Wednesday, March 12, 2025
3/12/2025
Project Summary The goal of mitotic cell division is to produce two cells from one and to ensure that each daughter cell inherits an exact copy of the original genetic material. When mitosis malfunctions, a common result is aneuploidy, in which cells contain the incorrect number of chromosomes. This impacts human health, since aneuploidy is a leading cause of birth defects and is implicated in cancer initiation and progression. The fidelity of mitosis depends on kinetochores, which are structures built at defined regions of chromosomes called centromeres. Kinetochores have several essential functions, including the following: (1) they connect chromosomes to spindle microtubules, (2) they regulate the strength of these connections, and (3) they ensure that cells do not exit mitosis if chromosomes are incorrectly attached to microtubules (via the spindle assembly checkpoint). Our lab focuses on understanding kinetochores, and specifically how they establish and regulate attachments to microtubules. Our lab also investigates how tumorigenesis results in kinetochore defects, and how these defects lead to cancer cell-specific vulnerabilities that can be exploited for cancer therapies. The first two projects in the proposal aim to uncover the mechanisms cells use to establish and regulate kinetochore-microtubule attachments in human cells. Based on our recent findings, we have generated new hypotheses for how the Aurora family of kinases (i.e., Aurora A, and Aurora B) impact kinetochore-microtubule attachment stability. A significant obstacle that has hindered progress in understanding how kinetochore kinases precisely regulate kinetochore-microtubule attachments is the lack of tools that permit unobtrusive tracking of the dynamics of these kinases and their activities at kinetochores with high spatial and temporal resolution. We have overcome this by developing methods to generate genetically-encoded, fluorescently-tagged, antibody-based probes that can be used to track these phenomena in living cells. We will use this approach to generate phosphorylation “sensors” that recognize active, phosphorylated forms of Aurora A and Aurora B kinases, as well probes directed to their target substate sites at kinetochores. We will use these tools – and generate new ones – to discover how kinetochore kinases regulate kinetochore-microtubule attachment stability throughout mitosis to ensure successful chromosome segregation. A related project will address how kinetochore-microtubule attachment status is communicated to the checkpoint. For this, we will employ our phospho-sensors, super-resolution imaging, and a newly-developed in vitro chromosome capture assay. Finally, we made the recent discovery that hyperactive signaling by the RAS and MAPK pathways over-stimulates kinetochore kinases to induce kinetochore defects and cancer cell-specific vulnerabilities in laboratory-transformed cells and glioblastoma tumor isolates. We aim to identify the targets of MAPK in mitosis and define new roles for RAS/MAPK signaling in dictating cancer vulnerabilities, with the long- term goal of devising strategies to target kinetochore dysregulation in cancer cells for therapeutic intervention.
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