Investigating the molecular mechanism of microtubule-generated tension in accurate chromosome segregation using Saccharomyces cerevisiae

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2023-08
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Bunning, Angela
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Gupta, Mohan
Yandeau-Nelson, Marna
McGrail, Maura
Johansen, Kristen
Walley, Justin
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Genetics, Development and Cell Biology
Abstract
Faithful segregation of chromosomes is a vital mitotic process that must happen every cell division cycle. During mitosis, chromosomes are replicated and bundled together before becoming attached to the mitotic spindle, a complex structure of microtubules. The mitotic spindle comprises microtubules, dynamic polymers that shorten and elongate by a process known as dynamic instability. These microtubules are organized by Spindle Pole Bodies (SPBs), known as centrioles in animal cells, that radiate the dynamic microtubules toward the center of the cells, where they can become connected to the sister chromatids. Once the chromosomes attach to the mitotic spindle and metaphase is achieved, the sister chromatids are pulled apart in anaphase. This metaphase-to-anaphase transition is important because it is an irreversible step in mitosis where sister chromatids separate. Each chromosome must become attached to microtubules at kinetochores, a complex of proteins that localize to the centromere of each chromosome. A mechanism known as the Spindle Assembly Checkpoint (SAC) delays anaphase onset until each kinetochore becomes attached to a microtubule. Once a dynamic microtubule becomes attached to a kinetochore, it exerts a pulling force on the kinetochore, a mechanical force known as tension. This tension force readies the kinetochore-microtubule attachment for anaphase. In some cases, kinetochores can become attached to microtubules where the tension cannot be generated. In this case, the tensionless kinetochore attachment must be corrected before anaphase proceeds. A conserved kinase known as Aurora B selectively phosphorylates tensionless kinetochores to induce detachment of tensionless kinetochore attachments. The cell is sensitive to both attachment and tension forces generated at kinetochores as signals of bipolar spindle formation which will lead to accurate chromosome segregation, but due to the intrinsic nature of the kinetochore-microtubule attachment, it has been difficult to understand the impact that strictly tension on the metaphase-anaphase transition. This work utilizes Taxol-sensitized budding yeast strains to manipulate kinetochore-microtubule attachments and tension status. This work documents detailed methods for working with these Taxol-sensitive yeast strains and assays developed to assess anaphase onset timing and SAC strength. Bub1, a conserved kinase involved in the SAC, is involved in delaying anaphase onset in the presence of low-tension kinetochores, but the mechanism behind this is unclear. This thesis addresses gaps in the field by investigating the role of Bub1 in mitotic signaling by generating a phosphoproteomic profile of the kinase domain in metaphase-arrested cells with unattached and tensionless kinetochores. We have found that Bub1 regulates cohesin proteins, specifically Pds5, needed for maintaining genome stability. This work also focuses on understanding molecular signaling due to differences in kinetochore conditions. Different classes of microtubule agents were used to manipulate kinetochore-microtubule conditions to create three distinct states: attached with tension (Control, mock treatment), unattached and tensionless (Nocodazole), and attached with low tension (Taxol). We generated a phosphoproteome of each condition and a kinase signaling network to reveal shared and unique signaling pathways impacted by kinetochore conditions. This work investigated tension as a molecular signal and what proteins are needed for tension sensing. Future directions must elucidate this signaling cascade and further clarify how these proteins work together to promote accurate chromosome segregation.
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