in pondwater; feeding of protozoans by sweepinging food-containing pondwater into their oral cavities; and clearance of respiratory mucus from the lungs by the ciliary beat of epithelial cells lining the trachea and bronchi. The axonemes of such cells are rooted in cellular structures called basal bodies, which are homologous to centrosomes, structures in which intracellular microtubular arrays (such as mitotic spindles) are rooted. Although basal bodies have been studied for more than a century, important questions about their structure, chemistry, formation, and function remain unanswered. These organelles form definite structural associations with various fibrillar complexes in the cell, one of which is the cage. The structure-function relationship of these fibrillar complexes has not received much attention from investigators. Recent work has shown actin to be present in the basal body cage complex of a ciliated protozoan, Tetrahymena (a useful model system for studying ciliary function), and on the basis of the presence of actin it was proposed that there is an actomyosin contractile machinery involved in the regulation of basal body position (and by implication, the direction of the movement). This proposal focuses on the identification of the putative myosin component. The implications of such a model go well beyond motility in protozoans.
