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  • ItemEmbargo
    Spn1, Spt4, Spt5, and Spt6 preserve chromatin structure over promoters and open reading frames
    (Colorado State University. Libraries, 2024) Tonsager, Andrew Jordan, author; Stargell, Laurie A., advisor; Hansen, Jeffrey C., committee member; Santangelo, Thomas, committee member; Argueso, Juan Lucas, committee member
    The eukaryotic chromatin landscape presents formidable nucleosomal barriers for processes that require access to DNA, such as transcription. These barriers are overcome through the action of many factors, including histone chaperones Spn1, Spt5, Spt6, and FACT and transcription elongation factor Spt4. However, it is poorly understood how each contributes to this process. To ascertain the role that these factors play on preserving chromatin structure over the genome, this thesis has utilized micrococcal nuclease digestion followed by sequencing (MNase-seq) to analyze chromatin protections in the yeast genome in cells expressing numerous mutant alleles of these factors. Extensive characterization of MNase-protected fragments in a wide range of sizes established that the essential histone chaperone Spn1 preserves both nucleosomal and subnucleosomal structures over both promoters and open reading frames across the genome. Additional analyses from existing MNase-seq datasets demonstrated the extent to which Spn1 and other RNAPII-associated factors maintain nucleosome features over genes of varied characteristics. The study of factors described in this thesis is performed in living cells, which have been genetically modified to express mutant alleles of chromatin factors. This thesis also describes a course-based undergraduate research experience (CURE) developed to introduce upper-level biochemistry students to techniques in yeast genome engineering in an authentic research setting.
  • ItemOpen Access
    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.  
  • ItemOpen 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.
  • 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.
  • ItemOpen Access
    Biochemical, biophysical and structural study of the nucleosome-MeCP2 complex
    (Colorado State University. Libraries, 2009) Yang, Chenghua, author; Luger, Karolin, advisor
    Methyl-CpG Binding Protein (MeCP2) is an abundant chromatin associated protein that is important in maintaining human health; mutations in this protein cause Rett Syndrome, a neurodevelopmental disease that is a common cause of mental retardation and autism in females. MeCP2 was initially identified as a protein that recognizes the genetic DNA methyl-CpG mark and it was thought to repress gene transcription by recruiting histone deacetylases. Recent studies show that MeCP2 can both repress and activate gene transcription. It also binds chromatin in the absence of the methylation mark, suggesting that its mode of action is more complex than previously assumed. The observation that MeCP2 compacts nucleosomal arrays in vitro and mediates silent chromatin loop formation in vivo suggests a novel mechanism by which MeCP2 regulates gene expression. To further characterize the interplay between MeCP2 and chromatin, it is important to understand the interactions between MeCP2 and nucleosomes, the fundamental component of chromatin. We used biochemical and biophysical approaches to study the interplay between MeCP2 and nucleosomes. Gel mobility assays showed that although MeCP2 can interact with a nucleosome with or without extra nucleosomal DNA, it has a higher affinity for nucleosomes with extra nucleosomal DNA. The N-terminal portion of human MeCP2 (amino acids 78-305) is sufficient to establish this interaction. Size-exclusion chromatography combined with multi-angle light scattering and fluoresecence resonance energy transfer (FRET) assays demonstrated that this interaction occurs at a 1:1 molar ratio and that MeCP2 brings the extra nucleosomal DNA ends in a closer proximity. Small angle X-ray scattering (SAXS) revealed the formation of a more compact complex when MeCP2 interacts with nucleosome with (versus without) extra nucleosomal DNA, indicating that the extra nucleosomal DNA is important in organizing the MeCP2-nucleosome complex. Our data suggest a model in which MeCP2 compacts chromatin by changing the extra nucleosomal DNA path. X-ray crystallography is also used to characterize the nucleosome-MeCP2 complex. Crystals of the nucleosomes with extra nucleosomal DNA in complex with MeCP2 were obtained and diffracted to 5.2 Å. Although MeCP2 dissociated from the crystals after soaking in cryo-protectant, the electron density map reveals the path of extra nucleosomal DNA which may be organized by MeCP2.
  • ItemOpen Access
    Molecular basis of yeast prion formation
    (Colorado State University. Libraries, 2009) Toombs, James A., author; Ross, Eric, advisor
    Amyloid fibers are highly organized protein aggregates that are associated with many fatal diseases. Prions represent a unique class of amyloid fibers that are distinguished by their infectivity and inheritability. In the yeast S. cerevisiae, there are several known prion forming proteins. Since the discovery of the first yeast prions in the early 1990s, they have provided a useful model system for studying the biology of prion proteins. While it has been determined that amino acid composition is important to prion formation, there has not yet been any quantitative study aimed at determining how composition promotes or inhibits prion formation. Without this knowledge, our understanding of the events that drive prion formation and our ability to identify new prion-forming proteins is severely limited. In this dissertation, we describe our experiments with the yeast prion protein Sup35p that have illuminated the sequence requirements for yeast prion formation. From these results, we conclude that: (i) amino acid composition, not primary sequence, is the major driving force behind yeast prion propagation, and (ii) prion formation occurs in domains characterized by relatively few prion promoting residues dispersed throughout an intrinsically disordered region.
  • ItemOpen Access
    Role of polyglutamylation in nucleosome assembly protein 1 (NAP1) function
    (Colorado State University. Libraries, 2008) Subramanian, Vidya, author; Luger, Karolin, advisor
    The organization of DNA into chromatin requires the systematic deposition of the histones onto the DNA template. Chromatin function requires the dynamic exchange of the histone components during replication and transcription. Deposition and exchange is mediated in part by a family of proteins generally referred to as histone chaperones. It has been shown recently that recombinant yeast NAP1 (yNAP1) is capable of promoting ATP-independent histone exchange and nucleosome sliding in vitro, and this ability is specifically attributed to the highly acidic C terminal tail of the protein. Drosophila NAP1 (dNAP1) has a shorter acidic C terminus than yNAP1. Preliminary data in the lab suggests that recombinant wild-type dNAP1 is incapable of this nucleosome dissociation. Native dNAP1 purified from Drosophila embryos, on the other hand, is capable of nucleosome dissociation. In this study we reveal the presence of a unique post-translational modification, polyglutamylation in native dNAP1, which restores the nucleosome dissociation function to recombinant dNAP1. We have also been able to identify two target sequences, as well as the number of glutamyl units associated with these modifications using mass spectrometric analysis (MALDI & MS/MS). The modification at the CTAD (C-terminal acidic tail domain) could compensate for the lesser amount of acidic amino acid in dNAP1 and may account for the gain in nucleosome dissociation function. The second polyglutamylation site is located at the NLS (Nuclear Localization Sequence) (based on the conserved core domain of yNAP1 and dNAP1).
  • ItemOpen Access
    Insulin and IGF-I prevent brain atrophy in diabetic rats independently of hyperglycemia
    (Colorado State University. Libraries, 2009) Šerbedžija, Predrag, author; Ishii, Douglas N., advisor
    The causation of brain atrophy associated with dementia in diseases such as diabetes and Alzheimer's Disease (AD) is very poorly understood. There are 20.8 million patients in US with diabetes, 4.5 million with AD, and the incidence of both is rising. Their combined annual treatment cost (direct and indirect) for patients with dementia exceed $148 billion. Reduced insulin and insulin-like growth factor (IGF) signaling is a common biochemical feature in brains of diabetic as well as AD patients. Rodents with knockout of brain neuronal insulin receptor display no change in glucose utilization, neurodegeneration, nor impaired learning/memory. However, IGFs may substitute for insulin or be required for its activity. The literature is virtually silent concerning the role of the widespread insulin receptors in the brain. It is possible that brain atrophy is the consequence of a concomitant decline in IGF as well as insulin levels. The causation of adult brain atrophy is understudied, and identification of the factors that help maintain normal brain mass may provide hints as to the causation of neurodegenerative disorders.
  • ItemOpen Access
    The influence of histone orthologues, histone variants and post-translational modifications on the structure and function of chromatin
    (Colorado State University. Libraries, 2008) Resch, Michael George, author; Hansen, Jeffrey C., advisor; Luger, Karolin, advisor
    Two meters of DNA is packaged into the nucleus of each eukaryotic cell in the form of chromatin. DNA wraps around a protein histone octamer to form a nucleosome, the fundamental repeating unit of chromatin. The highly basic histone octamer contains two copies each of H2A, H2B, H3 and H4 to form the protein core of the nucleosome. There is a dynamic interplay of accessibility which compacts DNA yet allows access for fundamental cellular processes like transcription and DNA replication. This thesis investigates how histone variants and post-translational modifications contribute to the level of chromatin compaction. I demonstrated that defined nucleosomal arrays made with histones from multiple species oligomerize at different concentrations of MgCl2. A comparison of endogenous and recombinant Drosophila melanogaster histone octamers showed that this is unlikely due to posttranslational histone modifications, but likely a result of subtle changes in the sequences constituting the histone tails and structured surface of the histone octamer. I investigated the effect of incorporation of the centromere specific H3 histone variant centromere protein - A (CENP-A) into nucleosomes and nucleosomal arrays. Despite the fact that CENP-A shares only 60% sequence homology within the structured domain of major-type H3 (15% in the N-terminal domain), CENP-A (together with the other three core histones) forms nucleosomes and condensed nucleosomal arrays comparable to major-type H3. Post-translational modifications (PTM) contribute to the regulation of chromatin structure. I have analyzed the effect of H3 lysine 56 acetylation on nucleosome structure and chromatin condensation. This modification was previously thought to disrupt nucleosome structure. I developed methods to enzymatically acetylate large amounts of H3 specifically at Lys 56, and demonstrated that histone octamers containing H3-K56Ac form canonical nucleosomes. However, nucleosomal array condensation is compromised by this particular PTM. Together, these studies suggest that even subtle variations in histone sequence or post-translational modifications result in differences in chromatin higher order structure.
  • ItemOpen Access
    Tax deregulation of host-cell proteins
    (Colorado State University. Libraries, 2008) Kim, Young-Mi, author; Nyborg, Jennifer K., advisor
    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus etiologically linked to an aggressive and generally fatal malignancy called adult T-cell leukemia (ATL) and to a chronic inflammatory neurological disease. Only a small percentage of infected individuals develop ATL following a prolonged latency period of up to 30 years post infection. The dominant mechanism of virus transmission in an infected individual is through clonal expansion of HTLV-1 infected cells. The HTLV-1-encoded protein Tax is the prominent player in promoting mitotic replication. Tax is also directly linked to malignant transformation and the etiology of ATL. Tax is a potent transcriptional activator that stimulates HTLV-1 viral gene expression. Three 21 base pair repeat enhancer elements called viral cyclic AMP response elements (vCREs), located in the HTLV-1 transcriptional control region, are critical to Tax-activated transcription. Tax associates with the vCREs through protein-DNA interactions and through protein-protein interaction with the cellular transcription factor cAMP response element binding (CREB) protein. Together this complex recruits the cellular coactivators CBP/p300. The role of Ser133 phosphorylated CREB in mediating Tax function in HTLV-1 transcription has long been controversial. Our data reveal that CREB phosphorylation is absolutely required for viral Tax transactivation. Consistent with this, Tax induces constitutively elevated levels of phosphorylated CREB in vivo and in vitro. We further investigated the mechanism of Tax-mediated CREB phosphorylation and uncovered a novel function of Tax: stimulation of CREB phosphorylation via the Ca2+/Calmodulin (CaM)-dependent protein kinase (CaM kinase) pathway to promote viral transcription. In addition to Tax-dependent CREB phosphorylation, we found that Tax upregulates B-cell lymphatic leukemia protein 3 (Bcl-3) and cyclin D1 expression, two key determinants of cell fate. Furthermore, Tax interacts with Bcl-3 in vivo and in vitro. Deregulation of these key host-cell proteins by Tax may contribute to the transformation of T-cells.
  • ItemOpen Access
    Mechanisms of RNA polymerase II-mediated transcription
    (Colorado State University. Libraries, 2007) Fletcher, Aaron Glenn Louis, author; Stargell, Laurie, advisor
    Transcription by RNA Polymerase II (RNAPII) is a critical step in controlling biological events such as cell growth, cell differentiation, response to environmental change, homeostasis, and disease. The regulation of transcription initiation of some genes is controlled at the level of TBP and RNAPII recruitment to the promoter. At such genes, the binding of TBP/RNAPII is the rate-limiting step for gene expression. Other genes already have TBP/RNAPII occupying the promoter before induction of gene expression, and the rate limiting step is no longer recruitment of TBP/RNAPII. These genes are collectively known as post-recruitment regulated genes. The yeast CYC1 gene is a post-recruitment regulated gene and serves as an excellent model for understanding the mechanism behind post-recruitment regulation. A TBP recruitment bypass screen was developed to investigate the mechanism of post-recruitment regulation. The results of the bypass screen revealed that SAGA and Mediator play important roles in post-recruitment regulation. Further analysis of SAGA uncovered a new function: that suggests SAGA is important in recruitment of Mediator to post-recruitment regulated genes. In addition to RNAPII and TBP, the CYC1 gene was found to have TFIIH, capping enzyme and serine 5 phosphorylation of the RNAPII C-terminal domain occupying the promoter in the uninduced condition. These results indicate that much of the Pre-Initiation Complex (PIC) occupies the CYC1 promoter in the uninduced state. In addition to PIC occupancy at CYC1, a conserved and essential protein, Spn1, is found to occupy the promoter during uninduced conditions. To further understand the role of this essential protein, genome localization studies and transcription profiling were performed. These studies suggest that in addition to playing an important role in post-recruitment regulation of gene expression, Spn1 may be involved in the transcription of ribosomal proteins. Taken together, this body of work contributes significantly to understanding the regulatory mechanisms of post-recruitment regulation.
  • ItemOpen Access
    Structural and functional studies on the chromatin and nucleosome binding proteins
    (Colorado State University. Libraries, 2007) Chodaparambil, Jayanth Velandy, author; Luger, Karolin, advisor
    The approximately two meters of eukaryotic DNA are compacted within the confines of the nucleus by hierarchical packing with an equal amount of histone proteins to form chromatin. The nucleosome is the fundamental repeating structural unit of chromatin. The nucleosome is the fundamental repeating structural unit of chromatin. Highly compacted DNA is very accessible to the transcription machinery. To understand the mystery behind the two opposing functions of the chromatin, it is essential for us to study nucleosome and chromatin structure in detail.
  • ItemOpen Access
    New functions of the SAGA complex in regulation of transcription by RNA polymerase II
    (Colorado State University. Libraries, 2008) Chen, Xu, author; Stargell, Laurie, advisor
    The yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex plays a role in Gal4-mediated transcriptional activation via delivery of TATA-binding protein (TBP) to Gal4-responsive promoters. Little is known about the impact of the sequence of the TATA element in the core promoter in this process. To investigate the SAGA complex regulatory function at different TATA element sequences, we compared a consensus element (TATA) to an off-consensus element (CATA) in the kinetics of Gal4-dependent gene activation, PIC occupancy, the requirement of SAGA components, and the histone acetylation state. We have found a new function of SAGA carried by subunits Gcn5, Ada2 and Spt8: TATA-element-censoring. This function enhances transcription driven by the consensus TATA element and represses transcription driven by off-consensus TATA elements. This functions works at both synthetic promoters and the endogenous GAL promoters. Via a genetic screen, Swi/Snf and RSC complexes were also identified with TATA-censoring function. Our study suggests that the new function involves TBP delivery, histone acetylation and histone eviction.
  • ItemOpen Access
    Post-initiation regulatory mechanisms of transcription in the Archaea
    (Colorado State University. Libraries, 2023) Wenck, Breanna Renée, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Osborne Nishimura, Erin, committee member; Wilusz, Carol, committee member
    Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. Archaeal systems contain simplified, basal regulatory transcription components and mechanisms homologous to their eukaryotic counterparts, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions. Many archaeal genomes are organized with histone proteins that resemble the core eukaryotic histone fold, which permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that impacts transcription regulation and gene expression. Despite such semblance between the eukaryotic and archaeal core histone folds, archaeal genomes lack the canonical N and C terminal extensions that are abundantly modified to regulate transcription in eukaryotic genomes. Much of what is known regarding factor-mediated transcription regulation in the Archaea is limited; however combined and continued efforts across the field provide tidbits of information, but many pieces are still missing. This thesis aims to i) delineate the role key residues within the histone-DNA complex and archaeal histone-based architecture and key residues within the histone-DNA complex have on the progression of the transcription apparatus, characterize factor-mediated transcription termination, and explore chromatin- and TFS-mediated regulatory effects on transcription via global RNA polymerase (RNAP) positions.
  • ItemEmbargo
    N-linked glycosylation is fundamentally linked to the surface expression of neuroligins
    (Colorado State University. Libraries, 2023) Cast, Thomas, author; Chanda, Soham, advisor; DeLuca, Jennifer, committee member; Di Pietro, Santiago, committee member; Tobet, Stuart, committee member
    N-linked glycosylation is one of the most prevalent forms of post-translational modification, decorating secreted and cell-surface transmembrane proteins as they are trafficked along the secretory pathway. While well-characterized in most tissues, non-canonical N-glycan diversification has been reported to occur in the central nervous system. Chapter 2 of this dissertation describes the importance of N-linked glycosylation for the neuroligin family of synaptic cell-adhesion molecules (NLGN1-4). NLGNs play a crucial role in regulating synaptic transmission strength by recruiting neurotransmitter receptors to synapses. Mutation of N-glycosylated residues increased retention of each NLGN isoform in the endoplasmic reticulum (ER), consequentially reducing their ability to interact with presynapses. Pharmacological inhibition of various stages of the N-glycan maturation pathway further revealed that only the initial transfer of the polysaccharide is essential for the surface expression of NLGN proteins. Chapter 3 characterizes a missense mutation identified in the NLGN4 gene of a patient with autism. This mutation, p.Arg101Gln (R101Q), is directly upstream of a conserved N-linked glycosylation site, which played a universal role for the surface localization of each NLGN isoform. Biochemical and cellular analysis revealed the NLGN4-R101Q variant to be immaturely glycosylated and mistrafficked, retained in the ER similarly to N-glycan site mutants. In neurons, the mistrafficked R101Q variant failed to reproduce the excitatory synaptogenic effects of NLGN4-WT, indicating an overall loss-of-function phenotype. Further, equivalent RQ mutations introduced in other NLGN isoforms mimicked the glycoprotein maturation and surface expression defects. Together, these findings reveal a profound overall significance of N-glycans for NLGNs and the conserved role of a specific N-linked glycosylation site for promoting the forward trafficking of NLGN protein.
  • ItemEmbargo
    Identification of the TPC2 interactome reveals TSPAN10 and OCA7 as key players in the biogenesis of melanosomes
    (Colorado State University. Libraries, 2023) Beyers, Wyatt, author; Di Pietro, Santiago, advisor; Amberg, Gregory, committee member; Santangelo, Thomas, committee member; Yao, Tingting, committee member
    Many specialized cell types gain their function through the generation of specialized organelles that make or store cell-specific biomolecules. A group of specialized organelles are called Lysosome Related Organelles (LROs) because they are derived from Golgi and endolysosomal compartments and their biogenesis depends on trafficking pathways and machinery shared with lysosomes, many have protein contents partially overlapping with lysosomes, and typically have low pH during stages of their maturation. One well-studied model LRO is the melanosome, the organelle in melanocytes and retinal pigment epithelial cells responsible for melanin pigment production in the eyes, hair, and skin, and defects in melanosome function lead to pigmentation diseases such as oculocutaneous albinism. Melanosome biogenesis is a complex process requiring ubiquitous membrane trafficking machinery to be repurposed for the differentiation of melanosomes from other endosomal compartments and specific delivery of melanosome synthesizing enzymes, Tyrosinase and Tyrosinase Related Proteins 1 and 2. Furthermore, correct melanosome maturation requires remodeling of the melanosome membrane, recycling of membrane trafficking machinery, generation of intraluminal amyloid fibrils with the correct structure for melanin packaging, tight pH control, as well as coordinated influx of copper, zinc, tyrosine, and cysteine for melanin synthesis. These processes require the temporospatial coordination of at least 100 known proteins, and probably dozens more remain undiscovered. In this dissertation, I present the discovery of new proteins involved in the biogenesis of melanosomes. Proximity biotinylation by promiscuous biotin ligase enzymes followed by biotin pulldown and mass spectrometry has emerged as a powerful technique for the identification of protein-protein interactions, protein complex determination, and identification of organelle membrane proteomes. I utilized the melanosome localized cation channel, TPC2, genetically fused with the BioID2 biotin ligase, to identify proteins in proximity to TPC2 at the cytosolic surface of melanosome membranes of MNT1 melanoma cells. Through mass spectrometry analysis of biotinylated proteins enriched through Streptactin pulldown, a TPC2 proximity interactome was identified comprising over 200 proteins. Subsequent fluorescence confocal microscopy analysis confirmed several proteins, including PLD1, SV2A, TSPAN10, and OCA7/C10orf11/LRMDA all colocalize highly with TPC2-EGFP, confirming they are new melanosome proteins. In follow-up functional studies, TSPAN10 and OCA7 were confirmed to be involved in pigmentation, with severe melanin depletion in TSPAN10 or OCA7 knockout MNT1 cells. TSPAN10 and OCA7 both influence the processing of the PMEL protein, which is required for correct melanosome ultrastructure and for melanin packaging. Further investigation of TSPAN10 revealed it functions with the pigmentation associated metalloproteinase, ADAM10, and is required for ADAM10 expression and localization to endosomal compartments. On the other hand, OCA7 was found to work with the melanosome localized Rab proteins, Rab32 and Rab38, and regulates the pH of melanosomes. Thus, the newly defined TPC2 interactome in melanocytes was proven as a valuable dataset that robustly identifies new melanosome proteins. Chapter 1 of this dissertation provides a broad overview of membrane trafficking pathways, as well as a synopsis of the specific proteins and pathways involved in melanosome biogenesis and homeostasis. Chapter 2 investigates the TPC2 interactome in MNT1 cells, and it characterizes TSPAN10 as a new player in melanosome biogenesis. Finally, Chapter 3 provides a characterization of the OCA7 protein associated with oculocutaneous albinism type 7 and investigates OCA7 function using a newly generated OCA7 knockout cell model.
  • ItemOpen Access
    Live-cell imaging uncovers the relationship between histone acetylation, RNA polymerase II phosphorylation, transcription, and chromatin dynamics
    (Colorado State University. Libraries, 2023) Saxton, Matthew Neeley, author; Stasevich, Timothy J., advisor; Nishimura, Erin O., committee member; Hansen, Jeffrey, committee member; Krapf, Diego, committee member
    Living cells are capable of turning a one dimensional strand of nucleic acids into a functional polypeptide. A host of steps and factors are involved in the process of transcription and translation, and understanding each of them is necessary for comprehending and characterizing life. While new technologies and assays have expanded our understanding of eukaryotic transcription, there is still much to be learned. In particular, single-molecule microscopy provides a powerful and versatile platform for studying the genesis of RNA with unparalleled spatiotemporal resolution (Chapter 1). First, we characterize the timing, kinetics, and occupancy of phosphorylated RNA polymerase II (RNAP2) using a single-copy HIV-1 reporter system. This work provides strong evidence for clusters of phosphorylated, initiating RNAP2 which is spatially separated from bursty, downstream RNA synthesis. It is found that RNAP2-Ser5-phosphorylation (Ser5ph) precedes RNA output by ~1 minute, and RNAP2 arrives at the locus in a phosphorylated state (Chapter 2). Then, we examine the spatial correlation between H3K27 acetylation and Ser5ph in living cells on the course of minutes to hours. Contrary to expectations based upon ChIP data, we find that the two signals are in fact spatially separated. This argues for a functional separation between transcriptional poising and initiation, likely aiding bursty behavior. Next, the dynamics of single chromatin-incorporated nucleosomes in the context of H3K27 acetylation and transcription initiation is determined with super-resolution single-molecule imaging. The physical movement of chromatin inside of H3K27ac and RNAP2-Ser5ph enriched regions is found to be significantly different, despite both marks being traditionally associated with transcriptionally active chromatin. (Chapter 3). Much of this work utilizes bead-loading in order to introduce proteins and DNA into living cells. A simple, effective, and cheap procedure, bead-loading is a highly effective and versatile technique that is generally underutilized. To facilitate communication of this process, a detailed protocol is included (Chapter 4). While this culmination of work furthers our understanding of cellular genetic expression and eukaryotic transcription, it also introduces many new questions that are promising areas of study. Fortunately, the combination of imaging technology and knowledge developed here provides promising new fronts for studying transcription in living cells (Chapter 5).
  • ItemOpen Access
    Sex dependent regulation of immune responses in ex vivo lung slices
    (Colorado State University. Libraries, 2023) Patlin, Brielle Honor, author; Snow, Chris, advisor; Tobet, Stuart, advisor; Chanda, Soham, committee member
    Sex differences in respiratory disease have been increasingly obvious over the last several decades ranging from asthma, to interstitial lung disease, to the common cold. One way that lung functions are dependent on sex is in their immune responses to disease. While there are many factors that contribute to the severity of immune responses and recovery from illness, neuroimmune signaling is an understudied aspect. This could be partially caused by the difficulty of studying individual neuronal circuits in the periphery in live animals and the lack of necessary cell types in organoid or cell line models. To study these processes in the lung, an organotypic model, known as a precision cut lung slice (PCLS) can be utilized to maintain intracellular and extracellular signaling. The first study herein addresses the role of the most prevalently produced neuropeptide in the lung on immune, neuronal, and epithelial populations in the lung. This abundant neuropeptide, calcitonin gene related peptide (CGRP), generates several sex dependent responses in PCLS. With CGRP treatment, number of B cells, size of neuroendocrine bodies (NEB), and surfactant protein C (SPC) granule are higher in female PCLS. However, the number of CGRP immunoreactive fibers in female PCLS is lower than in male PCLS. These sex related changes of lung cell behavior may partially explain some disease susceptibilities and are important factors to consider in pharmaceutical development for respiratory diseases. PCLS can be used to test pharmaceutically relevant substances and drug delivery systems. The recent pandemic has made it evident that better ways to deliver pharmaceuticals to the lungs are required. Regarding this problem, a focus on nasal drug delivery is important. The current leading technology in this type of delivery mechanism involves utilizing lipid nanoparticles and nasal administration. However, this is not the most efficient way to treat the lungs. Crystallized protein structures have begun to be used for purposes other than determining protein structure. In the second study included here, protein crystals were loaded with biologically relevant molecules to purposefully induce immune responses, without causing an immune response by themselves. This functionality has a variety of benefits, because a primary problem in respiratory disease is over activation of the immune response. In this study, crystals were customized by loading different molecules (e.g., lipopolysaccharide (LPS)) and the immune modulatory affects were observable in PCLS. This generated a sex dependent immune response in PCLS, which was less over time than slices treated with pure LPS indicating a differential response over 48 hours. Using protein nanocrystals for pharmaceuticals may provide new ways to target respiratory disease by nasal delivery with benefits over lipid nanoparticles.
  • ItemOpen Access
    Investigation of transcriptional dynamics in the Caenorhabditis elegans intestine gene regulatory network
    (Colorado State University. Libraries, 2022) Williams, Robert Thomas Patton, author; Osborne Nishimura, Erin, advisor; Wilusz, Carol, committee member; Hansen, Jeffrey, committee member; Santangelo, Tom, committee member
    ELT-2 is the major transcription factor required for the activation of Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos to promote development and persists after hatching through larval and adult stages. Though the sites of ELT-2 binding have been defined and the transcriptional changes that result from ELT-2 depletion described, the intestine-specific transcriptome profile over developmental time has not been characterized, in part because of the difficulty in isolating intestine from other tissues. To address this knowledge gap, we used Fluorescence Activated Cell Sorting (FACS) to enrich intestine cells and performed RNA-seq analysis at distinct developmental stages. By linking the transcriptome profiles to previous ELT-2 studies, we were able to gain new insight into the role of ELT-2 in the intestinal regulatory network throughout development. Correlation of ELT-2 binding to the intestine transcriptome data, revealed that only 33% of intestine-enriched genes were direct targets of ELT-2 binding in embryos, but that number increased to 75% by the L3 stage. This suggests additional transcription factors may promote intestine-specific transcription early in development. Consistent with this possibility, half of the ELT-2 direct target genes were not transcriptionally dependent on ELT-2 for appropriate expression (i.e. their expression was not impacted following ELT-2 depletion) implying that other factors may compensate in the absence of ELT-2. Among direct target genes that were affected by ELT-2 depletion, equal proportions were over and under-expressed thus ELT-2 can both activate and repress direct target genes. Both activated and repressed sets of ELT-2 target genes were enriched for defense response genes reinforcing recent findings demonstrating ELT-2 participating in mediating the immune response upon pathogen exposure. Fluorescent reporter assays demonstrated that expression of two direct targets of ELT-2 cebp-1 and ets-4 are indeed repressed by ELT-2. Moreover, we observed that ELT-2 repressed its own promoter in a negative feedback loop that regulates elt-2 gene expression. Together, our findings illustrate that ELT-2 contributes directly to roughly 20 – 50% of intestine-specific gene expression, that ELT-2 exerts both positive and negative regulatory control on its direct targets, and that our overall picture of the intestinal regulatory network is incomplete with more intestine specific transcription factors and mechanisms remaining to be discovered.
  • ItemOpen 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.