: Giardia intestinalis is a widespread and evolutionarily divergent intestinal parasite of humans and animals, and a member of one of the earliest diverging eukaryotic lineages. In general, there has been little work to develop molecular models of either cytoskeletal-mediated chromosome segregation or of parasite attachment to the host intestinal epithelium via a specialized ventral disc structure. The goals of this proposal are to establish the function of kinesin motors in mitosis and in the assembly and function of the ventral disc in Giardia. Kinesin-like proteins (klps) are distributed among roughly ten phylogenetic subclasses and are essential components of many cellular mechanisms including mitosis, flagellar motility, intracellular transport, and cell polarity. We have identified and cloned a total of twenty-four Giardia klps that group to each of the major kinesin subclasses as well as multiple novel Giardia kinesin lineages. We predict the functional conservation of klps in Giardia mitosis, as Giardia possesses at least one klp from each mitotic subclass. Conversely, we suggest that novel Giardia klps contribute to the unique contractile functions and assembly of the ventral disc, a novel cytoskeletal structure responsible for adherence to the host epithelium (or laboratory substrates). To test these hypotheses, we will first use GFP and epitope protein tagging approaches to localize gklps within the cell. Based on mitotic or disc-localization, we will use several methods of functional inhibition followed by assays of mitosis and disc function to monitor the cellular behavior and assess the contribution of select Giardia klps to both mitosis and to the assembly and function of Giardia-specific cytoskeletal structures. We will also use state of the art light and electron microscopy to characterize Giardia mitosis and ventral disc function/assembly to provide a structural baseline for the analysis of kinesin function in these processes. These cell biological analyses wilt permit us to define the role of kinesins in spindle assembly and function and chromosome segregation and develop a molecular model of Giardia mitosis. Furthermore, we will assess the functions of kinesins in ventral-disc assembly and attachment/detachment. These analyses of Giardia kinesin functions should provide novel therapeutic targets for anti-Giardia anti-kinesin compounds. ? ?
