UC Santa Cruz

The kinetochore has been viewed as playing a critical role in many aspects of chromosome dynamics as the cell progresses through mitosis. This includes chromosome congression and alignment on the metaphase plate and sister chromosome separation and segregation to the spindle poles. Defects in kinetochore function result in mitotic errors and aneuploidy. Thus, the finding that chromosome fragments lacking a kinetochore (acentrics) are capable of normal congression, sister separation and segregation is fascinating. My thesis takes advantage of a Drosophila system in which acentrics can be efficiently generated and their behavior analyzed through live fluorescence analysis. In Chapter 1, we review how cells transmit chromosomes through mitosis without canonical kinetochore-microtubule interactions. Decades of research and a collection of studies reveal that microtubule-based mechanisms and DNA-based “tethers” connecting the acentric to the main chromosome mass are responsible for proper segregation of acentrics. In Chapter 2, we discuss the mechanisms by which acentric sister chromatids remain paired and eventually separate from one another during anaphase. Taking advantage of Drosophila transgenic for the I-CreI endonuclease, we efficiently generate broken chromosome fragments lacking centromeres, thereby lacking kinetochores. By using a genetic screen and live analysis, we identify proteins responsible for the separation of acentric chromosomes. We conclude DNA catenations are responsible for keeping acentric sisters paired and Topoisomerase II activity and microtubule plus-end pushing forces are needed to resolve these catenations and separate the acentrics. In Chapter 3, we highlight the remarkable ability of acentrics to congress to the metaphase plate despite the absence of kinetochore-microtubule interactions. Through mutational and live cell analysis, we define the forces acting on chromosome arms and the role of the kinetochore in chromosome congression. Utilizing Drosophila with fluorescently tagged acentric X chromosomes, we find acentric chromosome congression relies on interpolar microtubules as well as polar ejection forces. Our studies also show the induction of the DNA damage response leads to a global reorganization of congressed chromosomes at metaphase. Overall, this dissertation reveals a previously unsuspected kinetochore-independent backup mechanism by which chromosome fragments are able to progress normally through the initial stages of mitosis. Just as the discovery of cell cycle checkpoints led to new insights into the origins of cancer and novel therapies, it is likely that these and future insights into the transmission of chromosome fragments will have a similar impact on basic and applied cancer research.