Date of Award
Fall 9-2023
Document Type
Thesis (Ph.D.)
Department or Program
Biochemistry and Cell Biology
First Advisor
Duane A. Compton
Second Advisor
Kristina M. Godek
Abstract
Cells progress through different stages of the cell cycle as they ultimately prepare to divide during mitosis. Mitosis relies on many interconnected networks to carry out the coordinated events required for faithful chromosome segregation. When a cell enters mitosis, chromosomes are highly condensed and sister chromatids are held together by cohesion. Kinetochores assemble at the constricted regions on each chromosome. The kinetochore is a large macromolecular structure composed of many proteins that form the binding site for microtubules of the mitotic spindle to attach. These kinetochore-microtubule (k-MT) attachments serve to align chromosomes at the center of the cell and are necessary to segregate DNA into the resulting daughter cells during anaphase. Because erroneous k-MT attachments can result in chromosome missegregation, this establishes the need for monitoring and correction mechanisms to ensure that proper bioriented attachments are favored. The rate-limiting step for error correction is microtubule detachment from kinetochores to permit microtubules with proper orientation to attach. However, the underlying mechanisms for this correction process are incompletely understood. Two general models include coordinated control of k-MT attachments on all chromosomes and targeted chromosome-autonomous control. Evidence indicates that early in mitosis, the stability of k-MT attachments is coordinately controlled by a mitotic cell cycle regulator, Cyclin A, likely as a regulatory subunit of Cyclin-dependent kinase (CDK). In addition to regulating k-MT attachment stability in early mitosis, it remains unknown if Cyclin A/CDK regulates other early mitotic events. In chapter two, I tested if Cyclin A is required for other key events in early mitosis. Using live and fixed cell-imaging approaches I demonstrate that the duration of mitosis is extended and that the localization of kinetochore proteins with documented roles in chromosome alignment are reduced in Cyclin A-depleted cells. The data generated here demonstrates that Cyclin A promotes multiple steps to ensure the efficient execution of events in early mitosis. Additionally, I contributed to efforts designed to test if there is feedback from k-MT attachment stability to molecular components with known roles in mitotic regulation. In chapter three, I used quantitative fluorescence microscopy to demonstrate that subtle changes in k-MT attachment stability alter the activity of Aurora B kinase at centromeres. This data complements other data generated with an Aurora B-substrate phosphorylation biosensor and demonstrates that Aurora B kinase activity responds to very subtle changes in k-MT attachment stability. Collectively, these data provide insight into how the complicated events in early mitosis are regulated to support faithful chromosome segregation during cell division.
Recommended Citation
Valles, Sarah Yvette, "Molecular Mechanisms Regulating Prometaphase and Metaphase States for Faithful Chromosome Segregation" (2023). Dartmouth College Ph.D Dissertations. 172.
https://digitalcommons.dartmouth.edu/dissertations/172