| The kinetochore is the protein structure which attaches spindle microtubules to centromeric DNA during mitosis. The protein composition of the kinetochore is widely conserved in vertebrate, yeast, and plant systems; in particular, the Ndc80 complex, Mis12 complex, and Knl1 complex (named the KMN network). Outside of these model systems, the composition of the kinetochore has not been experimentally studied thoroughly. In our effort to find kinetochore proteins in Giardia lamblia, we used a computational approach using already known kinetochore proteins in other organisms and the availability of newly sequenced protists in an attempt to expand upon previous knowledge. This data itself became a treasure trove of information, warranting further description and study. In this dissertation, I analyzed the phylogenetic profile of over fifty proteins encompassing fifteen sub-complexes of the kinetochore using data from over four hundred different species which represent a diverse sampling within all of the major Eukaryotic supergroups. I describe the conservation, or lack thereof, of these kinetochore proteins outside of the model systems in which they were discovered. From this data, I have shown that the core conservation of the KMN network lies within the microtubule binding layer of the kinetochore. Additionally, I show that the inner kinetochore, the proteins which link the microtubule binding layer to the DNA binding layer, does not show any significant conservation outside of Opisthokonts, and any conservation of these proteins seen can be explained by alternative functions for those proteins. I also expand on data of the human Ska complex and the yeast DASH complex, which are described as functional homologs though consisting of different sets of proteins, but both serve the function in helping the stabilization of the kinetochore / microtubule attachment. From this data, I show that the DASH complex has an ancient eukaryotic origin, and did not evolve specifically within the yeast lineage but also has existing homologs within stramenopiles. I also expand the presence of the Ska complex to other organisms outside of the metazoans. In my hunt to identify giardial kinetochore proteins, bioinformatics led me only to find Nuf2 and Spc25, both members of the ndc80 complex which has been shown to bind spindle microtubules in human and yeast systems. I GFP tagged these proteins and determined their localization either as foci within the nucleus during interphase, and in the case of Nuf2, as foci on the leading edge of migrating chromosomes during mitosis. The knockdown of these proteins using morpholinos results in missegregation of chromosomes, lagging chromosomes, and nuclear positioning defects, supporting their involvement in microtubule attachment at the kinetochore in Giardia. |
