Mountain Scholar :: Browsing by Author "Markus, Steven, committee member"

Browsing by Author "Markus, Steven, committee member"

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Open Access
A new dawn for Aurora B regulation: shining light on multiple discrete populations of Aurora B kinase at centromeres and kinetochores
(Colorado State University. Libraries, 2020) Broad, Amanda J., author; DeLuca, Jennifer G., advisor; Luger, Karolin, committee member; Markus, Steven, committee member; Yao, Tingting, committee member; Amberg, Greg, committee member
Cell division is a fundamental biological process that is essential for all eukaryotes to divide the replicated genome with high fidelity into individual daughter cells. Improper segregation of replicated DNA results in chromosome instability, a characteristic that is deleterious to most cells. Critical to the proper segregation of mitotic chromosomes is attachment to spindle microtubules, which are dynamic cytoskeleton filaments that drive the movement of chromosomes during mitosis. A complex network of proteins, collectively called the kinetochore, mediates microtubule attachments to chromosomes. Kinetochores are recruited to individual chromosomes through a specialized heterochromatin domain known as the centromere. Centromeric heterochromatin is comprised of both canonical, H3-containing nucleosomes as well as nucleosomes that contain the histone H3 variant CENP-A. Centromeres serve as a central point of organization in mitotic cells, recruiting both structural and regulatory kinetochore proteins to chromosomes. This extensive protein/DNA network ensures the accurate segregation of chromosomes by regulation of proper kinetochore-microtubule attachments in mitosis. Kinetochore-microtubule interactions are regulated by Aurora B kinase, which phosphorylates outer kinetochore substrates to promote release of erroneous attachments. Although Aurora B kinase substrates at the kinetochore are defined, little is known about how Aurora B is recruited to and evicted from kinetochores, in early and late mitosis, respectively, to regulate these essential interactions. We set out to determine how Aurora B kinase is regulated during mitosis. We found that, contrary to the current model, Aurora B kinase and the Chromosomal Passenger Complex are recruited to distinct regions within the centromere and kinetochore. Furthermore, we found that accumulation of Aurora B kinase at centromeres is independent from Aurora B localization and activity at outer kinetochores. These results lead us to hypothesize a new model for Aurora B kinase regulation. In the direct recruitment model, a population of the kinase is recruited directly to kinetochores in early mitosis, then as mitosis progresses and kinetochore-microtubule attachments are stabilized, architectural changes within the kinetochore result in the eviction of outer-kinetochore localized Aurora B kinase and the stabilization of kinetochore-microtubule attachments.
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 member
The 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.
Open Access
Characterization of biased partner choice in mitotic non-allelic homologous recombination of Saccharomyces cerevisiae
(Colorado State University. Libraries, 2023) Merriman, Sean, author; Argueso, Juan Lucas, advisor; Markus, Steven, committee member; Nishimura, Erin, committee member; Wiese, Claudia, committee member
Using yeast as a model in which to study copy number variation (CNV)-generating mutations, the J.L. Argueso lab has discovered that a specific region of S. cerevisiae genome (the right arm of chromosome 7; Chr7R) is much more susceptible to sustaining deletions as a translocation recipient than other apparently similar segments of the genome. Further, Chr7R acquires amplifications as a translocation donor less frequently than other chromosomes. To begin unraveling the cause of this unusual behavior, we evaluated the effect of several candidate genes involved in chromatin mobility and sister chromatid cohesion on the mutational spectra involving Chr7R. Our results suggest that regulatory factors of chromatin mobility or sister chromatid cohesion affect the outcomes of HR-mediated repair events at Ch7R. We are hopeful that our findings will open a window into the fundamental cellular processes that are responsible for CNVs found in eukaryotic genomes, and inform translational implications for modeling this class of mutation in cancer.
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 member
Chromosome 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.
Open Access
Detection and measurements of free ubiquitin in fixed cells and characterization of OTUB1 contribution to ubiquitin homeostasis
(Colorado State University. Libraries, 2020) Prada Gomez, Luisa Fernanda, author; Cohen, Robert, advisor; Di Pietro, Santiago, committee member; Markus, Steven, committee member; Tamkun, Michael, committee member
Post-translational modifications with Ubiquitin (Ub) have been found to participate in a wide range of cell functions, including protein degradation, endocytosis, regulation of gene expression and cell cycle progression. Therefore, regulation of free Ub levels is essential to ensure that enough Ub is available for conjugation, while excess Ub does not compete in the large number of processes that depend on binding to ubiquitinated proteins or polyUb. Not surprisingly, changes in Ub pool dynamics can affect the cell functions, and perturbations of free Ub levels have been reported to cause neurological and developmental disorders. Although there are techniques to measure Ub pools in vitro, visualization and quantification of free Ub inside individual cells has not been possible. One way to regulate the intracellular concentration of free Ub, is by means of Deubiquitinating enzymes (DUBs), however specific details about the regulatory mechanism are, in large part, unknown. Most studies about DUBs have focused on enzymatic activity and regulation in vitro, with only few reports on the regulation of Ub homeostasis in vivo. The role of OTUB1 in Ub homeostasis has been hypothesized because its catalytic activity is affected by the ratio of [Ub~E2] to [E2] in response to free Ub concentration. Interaction between OTUB1 and a subset of E2s can stimulate OTUB1 isopeptidase activity, whereas interactions with Ub~E2s can inhibit the ubiquitin transfer from the thioester Ub~E2 adduct. This dissertation describes the successful development of a technique to detect and quantify changes in free Ub levels in fixed cells using a high affinity binding protein. The method was used to quantify changes in Ub levels after proteasome and E1 inhibition and to establish the free Ub distribution in hippocampal neurons. It was shown also that OTUB1 activity is not directly involved in the regulation of free Ub levels under stress conditions. However, a new mechanism for regulation of UBE2D expression levels dependent on OTUB1 was identified. This mechanism is independent of proteasomal degradation and could possibly involve translational regulation.
Embargo
DNA replication in the environmental extremes
(Colorado State University. Libraries, 2024) Liman, Geraldy Lie Stefanus, author; Santangelo, Thomas J., advisor; Markus, Steven, committee member; Schauer, Grant, committee member; Sloan, Daniel, committee member
DNA replication is an essential biological process across all life on Earth. For the prokaryotic Archaea domain, which contains organisms that can thrive in inhospitable environments like hydrothermal vents or salt deposits in the Dead Sea, the cell machinery for these conserved processes have acclimated over the course of evolution to encourage survival. While the origin of replication (ori), a predetermined position within the genome where DNA replication starts, is conserved in all Domains, its significance is not equal between them. Surprisingly, the model hyperthermophilic archaeon, T. kodakarensis, replicates its genome without relying on origin-dependent replication (ODR), and instead, relies mostly on recombination-dependent replication (RDR). In fact, the ori in T. kodakarensis is dispensable from the organism without much phenotypic consequence. Although dispensable, ori persists after millions of years of evolution in this organism, suggesting some functional significance under certain conditions. Not to mention, archaeal replisomes are comprised of unique components that are distinct from the other two domains of life, though surprisingly more similar to those found in Eukarya. Central to all replisomes is the activity of the DNA polymerase (DNAP). Most archaeal organisms, except for the Creanarchaea, encode two main replicative DNAPs, the eukaryotic-like B-family DNAP (PolB) and the archaeal-specific D-family DNAP (PolD). In T. kodakarensis, PolD is the essential replicative DNAP while PolB is dispensable. This thesis aims to (1) characterize the activity and regulation of RadA, the main recombinase in Archaea, (2) characterize the exaptation of inteins to regulate DNA replication, (3) delineate the in vivo function(s) of PolB. Furthermore, I hope to further characterize DNA replication in the context of evolutionary biology and how it relates to the three Domains of life.
Open Access
Investigating mitotic vulnerabilities that arise upon oncogenic cell transformation
(Colorado State University. Libraries, 2020) Shirnekhi, Hazheen K., author; DeLuca, Jennifer G., advisor; Markus, Steven, committee member; Nishimura, Erin, committee member; Bailey, Susan, committee member
During mitosis, cells must accurately divide their duplicated chromosomes into two new daughter cells. This process is highly regulated and much of this regulation is centered around kinetochores. Kinetochores are large proteinaceous structures built upon centromeric heterochromatin that must form stable, load-bearing attachments to microtubules (MTs) emanating from the spindle poles. Failure to undergo high-fidelity cell division can result in aneuploidy and even progress to continued mis-segregation in a phenomenon known as chromosome instability (CIN). As aneuploidy results from defective pathways in mitosis, it is important to characterize the changes cancer cells exhibit in their mitotic machinery, with the goal of identifying targets for therapeutic potential. Here, we utilize a human papillomavirus cell culture model system to determine how expression of E6 or E7, two viral transforming proteins, influences mitosis. We find that E6-expressing cells exhibit a weakened spindle assembly checkpoint (SAC) and an increased incidence of pole-associated chromosomes. This combination of mitotic errors allows some of these cells to exit mitosis in the presence of improper kinetochore-MT attachments, leading to aneuploid daughter cells. Defective mitotic processes in cancer cells provide a means of differentiating them from healthy cells, which may be important in developing new effective cancer therapeutics. Through two independent cancer lethality screens, the mitotic proteins BuGZ and BubR1 were identified as essential for Glioblastoma Multiforme cancer cell survival but dispensable for healthy neural cell survival. We characterize the important chaperone-like role BuGZ plays in mitosis to examine its apparent dispensability in healthy cells. BuGZ aids in the kinetochore loading of Bub3, which in turn is needed for the kinetochore loading of proteins with important roles in kinetochore-MT attachment and in spindle assembly checkpoint signaling. BubR1's cancer lethality has also been previously described in Glioblastoma cells. BubR1 is needed to recruit the PP2A phosphatase to kinetochores, where it stabilizes kinetochore-microtubule attachments. We identify the HEC1 tail as a substrate for the BubR1-recruited population of PP2A, and we demonstrate that kinetochore-microtubule attachment defects in BubR1 depleted cells can be rescued with a phospho-null HEC1 mutant. This work identifies important changes in the mitotic machinery of transformed cells, providing potential pathways to target for therapeutics that may apply to many different cancers.
Open Access
Regulation of actin capping protein during clathrin-mediated endocytosis
(Colorado State University. Libraries, 2022) Lamb, Andrew, author; Di Pietro, Santiago, advisor; Krapf, Diego, committee member; Markus, Steven, committee member; Peersen, Olve, committee member
Clathrin-mediated endocytosis (CME) is a major endocytic pathway that is essential in all eukaryotic cells. In the budding yeast S. cerevisiae, polymerization of actin into a branched network is critical to provide the force necessary for membrane invagination during CME. Polymerization of this branched actin network is a highly regulated process, reliant on a multitude of endocytic factors for proper formation. A key regulator is actin capping protein (CP), which binds to the barbed end of actin filaments with high affinity to prevent the loss or addition of actin subunits. While regulation of CP by proteins containing a capping protein-interacting (CPI) motif has been demonstrated in higher eukaryotes, it has not been described in yeast or during endocytosis. Here, we identify and dissect the roles of three CPI motif-containing endocytic factors, Aim21, Bsp1 and Twf1, in CP regulation. Aim21 was the first CPI motif we identified, and the first CPI motif described in yeast. Together with its binding partner Tda2, Aim21 binds to CP through its CPI motif with nanomolar affinity. We demonstrate that Tda2 functions as a dimerization engine for Aim21, bringing two molecules of Aim21 together to form a hetero-tetrameric complex that we term the Tda2-Aim21 complex. Formation of the Tda2-Aim21 complex is essential for a strong interaction with CP, as Aim21 alone binds to CP with more than a 10 fold weaker affinity. Mutating the CPI motif of Aim21 in the yeast genome leads to a recruitment defect in CP and an over-accumulation of F-actin at CME sites, suggesting Aim21 aids in the recruitment of CP to endocytic sites. The little-studied endocytic factor, Bsp1, displays the same phenotype when its CPI motif is mutated in yeast. In addition, the Bsp1 and Aim21 CPI motifs allosterically inhibit the capping function of CP during in vitro actin polymerization assays. When mutations to both the Aim21 and Bsp1 CPI motifs are combined in yeast, CP localization to CME sites is severely reduced, demonstrating that Aim21 and Bsp1 have redundant functions during yeast CME in recruiting a transiently active CP to cortical actin patches. In contrast, the well-conserved actin disassembly factor, twinfilin (Twf1), is not important for recruitment of CP, but is itself reliant on its interaction with CP to localize to CME sites. While the CPI motifs of Aim21 and Bsp1 inhibit the capping function of CP, the Twf1 CPI motif has no effect, despite binding to CP with nanomolar affinity. Mutation of the Twf1 CPI motif results in an accumulation of CP and F-actin at endocytic sites, suggesting that it functions downstream of CP recruitment to recycle CP and actin network components. Together, these findings shed light on how CPI motifs regulate CP in in a step-wise manner during yeast endocytosis.
Open Access
Strange translation - investigating ires-mediated and codon non-optimal translation dynamics at the single mRNA level in living cells
(Colorado State University. Libraries, 2021) Koch, Amanda Lynn, author; Stasevich, Timothy, advisor; Munsky, Brian, committee member; Markus, Steven, committee member; Peersen, Olve, committee member; Wilusz, Jeffrey, committee member
With the advent of Nascent Chain Tracking (NCT), a technique used to visualize single-molecule events of translation in living cells, answering detailed questions about how, when, and where translation is occurring in living cells is possible. Since its publishing debut in 2016, NCT has provided a wealth of information about translation initiation and elongation dynamics, subcellular localization, translation site structure, and reaction to stress for both canonical and non-canonical translation in living cells. Here, we slightly modify the NCT assay to quantify translation dynamics when a ribosome is recruited to an mRNA in a non-canonical fashion and when a ribosome encounters codon non-optimal stretches on a transcript. The first step of translation requires a primed ribosome to be recruited to a readied mRNA. Canonically, this recruitment takes place on the 5' cap of an mRNA and is termed cap-dependent initiation. However, some eukaryotic messages and many viral RNAs use an internal ribosome entry site (IRES) to recruit ribosomes and initiate translation in a cap-independent manner. Specifically, viruses use IRES elements to hijack host ribosomes to translate viral proteins and properly propagate in host cells. While well- studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5'-cap, the other from the Encephalomyocarditis Virus IRES. When combined with a pair of complementary probes that bind the epitopes co-translationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap- dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states. Once the ribosome has been recruited to the mRNA and a start codon located, the ribosome will begin decoding the mRNA in nucleotide triplets or codons to ultimately create a protein. In some cases, the ribosome encounters a codon that it cannot decode efficiently. The relationship between codons and ribosome efficiency is termed codon optimality. It has been shown that codon non-optimal mRNA are less stable in cells. However, little is known about the effects of codon non-optimality on translation kinetics and overall translation regulation. In an ongoing collaboration with the Rissland group, we use bulk assays and NCT to address unanswered questions about how codon non- optimality leads to translation regulation along with mRNA instability. Thus far, we have evidence to support that translation repression is occurring in codon non-optimal conditions through inhibition of ribosome initiation and slower elongation. Further investigations of exact translation repression mechanisms are ongoing.

Browsing by Author "Markus, Steven, committee member"

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