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Abstract
Cilia are microtubule-based organelles adapted to multiple functions in eukaryotic organisms, including locomotion, feeding, movement of extracellular fluid, and various sensory functions. The highly conserved ultrastructure of the cilium is assembled from over 250 proteins which are delivered from the cytoplasm to their sites of incorporation in the growing organelle. This transport is tightly regulated to coincide with the cell cycle, maintain proper ciliary length and number, and ensure that cilia can regenerate if damaged or lost. Intraflagellar transport (IFT), the movement of protein raft particles within cilia and flagella, has been implicated in ciliary assembly in multiple organisms, but the specific function of IFT remains unknown. The results presented here explore the mechanisms and significance of IFT in the ciliate, Tetrahymena thermophila. In the first study, genes encoding two motor subunits, KIN1 and KIN2, of the kinesin-II molecular motor, were cloned and disrupted in Tetrahymena. Disruption of either subunit alone resulted in mild phenotypic defects. Disrupting both genes led to a complete loss of cilia and an inability to undergo normal cytokinesis. Localization studies revealed that kinesin-II accumulated in actively assembling cilia. Multiple results of this study lead us to propose that the cytokinesis defects in the kinesin mutants were a secondary effect caused by loss of motility. In the second study, the connection between ciliary motility and cytokinesis was examined further. This study describes a type of motility, which we call "rotokinesis", where the posterior daughter of a dividing cell rotates unidirectionally around the membrane bridge connecting the two daughters. We propose that this rotation increases the efficiency of cytokinesis in cells with wildtype motility. In the final study, RFT1, a homologue of one of the IFT raft proteins, was cloned and disrupted. The RFT1 null phenotype is identical to the kinesin-II null phenotype, suggesting these genes are in the same pathway. Excitingly, we isolated RFT1 suppressors which assemble intermediate length cilia in a temperature-sensitive and cell density-dependent manner, suggesting that RFT1 is not absolutely required for transport of ciliary structural components. This study provides the first evidence that IFT rafts may have a signaling function.