Dissecting the molecular mechanisms of chromosome condensation and spindle assembly using Xenopus egg extracts

Spindle assembly and chromosome condensation are essential mitotic processes that have evolved to ensure the faithful segregation of the genome during cell division. In order to fully understand these complex events, the functional contribution of individual cellular factors to spindle assembly and chromosome condensation must be defined. While significant progress has been made in these fields, much remains to be discovered. The goal of my research was to utilize Xenopus egg extracts to further characterize spindle assembly and chromosome condensation at the molecular and mechanistic levels.; In my first project, I investigated the role of the Regulator of Chromosome Condensation (RCC1) in mitotic chromosome condensation. RCC1 is an abundant chromatin-associated protein that binds directly to core histories. Additionally, as the guanine nucleotide exchange factor (GEF) for the small GTPase Ran, RCC1 generates a gradient of RanGTP around mitotic chromatin that locally activates spindle regulators. We hypothesized that RCCI could regulate chromosome condensation at either a structural level or via spatial regulation of condensation factors by the RanGTP gradient. While RCCI and a RanGTP gradient were found to be essential for nuclear assembly in interphase they were not required for chromatin condensation in metaphase extracts.; I next set out to identify and characterize microtubule-associated proteins (MAPs) that could be regulated by RanGTP during mitotic spindle assembly. A number of MAPs that contribute to spindle assembly are locally released from import receptors and activated around mitotic chromatin by a gradient of RanGTP. Xenopus nuclear factor 7 (Xnf7) was identified from X. laevis egg extracts as a mitotic MAP that binds to the importin alpha/beta complex.; I then returned to the topic of mitotic chromosome condensation when we set out to address the contribution of embryonic linker histone H1 to mitotic chromosomal architecture. Immunodepletion of histone H1 caused assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned and exhibited poleward movement, long and tangled chromosomes could not be segregated in anaphase.; My final graduate project focused on pioneering the use of Xenopus tropicalis egg extracts for in vitro assays, biochemistry and studying mechanisms that govern spindle length. These extracts support an array of functional assays commonly used in X. laevis extracts. (Abstract shortened by UMI.)...