Davis, Trisha NDavidson, Eliott2014-02-242014-02-242014-02-242013Davidson_washington_0250E_12583.pdfhttp://hdl.handle.net/1773/25102Thesis (Ph.D.)--University of Washington, 2013Cells carrying out mitosis must ensure that each daughter cell receives a full complement of chromosomes. As part of this process, multiprotein complexes called kinetochores link centromeric DNA to microtubule plus ends. This attachment allows cells to carry out two tasks that are essential for proper chromosome segregation. It couples chromosome position to microtubule plus end dynamics to allow maneuvering of chromosomes. It also senses whether chromosomes have been correctly attached to the spindle, correcting errors and delaying the cell cycle as necessary. That the kinetochore can maintain attachment to a microtubule plus end that is constantly polymerizing and disintegrating under its grip is one of the kinetochore's more extraordinary feats. Why evolution has chosen to utilize the plus-end, one of the cell's most ephemeral structures, as the site of its crucial chromosomal attachments is unknown. My lab previously isolated a <italic>Saccharomyces cerevisiae</italic> kinetochore mutant, <italic>DAM1-765</italic>, whose kinetochores retain their initial lateral attachment to the microtubule lattice instead of maturing into end-on attachments. Despite this fundamental defect in spindle organization, <italic>DAM1-765</italic> cells proliferate nearly as rapidly as wild-type. To determine how end-on kinetochore-microtubule attachments benefit the cell, I performed a synthetic lethal screen against <italic>DAM1-765</italic>. This screen produced mutant alleles of the EB1 homolog Bim1, CLIP-170 homolog Bik1, and the kinesin-14 subunits Kar3 and Cik1. Cells possessing these mutations fail to properly bundle spindle microtubules and struggle to biorient chromosomes, phenotypes which are exacerbated in the presence of <italic>DAM1-765</italic>. The presence of a kinetochore capping the plus end of a kinetochore microtubule appears to assist in the proper development of interpolar microtubules. In the absence of such a cap, spindle microtubules are poorly organized, and spindles are short and subject to collapse. Three proteins identified in my synthetic lethal screen, Bim1 and Kar3/Cik1, are believed to be involved in bundling and regulating interpolar microtubules. It is unknown, however, how these proteins accomplish these tasks when they are only known to be able to bind to a single microtubule. I have identified a potential Bim1 binding motif, SxIP, on Cik1's N-terminus. This motif is highly conserved among kinesin-14s. To determine the role this motif plays in kinesin-14 function on the mitotic spindle, I mutated this motif to alanine residues and assayed the mutation's effect on spindle structure via fluorescence microscopy. The motif is important for recruitment of Cik1 to the spindle and for maintenance of proper spindle length in both metaphase and anaphase. The existence of a greater Bim1/Kar3/Cik1 complex capable of binding to two microtubules (one via Bim1's calponin homology domain, and one via Kar3/Cik1's motor and motor homology domains) could explain how Bim1 and Kar3/Cik1 are able to bundle interpolar microtubules together. This work confirms that Bim1, Kar3, and Cik1 are important bundlers and regulators of interpolar microtubules and raises the possibility that Bik1 may be involved in this process as well. It suggests a mechanism by which Bim1 and Kar3/Cik1 may carry out these tasks. It also demonstrates that regulation of the different cohorts of spindle microtubules is intimately linked; interpolar microtubules rely on the proper maturation of kinetochore microtubules to properly develop themselves.application/pdfen-USCopyright is held by the individual authors.kinesin; microtubule; mitosis; spindle; yeastMolecular biologyCellular biologyGeneticsmolecular and cellular biologyThesis