Repository logo

Browsing by Author "Nishimura, Erin, committee member"

Now showing 1 - 5 of 5
  • Thumbnail Image
    ItemOpen 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.
  • Thumbnail Image
    ItemOpen Access
    Elucidating translation control of Argonaute in live cells by developing a tetherable biosensor with single-mRNA resolution
    (Colorado State University. Libraries, 2021) Cialek, Charlotte A., author; Stasevich, Timothy, advisor; Montgomery, Taiowa, advisor; Hoerndli, Fred, committee member; Nishimura, Erin, committee member; Ross, Eric, committee member
    Translation is an essential step for all living beings. It stands as the final hurdle of converting our genetic code into functional protein products. A culmination of specific factors heavily regulates what, when, and how much of a given peptide product is translated (Chapter 1). Though many technological advancements have expanded our understanding of translational control, they have often opened many new questions due to the high complexity of this process. Recently, a technique called Nascent Chain Tracking (NCT) was able to image translation with single molecule resolution in living cells (Chapter 2). NCT-based technologies have overcome some of the limitations of conventional in vivo and in vitro approaches to study translation. Though these NCT-based technologies accomplished detection of heterogeneity of translation dynamics, they were not capable of studying how specific factors mediate translational control. The process of translation can be controlled by small RNA silencing pathways that restrict or completely block protein production. Of these, microRNAs (miRNAs) direct translational repression and mRNA decay by guiding the enzyme Argonaute and its associated proteins to partially complementary sequences on target mRNAs (Chapter 1). The dynamics of miRNA-mediated gene silencing, and in particular the role of Argonaute on translation, remain difficult to interpret due to pathway's complexity, long (minutes-to-hours) timescale, and conflicting results from different studies. To address these problems, we developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on translation and subcellular localization of individual reporter mRNAs in living cells (Chapter 3). Translation and Tethering (TnT) is a tethering-based single molecule reporter that simultaneously monitors translation and Ago2-tethering status in live human cells. Since this technique is microscopy-based, its readout includes valuable subcellular localization and intensity information over timeframes ranging from seconds to hours, which describe when, where, and how much translation and tethering is occurring per single-mRNA. Finally, to simplify using this multi-construct, multi-probe system, we adapted a cell loading technique, called bead loading, to introduce TnT plasmids and proteins simultaneously into adherent cells (Chapter 4). Our TnT system reflects endogenous miRNA-mediated gene silencing when we compared it to natural miRNA-target site recruitment. Using the TnT system, we find that Ago2 association leads to progressive repression of translation at individual mRNA. The timescale of silencing was similar to that of translation, consistent with a role for Ago2 in blocking translation initiation and subsequent runoff of the ribosomes already engaged in translation elongation. At early timepoints, we observed occasional brief bursts of translational activity at Ago2-tethered mRNAs undergoing silencing, suggesting that translational repression may initially be reversible. At late timepoints, Ago2-tethered mRNA were redirected into P-bodies where they remained translationally silenced for 10+ hours, which was the duration of the experiment. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single molecule level (Chapter 5). Furthermore, due to the adaptability of the TnT system, it will likely have wide-ranging application in studying RNA-protein interactions more generally.
  • Thumbnail Image
    ItemOpen 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.
  • Thumbnail Image
    ItemOpen Access
    Modulating translation dynamics with tunable optogenetic protein recruitment
    (Colorado State University. Libraries, 2024) Fixen, Gretchen M., author; Stasevich, Timothy, advisor; Nishimura, Erin, committee member; Chung, Jean, committee member
    Genes encoded in our DNA are fundamental to human health and well-being. Their imperative role requires tight regulation throughout their journey to becoming functional proteins. These regulations, when disrupted, have been linked to many neurodegenerative disorders and cancers, stressing the importance of deconvolving their components. Translation is one of the final steps in this journey that has been extensively explored, resulting in a recent technique developed known as nascent chain tracking (NCT) coupled with MS2 stem loop tagging. Using this technique, we are able to track translation dynamics in real-time and in live cells. Despite this, there are still limitations in spatially and temporally tracking the recruitment of translation effectors to translation sites and accurately measuring these dynamics. With the incorporation of optogenetic blue-light-sensitive proteins, we can generate inducible biomolecular condensates that recruit green fluorescent protein (GFP)-tagged proteins and our reporter mRNAs. Using this controlled test-tube-like environment, we can discover the direct effects ribosomal quality control proteins have on translation dynamics. A main quality control pathway involves ZNF598, GIGYF2, and 4EHP proteins that mediate translation control during ribosome stalling. We discovered that both GIGYF2 and 4EHP can be recruited to these clusters and co-localize with our active translation sites in live cells. Further exploration found that 4EHP alone cannot fully cause translation inhibition with our system. Despite this, we do see translation initiation occurring over time due to complex formation with HIF-2∝. However, GIGYF2 has distinct effects on these kinetics that are variable. This tool, when optimized, will be able to describe different proteins' effects on translation kinetics in an isolated environment in live cells.
  • Thumbnail Image
    ItemOpen Access
    The cardiac jelly extracellular matrix contributes to valve development and overall cardiac function
    (Colorado State University. Libraries, 2022) Ostwald, Paige, author; Garrity, Deborah, advisor; Bark, David, committee member; Bedinger, Patricia, committee member; Nishimura, Erin, committee member; Peers, Graham, committee member
    Nearly 2.6 million infants are born every year with a congenital cardiac anomaly across the entire globe. Congenital heart defects (CHDs) within the valve occur in over 50% of cases. 56% of heart defects have an unknown etiology, illuminating the need for continuous research on heart development and potential causes. Before the valve is a mature structure with established leaflets, the heart forms two endocardial cushions that press together to occlude blood flow between chambers. The cushions are composed of an extracellular matrix called the cardiac jelly (CJ). Previous studies have found evidence of the vital role the cardiac jelly plays within the developing valve for structure, genetic signaling and cell organization. Here, we present a specific role the cardiac jelly plays in valve function and overall cardiac output. To alter the cardiac jelly, we used a morpholino approach in a zebrafish model to increase, decrease and structurally compromise the cardiac jelly. By doing so, we found decreased valve cell differentiation with decreased CJ and increased valve cell differentiation with increased CJ. Using high-speed video technology, we also found decreased valve opening regardless of cardiac jelly alteration, resulting in reduced overall cardiac function. Our results suggest that the function of the endocardial cushions relies on an appropriate presence of CJ. We next investigated just how the cardiac jelly may be altered during development. To do so, we exposed zebrafish embryos to hyperglycemic conditions during the initial and critical heart development period. We found that when embryos absorb over 1.5-fold more D-glucose due to high-glucose conditions, they exhibit significant alterations to CJ width. Altered CJ due to hyperglycemic conditions affected valve differentiation, valve opening, and cardiac function, particularly when embryos have absorbed over a 2-fold increase of glucose. Together, these results show the structural role of the cardiac jelly to support endocardial cushion opening which will supply enough oxygenated blood to the embryo.