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Browsing by Author "Stasevich, Tim, committee member"

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    Anomalous diffusion of mRNA in the cytoplasm of HeLa cells
    (Colorado State University. Libraries, 2024) Roessler, Ryan, author; Krapf, Diego, advisor; Stasevich, Tim, committee member; Prasad, Ashok, committee member
    Information about the diffusive motion of RNA would provide insights into intracellular structures and functions, as well as gene expression and genetic regulation. We study the motion of individual messenger RNA molecules in the cytoplasm of HeLa cells. RNAs are imaged in live cells via total internal reflection (TIRF) microscopy. In order to visualize individual RNA molecules expressing the MYH9 gene, they were labeled via MS2 stem loops bound to coat proteins tagged with the HaloTag-JF646 fluorophore. We then used single-particle tracking to obtain trajectories of individual molecules. Trajectories were analyzed in terms of their mean-squared displacement (MSD) and power spectral density (PSD). We observed non-ergodic, subdiffusive behavior, with statistics that depend on observation time, i.e., aging. Additionally, we observe stochastic switching between two mobility states with an order of magnitude difference in diffusivity. This switching process is responsible for the aging nature of the system. When compared to the cytoplasmic motion of synthetic nanoparticles, the analysis of RNA trajectories gives rise to discrepancies that raise questions about specific intracellular interactions.
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    Expanding and evaluating sense codon reassignment for genetic code expansion
    (Colorado State University. Libraries, 2017) Biddle, C. William, author; Fisk, John D., advisor; Ackerson, Chris, committee member; Henry, Charles, committee member; Stasevich, Tim, committee member
    Genetic code expansion is a field of synthetic biology that aims to incorporate non-canonical amino acids (ncAAs) into proteins as though they were one of the 20 "natural" amino acids. The amino acids which naturally make up proteins are chemical limited, and ncAAs can carry new chemical functionality into proteins. Proteins are of interest because they are simple to produce with good consistency and have immense potential due to the diversity of structure and function. Incorporating ncAA into proteins expands the scope of function of proteins even further. Two methods have been widely used for genetic code expansion, global amino acid replacement and amber stop codon suppression. Global amino acid replacement exchanges one of the natural amino acids for a ncAA, producing an altered 20 amino acid genetic code. Amber stop codon suppression incorporates ncAA in response to the UAG stop codon making a 21 amino acid genetic code, but is limited in incorporation efficiency and producing proteins with multiple instances of a ncAA is challenging. We wanted to use a third genetic code expansion system called sense codon reassignment which has not been widely employed at all but should enable multisite incorporation of ncAAs. When the work presented in this dissertation was started, a single report of sense codon reassignment existed in the literature. We set out to improve and expand sense codon reassignment for the incorporation of multiple copies of ncAAs into proteins. We quickly discovered disparities in what was known regarding the variables that could be used to manipulate genetic code expansion, and the focus of our work shifted to systems for improving sense codon reassignment using quantitative measurements. The first chapter of this dissertation is an introduction to genetic code expansion and the processes of translation and gene expression that are likely involved or could be involved in genetic code expansion. The three following chapters will build upon the fundamentals described in Chapter 1. The second chapter is a complete story about how a screen to quantify sense codon reassignment was developed. The fluorescence based screen was used in a high throughput fashion to screen a directed evolution library of variants for increased sense codon reassignment efficiency at the Lys AAG sense codon. While evaluating various sense codons for potential reassignment efficiency, the AUG anticodon was found to be incapable of discriminating between the CAU and CAC codons. This was anomalous relative to the other anticodons we tested. Chapter 3 describes how unintended modifications to an engineered tRNA were identified and then how the fluorescence based screen was used to engineer the tRNA further for increased sense codon reassignment efficiency and to avoid the unintentional modification. Most applications of genetic code expansion rely on modifications to tRNAs but few reports actually consider them, The final chapter of this dissertation is a manuscript in preparation describing the reassignment of a rare sense codon to incorporate ncAAs. The chapter focuses on how improvements made in a system specific for an amino acid can be transferred to systems specific for other ncAAs. Over 150 different ncAAs have been incorporated into proteins using genetic code expansion technologies, but the extent to which the various systems are combinable has barely been evaluated. This dissertation is a story about developing sense codon reassignment to functional levels and quantifying the effects of different variables along the way.
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    mRNA localization in Caenorhabditis elegans embryogenesis
    (Colorado State University. Libraries, 2021) Parker, Dylan M., author; Osborne Nishimura, Erin, advisor; Ben Hur, Asa, committee member; Montgomery, Tai, committee member; Stasevich, Tim, committee member; Santangelo, Tom, committee member
    From guiding cell specification to regulating protein output, post-transcriptional regulation of mRNA is essential for life. As a result, many mechanisms underlying post-transcriptional regulation are highly conserved across the kingdoms of life. As the spatial resolution of microscopy and sequencing assays has increased, mRNA localization has emerged as a prevalent form of post-transcriptional regulation directing various cellular processes. Perhaps most notably, our understanding of post-transcriptional mRNA regulation and cellular function as a whole has been revolutionized by the discovery that many well-studied mRNA foci, such as germ granules, P-bodies, and stress granules, do not follow the lock-and-key principle of stoichiometric complex formation, but are actually phase-separated, biomolecular condensates. Due to their liquid-like nature, biomolecular condensates can aggregate or disperse component transcripts and proteins with exquisite environmental and temporal sensitivity. As a result, biomolecular condensates can regulate myriad processes as varied as co-translationally organizing protein components for complex assembly (Budding yeast translation factor mRNA granules), reinforcing translation inhibition (Germ granules) or activation (Neuronal granules), and facilitating the organization of other organelles (Axonemal dynein foci/kl-bodies). While an influx of studies have provided insights into the function of well-studied and novel biomolecular condensates alike, much remains unknown. What factors govern assembly and disassembly of condensates? How do they interact with one another? Is condensation the cause or consequence of the functional regulation of any particular mRNA? To begin to answer these questions, this thesis defines Caenorhabditis elegans as a model organism for exploring mRNA localization, its mechanisms, and its functions with a focus on condensate transcripts. Thus, the discoveries made have contributed to the fields of post-transcriptional gene regulation, mRNA localization, and condensate biology by elucidating mechanisms of localization, improving on methods of observing localization patterns, and establishing C. elegans as a tractable model for exploration of mRNA localization.
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    Photoelectrochemical microscopy studies of transition metal dichalcogenides nanoflakes: addressing open questions of structure-function relationships
    (Colorado State University. Libraries, 2022) Van Erdewyk, Michael, author; Sambur, Justin, advisor; Krummel, Amber, committee member; Henry, Charles, committee member; Stasevich, Tim, committee member
    Transition metal dichalcogenides (TMDs) are exciting materials for applications in solar energy conversion. However, to advance technologies that leverage these materials, a strong understanding of fundamental photoelectrochemistry and related processes is necessary. Photoelectrochemical microscopy methods are well poised in this aspect. Methods like scanning photoelectrochemical microscopy allow for the excitation of small, localized region of a material with a focused laser and the subsequent measurement of the photocurrent. The measured photocurrent can be related to the position of the laser and the physical attributes of the material surface at the location, and variations in the photocurrent across the surface can be tracked. In this way, the technique offers insight into how different surface motifs affect the photoelectrochemical behavior of the material. This method can be combined with other spectroscopies, such as photoluminescence or Raman, to can further understanding about the studied material. The following work details the use of photoelectrochemical microscopy methods to answer questions relating to both the structure and underlying properties of mechanically exfoliated TMD nanoflakes.
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    Protein engineering therapeutic strategies and tools
    (Colorado State University. Libraries, 2019) Ta, Angeline Ngoc, author; Snow, Christopher, advisor; Henry, Chuck, committee member; Kennan, Alan, committee member; Stasevich, Tim, committee member
    Proteins have become an important tool for research development and therapeutics. Proteins complement the use of small molecules as well as overcome challenges that small molecules cannot. The contrasting difference of their diverse functional and structural properties allows for complex processes like molecular recognition and catalysis. Through loops, turns, helixes, and sheets, these structural motifs provide a protein with shape and electrostatics to achieve a particular function. Overall, I describe here two examples of functional proteins where the protein's complex structure plays an important role in the development of new strategies and tools for therapeutics. The first part of this dissertation shows the effects of increased antibody recruitment on targeted cell death through the use of an immunotherapeutic cocktail of cell surface HER2 receptor binding proteins. The second part of this dissertation describes the use of a protein's chiral environment to develop a new artificial metalloenzyme that selectively catalyses synthesis of the most common N-heterocycle found in FDA approved pharmaceuticals.
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    Reexamining the role of linker histones beyond 30 nm fibers in a complex chromatin environment
    (Colorado State University. Libraries, 2024) Kuerzi, Amanda, author; Hansen, Jeff, advisor; Stargell, Laurie, committee member; Stasevich, Tim, committee member; Mykles, Donald, committee member
    Eukaryotic cells store DNA in the cell nucleus in the form of chromatin. Chromatin is composed of nearly equal parts proteins and DNA. It is both highly compacted and organized into discrete domains within the nucleus. However, the manner in which chromatin is compacted, and domains are organized, remains elusive. The primary players in chromatin compaction are core histones, which bind DNA to form the nucleosome and the basis for 10 nm fibers. Linker histones also play an important role in chromatin compaction. Previous work showed that linker histones are important for the formation of 30 nm structures. 30 nm structures were long held to be folding intermediates for repressive chromatin domains. However, there is little evidence for these structures in most eukaryotic cell types. Instead, chromatin appears to be composed of an interdigitated 10 nm fibers in both repressive and accessible chromatin types. The role of linker histones in 10 nm fibers is not well characterized. Previous work showed that linker histones stabilized 30 nm structures, rendering them inaccessible to binding by additional proteins. In the following, we investigate the behavior of linker histones in an interdigitated 10 nm fiber environment. We use an in vitro model called "condensates" to mimic the formation of 200 nm chromatin domains. We find that linker histones stabilize these condensates by cross-linking chromatin fibers. Importantly, we show that the presence of linker histones does not preclude binding by additional proteins. Linker histones readily bind condensates in ratios above an expected one linker histone per nucleosome. Additional binding by linker histones suggests that 10 nm fibers provide a complex environment in which linker histones dynamically interact with both nucleosomes and linker DNA.  
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    Relationships between hydrogen bonds and halogen bonds in biomolecular engineering
    (Colorado State University. Libraries, 2019) Hartje, Rhianon Kay Rowe, author; Ho, P. Shing, advisor; Reynolds, Melissa, committee member; Snow, Christopher, committee member; Stasevich, Tim, committee member; Woody, Robert, committee member
    In this dissertation, we will explore the interconnectedness between halogen bonds (X-bonds) and hydrogen bonds in rational biomolecular engineering efforts. As X-bonds are not readily designed into biomolecules, we aim to show how they can be advantageous for molecular design. We will begin by considering how X-bonds compare to H-bonds and show how the two can work in harmony to provide enhanced stabilizing potential. In two unique protein engineering efforts we will show 1) how the X-bond can be just as specifying in terms of molecular assembly as compared to the H-bond, and 2) how it can coordinate with the H-bond to increase protein stability. One study shows the specifying potential the X-bond possesses in terms of coiled-coil assembly. While the study points to a direct application of a sensing probe, the scope of the work will aid others using coiled-coils for materials purpose, designing protein interfaces or potential ligand binding sites. In the other protein engineering study, we will survey how a protein with an intrinsically disordered region responds to hydrogen enhanced halogen bond engineering. We show how we can drastically increase the thermal stability of the protein through minimal change to its primary sequence. This study lends itself to exploring bigger structure-function questions and how the stabilizing capacity of halogen bonds fits into this. Through this work we aspire to show how useful X-bonds can be for biological engineering efforts by exhibiting their specifying and stabilizing characteristics in these settings.
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    The DXO decapping exonuclease is a restriction factor for RNA viruses
    (Colorado State University. Libraries, 2019) Lynch, Erin R., author; Geiss, Brian, advisor; Wilusz, Jeffrey, committee member; Perera, Rushika, committee member; Stasevich, Tim, committee member
    Cellular RNA exonucleases, such as XRN1 and DXO, aid in the destruction of defective cellular mRNAs and help maintain overall cellular health. The RNA decay system, however, also serves another purpose – degrading viral RNAs. The XRN1 exonuclease is known to be a major antagonist of RNA virus genomes, but the role of other cellular RNA decay enzymes in controlling viral infection is less clear. The cellular 5' decapping exonuclease DXO is able to recognize, de-cap, and degrade RNAs lacking 2'-O-methylation on the first nucleotide after the 5' cap, helping the cell to discriminate self from non-self RNAs. Preliminary data we have developed indicate that flaviviruses and alphaviruses replicate to much higher levels in DXO deficient cells than in cells containing DXO, indicating that DXO may also act as a cellular viral restriction factor. Interestingly, flavivirus genomes contain a 5' cap that is generally 2'-O-methylated at the first base of the transcript, providing a potential mechanism to evade DXO degradation. Overall, our results indicate that the DXO decapping exonuclease helps control the replication of positive strand RNA viruses in cells and represents a new viral restriction factor.
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    Translation-dependent mRNA localization in the Caenorhabditis elegans embryo
    (Colorado State University. Libraries, 2022) Winkenbach, Lindsay P., author; Osborne Nishimura, Erin, advisor; Wilusz, Carol, committee member; Stasevich, Tim, committee member; Di Pietro, Santiago, committee member
    Though each animal cell contains the same genetic information, cell-specific gene expression is required for embryos to develop into mature organisms. Embryos rely on maternally inherited components during early development to guide cell fate specification. In animals, de novo transcription is paused after fertilization until zygotic genome activation. Consequently, early embryos rely on post-transcriptional regulation of maternal mRNA to spatially and temporally regulate protein production. Caenorhabditis elegans has emerged as a powerful developmental model for studying mRNA localization of maternally-inherited transcripts. We have identified subsets of maternal mRNAs with cell-specific and subcellular patterning in the early C. elegans embryo. Previous RNA localization studies in C. elegans focused on maternal transcripts that cluster in the posterior lineage and showed mRNA localization occurs in a translation-independent manner through localization sequence elements in the 3'UTR. However, little is known about the mechanisms directing RNA localization to other subcellular locales in early embryos. Therefore, we sought to understand the localization of maternal transcripts found enriched at the plasma membrane and nuclear periphery, erm-1 (Ezrin/Radixin/Moesin) and imb-2 (Importin Beta), respectively. In this thesis, I characterize two different translation-dependent pathways for mRNA localization of maternal transcripts at the plasma membrane and nuclear periphery. I identified the PIP2-membrane binding region of the ERM-1 proteins is necessary for erm-1 mRNA localization while identifying additional membrane localized maternal transcripts through the presence of encoded PIP2-membrane binding domains. Additionally, I observed that mRNA localization patterns can change over developmental time corresponding to changes in translation status. For imb-2 mRNA localization, I found localization to the nuclear periphery is also translation-dependent. Through recoding the imb-2 mRNA sequence while maintaining the translated peptide sequence using alternative codons, I found both localization and transcript stability additionally depends on mRNA sequence context. These findings represent the first report of a translation-dependent localization pathway for two maternally-inherited transcripts in C. elegans and demonstrate the utility of C. elegans as a model for studying translation-dependent mRNA localization during development.