Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 100).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 30-37).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [69]-76).

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb 10, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 163-179).

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

The overall objective of this study was to identify novel proteins of the nuclear matrix in order to contribute to a better understanding of nuclear structure and organization. To accomplish this, a monoclonal antibody specific for the nuclear matrix was used to screen a human λgt11 expression library. Several cDNAs were isolated, cloned, sequenced, and shown to represent NuMA, the nuclear mitotic spindle apparatus protein. Further characterization of the gene and RNA was undertaken in an effort to obtain information about NuMA. The NuMA gene was present at a single site on human chromosome 11q13. Northern and PCR analysis of NuMA mRNA showed a major 7.2 kb transcript and minor forms of 8.0 and 3.0 kb. The minor forms were shown to be alternatively spliced although their functional significance is not yet understood. Immunofluorescence microscopy demonstrated that NuMA oscillates between the nucleus and the microtubule spindle apparatus during the mitotic cell cycle. NuMA appeared as a 200-275 kDa protein detectable in all mammalian cells except human neutrophils. To determine whether NuMA's changes in intracellular distribution correlated with post-translational modifications, the protein's phosphorylation state was examined through the cell cycle using highly synchronized cells. NuMA was a phosphoprotein in interphase and underwent additional phosphorylation events in mitosis. The mitotic phosphorylation events occurred with similar timing to lamin B (G2/M transition) and were concomitant with NuMA's release from the nucleus and its association with the mitotic spindle. However, the mitotic phosphorylation occurred in the absence of spindle formation. Dephosphorylation of NuMA did not correlate with reassociation with the nuclear matrix but occurred in two distinct steps after nuclear reformation. Based on the timing of these events, phosphorylation may playa role in nuclear processes. In conclusion, the work in this dissertation identified NuMA, a nuclear matrix protein and showed that it is phosphorylated during the cell cycle and may be important for nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.

The proper transmission of genetic materials into progeny cells is crucial for maintenance of genetic integrity in eukaryotes and fundamental for reproduction of organisms. To achieve this goal, chromosomes must be attached to microtubules emanating from opposite poles in a bi-oriented manner at metaphase, and then should be separated equally through proper spindle elongation in anaphase. Failure to do so leads to aneuploidy, which is often associated with cancer. Despite the presence of a safety device called the spindle assembly checkpoint (SAC) to monitor chromosome bi-orientation, mammalian cells frequently possess merotelic kinetochore orientation, in which a single kinetochore binds microtubules emanating from both poles. Merotelically attached kinetochores escape from the surveillance mechanism of the SAC and when cells proceed to anaphase cause lagging chromosomes, which are a leading cause of aneuploidy in mammalian tissue cultured cells. The fission yeast monopolin complex functions in prevention of mal-orientation of kinetochores including merotelic attachments during mitosis. Despite the known importance of Cdk1 activity during mitosis, it has been unclear how oscillations in Cdk1 activity drive the dramatic changes in chromosome behavior and spindle dynamics that occur at the metaphase/anaphase transition. In two separate studies, we show how dynamic Cdk1 phosphorylation regulates chromosome segregation. First, we demonstrate that sequential phosphorylation and dephosphorylation of monopolin by Cdk1 and Cdc14 phosphatase respectively helps ensure the orderly execution of two discrete steps in mitosis, namely sister kinetochore bi-orientation at metaphase and spindle elongation in anaphase. Second, we show that elevated Cdk1 activity is crucial for correction of merotelic kinetochores produced in monopolin and heterochromatin mutants.

DNA damage induces mitotic exit delays through a process that requires the spindle assembly checkpoint (SAC), which blocks the metaphase to anaphase transition in the presence of unaligned chromosomes. Using time-lapse confocal microscopy in syncytial Drosophila embryos, we show that DNA damage leads to arrest during prometaphase and anaphase. In addition, functional GFP fusions to the SAC components MAD2 and Mps1, and the SAC target Cdc20 relocalize to kinetochore through anaphase arrest, and a null mad2mutation blocks damage induced prometaphase and anaphase arrest. We also show that the DNA damage signaling kinase Chk2 is required for damage induced metaphase and anaphase arrest, and that a functional GFP-Chk2 fusion localizes to kinetochores and centrosomes through mitosis. In addition, in the absence of Chk2, we find that DNA damage sufficient to fragment centromere DNA does not delay mitotic exit. We conclude that DNA damage signaling through Chk2 triggers Mad2-dependent delays in mitotic progression, both before or after the metaphase-anaphase transition.