Browsing by Author "DeLuca, Jennifer, advisor"
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Item Open Access Aurora A kinase phosphorylates serine 62 on Hec1 to affect mitotic kinetochore microtubule interactions(Colorado State University. Libraries, 2024) Sparrow, Sarah, author; DeLuca, Jennifer, advisor; Markus, Steven, committee member; Bailey, Susan, committee memberThe Hec1 protein plays an important role in ensuring successful chromosome segregation during cell division. Its 80 amino acid, unstructured, "tail" region is critical for kinetochore-microtubule attachment regulation, which is mediated through Aurora kinase phosphorylation. At least nine phosphorylation target sites within this domain have been identified, including the recently confirmed target site, serine 62 (S62). However, the functional significance of phosphorylation of this residue remains elusive. Here, we selectively target Aurora A and Aurora B kinase protein activities using the inhibitors MLN8054 and ZM447439, respectively, and study their effects on the dynamics of serine 62 phosphorylation in the Hec1 tail. Utilizing immunofluorescence, we demonstrated that inhibition of Aurora A kinase activity leads to a significant reduction in phosphorylation levels at serine 62. Additionally, using phospho-null mutants, we studied the effect of serine 62 phosphorylation on the creation of stable, tension-generating kinetochore-microtubule attachments by measuring the distance between sister kinetochores. Our findings reveal that alterations in serine 62 phosphorylation status result in subtle changes in interkinetochore distances showcasing the functional relevance of this phosphorylation event in regulating kinetochore-microtubule attachments. Furthermore, under conditions of nocodazole-induced mitotic arrest, we observe a marked decrease in phosphorylation at serine 62 suggesting a microtubule dependent regulation of this phosphorylation. These findings provide evidence supporting the role of Aurora A kinase in phosphorylating serine 62 of the Hec1 tail and shed light on the regulation of this critical post-translational modification during mitosis.Item Open Access Characterizing the role of the Hec1 tail domain at the kinetochore-microtubule interface in human cells(Colorado State University. Libraries, 2020) Wimbish, Robert T., author; DeLuca, Jennifer, advisor; Markus, Steven, committee member; Reddy, Anireddy, committee member; Ross, Eric, committee memberChromosome segregation is powered by interactions between the mitotic spindle and kinetochores. Kinetochores – large, protein-rich machines built on the centromere of each sister chromatid – must bind to spindle microtubules and harness the forces from their dynamic instability to drive chromosome movement. This interaction must be robust enough to ensure chromosomes remain bound to the growing and shrinking microtubule polymers, yet must also be reversible: incorrectly oriented kinetochore-microtubule attachments can cause chromosome mis-segregation leading to aneuploidy, which can be catastrophic for the newly formed cell. Thus, cells must be able to actively regulate the strength with which kinetochores bind to spindle microtubules – such a regulatory scheme ensures that incorrect attachments can be released, and correct attachments can be preferentially stabilized. The direct linkage between kinetochores and microtubules is the highly conserved, kinetochore-anchored NDC80 complex. This complex is also an effector of attachment strength regulation; specifically, the N-terminal "tail" region of the NDC80 complex subunit Highly expressed in cancer 1 (Hec1) is a target for phosphorylation by the Aurora family of kinases, which ultimately weakens kinetochore-microtubule attachments. Here, we investigate the molecular basis for kinetochore-microtubule attachment regulation in human cells. We find that Hec1 tail phosphorylation regulates kinetochore-microtubule attachments independently of the spindle and kinetochore associated (Ska) complex, a critical factor for attachment stability, contrary to previous reports that the two pathways are functionally coupled. We additionally map the domains of the NDC80 complex required for its coordination with Ska complexes to strengthen attachments. We also find that the Hec1 tail domain is dispensable for the initial formation of kinetochore-microtubule attachments, but provide evidence it plays a role in force generation. We further interrogate this role and how phosphorylation of the tail regulates attachment formation and force generation, and find that the length requirements for these functions of the tail are different. Moreover, we demonstrate that the phospho-regulatory pathway for attachment regulation is deficient for short tails, suggesting a new model for the means by which attachments are regulated. Together these results provide novel insight into how attachments between chromosomes and the spindle are formed and regulated, and how errors in this process can lead to chromosome mis-segregation.Item Open Access Evaluating the effect dynein and related proteins exhibit on the spindle assembly checkpoint and kinetochore(Colorado State University. Libraries, 2019) Biebighauser, Tyler, author; Markus, Steven, advisor; DeLuca, Jennifer, advisor; Di Pietro, Santiago, committee member; Hoerndli, Fred, committee memberTo ensure that cell division is faithfully carried out without causing genetic errors, eukaryotic cells have evolved several conserved checkpoints during mitosis. One such checkpoint, the Spindle Assembly Checkpoint (SAC), blocks the cell from progressing through metaphase until all chromosomes have become bi-oriented by microtubules. Only once this occurs can the cell progress into anaphase to separate the sister chromatids. Errors in this checkpoint have been linked with aneuploidy, which itself is linked with oncogenesis. Naturally there are many layers of regulation within the SAC, most of which are associated with a proteinaceous structure on the sister chromatid – the kinetochore. The molecular motor dynein, and its kinetochore localized co-factors play several roles in this regulation. In one of these roles, dynein strips away kinetochore localized signal proteins upon microtubule bi-orientation, to weaken the strength of the SAC. We initially set out to test whether this process of SAC stripping has further levels of regulation, or if all dynein requires to strip these signal proteins is the presence of a microtubule. We used in-vitro motility assays to investigate whether dynein's motility along microtubules is changed depending on the length of its kinetochore localized cargo adapter, spindly. We purified truncated versions of spindly to test if it undergoes regulation analogous to other dynein cargo adapters. These in-vitro motility assays showed no difference in dynein motility past a certain length required to confer motility. Interestingly, we observed that some of the shorter spindly truncations undergo phase separation both in-vitro in the right conditions and in-vivo when transfected into HeLa cells. We postulate that this phase separation could have implications in a process called fibrous corona expansion, which occurs on a kinetochore that has spent a long time in prometaphase without attaching to a microtubule. In total these studies shed light on the nature of interactions at the kinetochore, and the complexity of regulation as it pertains to dynein mediated kinetochore stripping.Item Open Access Investigating the role of kinetochore dynein-dynactin in spindle assembly checkpoint function(Colorado State University. Libraries, 2020) Hodges, Amy Lauren, author; DeLuca, Jennifer, advisor; Markus, Steven, advisor; Bailey, Susan, committee memberWhen a cell divides it is essential that its chromosomes are equally divided into two new daughter cells, thus ensuring that each cell receives an identical copy of genetic information. The importance of this process is emphasized by the fact that a hallmark of cancer cells is erroneous chromosome segregation, leading to uncontrolled proliferation. A key cellular structure involved in maintaining genomic integrity is the kinetochore, a large proteinaceous structure that assembles upon centromeric chromatin during cell division. This complex structure is involved in linking mitotic chromosomes to spindle microtubules, as well as detecting and correcting erroneous kinetochore-microtubule attachments to ensure faithful chromosome segregation. Monitoring of kinetochore-microtubule attachments is carried out by the spindle assembly checkpoint (SAC), a surveillance system that generates a "wait anaphase" signal at unattached kinetochores, with the goal of delaying cell division until every kinetochore has attached to a spindle microtubule. The checkpoint signal is propagated by SAC effector proteins that accumulate at the outer surface of unattached kinetochores during mitosis. In metazoan cells, the minus end-directed motor protein cytoplasmic dynein-1 (dynein) is known to facilitate eviction of SAC effectors from the kinetochore upon stable microtubule attachment, effectively silencing the checkpoint and allowing for anaphase progression. It has been suggested that dynein-mediated eviction of checkpoint proteins is dependent on dynein's microtubule-based motor activity, with the prevailing model depicting SAC effectors transported as cargo toward the poles by the dynein motor along spindle microtubules. However, data supporting this model is lacking and the process is poorly understood. Here we have identified a subset of SAC effectors that require dynein for their removal from the kinetochore upon stable microtubule attachment. Additionally, we have generated a CRISPR cell line in which dynein is endogenously tagged, allowing us to characterize activity of kinetochore dynein in a manner not previously possible. Using this cell line in conjunction with small molecule-based inhibition of mitotic processes, we sought to investigate the role of spindle microtubules in dynein-mediated SAC silencing. Interestingly, our data show that dynein-mediated removal of key checkpoint proteins from kinetochores can occur in the complete absence of microtubules, suggesting a motility-independent role for the dynein motor in SAC silencing.Item Open Access Mapping the recruitment pathways of core spindle assembly checkpoint proteins(Colorado State University. Libraries, 2017) Mallal, Daniel, author; DeLuca, Jennifer, advisor; Markus, Steven, advisor; Spencer, John, committee memberThe Spindle Assembly Checkpoint (SAC) is a vital regulatory pathway in eukaryotic cells to ensure proper division of duplicated chromosomes such that each daughter cell receives a complete and equal copy of genetic material. The SAC specifically ensures that kinetochores form proper attachments to spindle microtubules by preventing anaphase until every chromosome is bi-oriented and attached at each pair of kinetochores to microtubules emanating from opposite spindle poles. The SAC is a highly regulated and intricate network of proteins which allows for a robust inhibitory signal to be produced in the presence of erroneous attachments, halting cells in anaphase allowing for error correction. An important set of interactions occurs surrounding the proteins Bub1, BubR1, BuGZ binding to Bub3 mediated through a GLEBS domain binding Bub3. The precise nature of the interplay between these proteins binding to Bub3 is rather unclear and requires further characterization. Here we set out to characterize the direct recruitment sufficiency of each of these proteins. In order to distinguish the direct recruitment sufficiency of each individual protein, we targeted Bub1, BubR1, Bub3, and BuGZ individually to an ectopic site on chromosomes away from the kinetochore. We find that Bub1, BubR1, and Bub3 are sufficient to recruit each other as well as BuGZ, however BuGZ is only able to recruit Bub3 indicating that the Bub3-BuGZ GLEBS interaction is the strongest of the three. Interestingly, we also find that BuGZ is able to recruit Bub3 less efficiently in mitotic cells, suggesting a regulatory mechanism that decreases the affinity of BuGZ for Bub3 as cells transition into mitosis. Together, these data support a model in which BuGZ is exchanged for Bub1 to bind Bub3 at kinetochores in mitosis to promote efficient SAC signaling.Item Open Access Mediation of kinetochore-microtubule interactions through the Ndc80 complex component Hec1(Colorado State University. Libraries, 2011) Guimaraes, Geoffry J., author; DeLuca, Jennifer, advisor; Bamburg, James, committee member; Peersen, Olve, committee member; Reddy, A. S. N., committee memberTo view the abstract, please see the full text of the document.Item Open Access Telomere length as a biomarker of exposure to indoor woodstove smoke in rural Honduras: a feasibility field study(Colorado State University. Libraries, 2017) Altina, Noelia, author; DeLuca, Jennifer, advisor; Bailey, Susan, advisor; Ross, Eric, committee member; Clark, Maggie, committee memberTelomeres, the natural ends of linear chromosomes, are important for maintaining genome stability. Telomere length is an inherited trait influenced by a host of lifestyle and environmental factors, which have been shown to accelerate the rate of telomere shortening, and thus of aging. Indoor air pollution is one of the environmental factors known to influence the length of telomeres. It has been reported that people exposed to this kind of contamination, have an increased risk for pulmonary diseases, cardiovascular diseases and cancer. The accumulation of evidence correlating telomere length with different diseases and chronological age supports the use of short telomere frequency as an informative biomarker of general health status and aging. Epidemiological studies suggest that increased frequencies of nuclear aberrations (micronuclei, buds) are also correlated with exposure to air pollution.Item Open Access The kinetochore protein KNL1 links kinetochore-microtubule attachment and checkpoint signaling during mitosis(Colorado State University. Libraries, 2014) Caldas, Gina V., author; DeLuca, Jennifer, advisor; Allen, Christopher, committee member; Di Pietro, Santiago, committee member; Peersen, Olve, committee member; Prenni, Jessica, committee memberMitosis is the phase of the cell cycle in which replicated chromosomes physically separate, resulting in the formation of two genetically identical daughter cells. This process is not only essential for the development of a single fertilized cell into a multicellular organism, but also for replacement of damaged and dying cells during the span life of an organism. The distribution of chromosomes during mitotic cell division requires accurate yet dynamic attachment between the plus-ends of spindle microtubules (MTs) and kinetochores, which are protein structures assembled at the centromeric region of replicated chromatids. The tightly regulated connection between kinetochores and MTs allows for chromosome congression to the metaphase plate and subsequent separation of the replicated chromosomes during anaphase. Not surprisingly, the inability of cells to resolve erroneous kinetochore-MT attachments results in missegregation of chromosomes, which is linked to uncontrolled cell proliferation and cancer. Thus, proper kinetochore-MT attachment during cell division is essential for the maintenance of genetic integrity. Despite a growing understanding of the identity of proteins that compose the kinetochore and the processes for which they are required, the precise functions of many kinetochore proteins are still unknown. KNL1, a large kinetochore scaffolding protein, contributes to several signaling pathways coordinated by the kinetochore. Yet, how KNL1 recruits its various binding partners to the kinetochore, and whether KNL1 directly or indirectly modulates protein function during mitosis are unresolved questions. In this dissertation, I examine the function of KNL1 in the regulation of kinetochore-MT attachment and determine the regions of KNL1 required for the accumulation of an array of kinetochore proteins. By loss of function analyses using a set of KNL1 mutants, combined with functional assays in cells, I demonstrate that the KNL1 N-terminus is essential for Aurora B kinase activity at kinetochores and for correct kinetochore-MT dynamics. Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore-microtubule (MT) turnover and preventing premature stabilization of kinetochore-MT attachments. Therefore, KNL1 is required for correct Aurora B-mediated kinetochore-MT attachment regulation during mitosis. I provide evidence that the KNL1 N-terminus influences Aurora B activity by mediating the activity of Bub1 kinase, a kinetochore protein required for the spindle assembly checkpoint (SAC). The SAC mediates amplification of an inhibitory signal to prevent mitotic exit until all chromosomes are correctly attached to MTs. Although the SAC is known to be tightly coupled to kinetochore-MT attachment, how such coupling occurs at the kinetochore is a major unanswered question. The finding that KNL1 mediates Aurora B activity through Bub1 establishes KNL1 as a key integrator of multiple signaling pathways at the kinetochore. Finally, I determine the regions of KNL1 required for the accumulation of several kinetochore proteins, providing a broad view and better understanding of kinetochore organization inside the cell. Overall, results from these studies establish KNL1 as a central organizer of kinetochore architecture and function, and demonstrate the direct influence of this scaffolding protein on kinetochore-mediated regulatory processes during mitosis.