Cell division (mitosis) is a central process in all living organisms. Defects in its regulatory systems cause genome imbalances and are intimately linked to neoplastic transformation and tumor progression. Cell division requires the precise duplication of chromosomes and their partitioning between two daughter cells. Thus, for successful mitosis, a precise coordination of many morphological changes in time and space has to be accomplished. After nuclear envelope breakdown and condensation of the replicated DNA, the individual chromosomes (sister chromatids) assemble their kinetochores. The kinetochore is a key structure composed by a network of proteins that forms at a single site on each chromatid. The kinetochore physically connects the chromatids to an array of microtubule and associated proteins that make up the so called "mitotic spindle" and ensures that chromatids are positioned and split correctly as the spindle pulls them apart. Kinetochore attachment is a stochastic process and as such prone to errors, which might result in chromosome miss-segregation. Therefore, a surveillance mechanism known as the spindle assembly checkpoint ensures that sister chromatids segregation in anaphase does not take place before all chromosomes are properly aligned at the metaphase plate, when all kinetochores are attached to spindle microtubules and hence under tension. In addition to elucidating the basic mechanisms of chromosome segregation, studies of the kinetochore are proving relevant to cancer etiology and treatment. While severe problems in chromosome segregation cause cell death, minor errors result in aneuploidy, which is typical of most human tumors and has been suggested to promote tumorigenesis for more than a century. Therefore, it is important to define the basic molecular mechanisms by which kinetochore proteins normally operate to ensure the error-free segregation of chromosomes. Our long-term goal is to understand, first, how chromosome segregation and progression through mitosis are regulated in time and space and, second, what mechanisms give rise to the chromosomal instability that is typical of tumor cells. Although it has been proposed some time ago that chromosomally unstable cancer cells possess a weakened mitotic checkpoint, this view has recently been challenged and new evidence indicates that most aneuploid cells have a functional checkpoint. Also, mutations in genes encoding spindle assembly checkpoint proteins appear to be surprisingly rare in aneuploid tumor cells. Thus, a more recent hypothesis posits that aneuploidy and chromosomal instability reflect an inherent defect in the ability of cells to correct erroneous kinetochore-microtubule attachments. Central to the machinery required for establishing stable kinetochore-microtubule attachments is the Ska (for Spindle and kinetochore-associated) complex, which was originally discovered in our laboratory. The present proposal thus focuses on elucidating the role of the Ska protein complex in generating stable kinetochore-microtubule attachments during mitosis in human cells. Specifically, we pursue two main goals: first, we aim for a comprehensive understanding of Ska function by identifying Ska interaction partners and studying the regulation of the Ska complex, with a focus on phosphorylation. Second, we propose to analyze the abundance, stochiometry and dynamics of endogenous Ska proteins in both normal cells and tumor cells. Collectively, these studies should allow us to determine whether protein imbalances in key regulators of kinetochore-microtubule attachments are a likely root cause of chromosome instability in cancer cells.