Mountain Scholar :: Browsing by Author "DeLuca, Jennifer, committee member"

Browsing by Author "DeLuca, Jennifer, committee member"

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Open Access
Analysis of equine zygote development after intracytoplasmic sperm injection
(Colorado State University. Libraries, 2016) Ruggeri, Elena, author; Carnevale, Elaine, advisor; Clay, Colin, advisor; Albertini, David, committee member; DeLuca, Jennifer, committee member; Seidel, George, committee member
Intracytoplasmic sperm injection (ICSI) is an established and widely used method to achieve oocyte fertilization in equine reproductive assisted technologies. However, not all the oocytes fertilized by ICSI undergo cleavage and develop into viable embryos. Limited knowledge on equine zygote development after ICSI is available, and reasons why developmental failure occurs after ICSI have been only partially studied and need further investigation. Fertility decline and early embryo loss is associated with maternal aging in the mare, and it is concomitant with reduced oocyte quality. Relatively little is known about the effect of maternal aging and zygote developmental failure or success in the mare. Effects of in vitro maturation of the oocyte or zygote development in the mare still need to be clarified and further studied. The overall objective of this dissertation was to study equine zygote development after ICSI using confocal microscopy. Objectives were to: (1) compare cytoskeletal and nuclear changes during progression of equine zygote development after ICSI for in vivo versus in vitro matured oocytes; (2) compare changes in cytoskeletal and chromosomal configurations after ICSI between oocytes from young and old mares to define maternal-aging related alterations; (3) determine cytoskeletal and nuclear alterations associated with fertilization failure in ICSI-produced presumptive zygotes in young and old mares; (4) determine cell-aging and cell donor-aging effects on cytoskeleton and chromatin configurations. Specifically, in our studies we evaluated the tubulin and actin cytoskeleton, chromatin, and kinetochores/centromeres. Immunostaining and confocal imaging of the equine zygotes was performed using a spinning disk confocal microscope. After ICSI, five distinct events of development were observed with no major differences over time whether oocytes matured in vivo or in vitro. Oocytes matured in vivo appeared to reach the pronucleus stage earlier after ICSI compared to in vitro matured oocytes. Abnormal phenotypes associated with fertilization failure were more significant in oocytes matured in vitro than in vivo. When ICSI was performed in oocytes from young and old mares, similar stages of zygote development were observed, and the number of zygotes reaching the pronucleus stage was similar between the two age groups. Nucleolus like bodies, sites of ribosomal RNA involved in embryonic genome activation, were observed only in zygotes at the pronucleus stage from young mares; no nucleolus-like bodies were observed in pronuclei of zygotes from old mares. Pronuclei morphology, based on CREST staining, and DNA localization, also differed between pronuclei of young and old mares. Actin vesicles were observed significantly more often within zygotes from old mares compared to young mares during all stages of zygote developmental progression. When potential zygotes were analyzed after failure of cleavage after ICSI, actin vesicles were greater in area, perimeter and number in oocytes from old mares than those from young mares. Tubulin cytoskeletal multiasters were associated with cell aging and with increased interval after ICSI for young mares but not old mares. In conclusion, zygotes produced from oocytes matured in vivo versus in vitro or collected from young and old mares went through similar stages of development, with pronuclei attainment appearing to be a crucial event in zygote development. Actin vesicles were a major cytoskeletal difference associated with oocyte origin and a potential factor involved in developmental failure of the oocyte. Confocal microscopy and image analysis were novel methods used to describe the equine zygote development and allowed us to elucidate the cytoskeletal and nuclear remodeling events that follow fertilization after ICSI in the mare.
Open Access
Cargo induced recruitment of the endocytic adaptor Sla1 and the role of Sla1-clathrin binding in endocytosis
(Colorado State University. Libraries, 2018) Tolsma, Thomas O., author; Di Pietro, Santiago, advisor; Ross, Eric, committee member; DeLuca, Jennifer, committee member; Reist, Noreen, committee member
Clathrin-mediated endocytosis is a highly dynamic process that is essential in all eukaryotes. This process is utilized for a number of functions including the uptake of extracellular nutrients, manipulation of the plasma membrane content, downregulation of cell signaling pathways, and viral entry. While differences in protein composition, sequence, and structure do exist between species for this process, many core protein functions and the mechanistic steps involved in endocytic vesicle formation and internalization are highly conserved. This has allowed findings from one species to be applicable to another. For this reason Saccharomyces cerevisiae has been characterized as a highly useful model organism for studying and identifying key proteins and conserved mechanisms in clathrin-mediated endocytosis that are found in all eukaryotes. In yeast, roughly 60 proteins have been identified as being part of the endocytic machinery. Clathrin-mediated endocytosis begins with the recruitment of early endocytic proteins that establish the site of endocytosis. This includes scaffolding and coat proteins, such as clathrin, that aggregate at the plasma membrane through interactions with lipids, protein cargo, and other components of the endocytic machinery. This is followed by recruitment of other late coat proteins that further prepare the site for internalization. Following coat formation the mobile phase of membrane invagination is initiated by the recruitment of the actin polymerization machinery. Actin polymerization then generates an inward force at the site of endocytosis that causes invagination of the plasma membrane. The invagination is then separated from the plasma membrane through the recruitment of scission proteins that pinch off the endocytic vesicle. Lastly the internalized vesicle undergoes a process of coat protein disassembly before being targeted to its proper destination in the cell. While much of this process has been well characterized, significant gaps in our understanding of how different steps in endocytic progression are coordinated and how endocytic proteins function still exist. Using a combination of yeast genetics, fluorescent microscopy, electron microscopy, and biochemistry we have furthered our understanding of clathrin-mediated endocytosis, focusing on the role adaptor-clathrin and adaptor-cargo binding plays in formation and progression of the endocytic process. Our work began by focusing on the role of the adaptor protein Sla1, a clathrin and cargo binding protein that serves essential roles in endocytosis. It was previously established that Sla1 binds clathrin through a variable clathrin box of sequence LLDLQ. Loss of clathrin binding by mutation of this clathrin box has a dramatic effect on endocytosis such as an increased patch lifetime of Sla1 at endocytic sites, and dramatic defects in internalization of endocytic protein cargo. While these experiments demonstrated the importance of Sla1-clathrin binding in endocytosis, they did not explain why Sla1-clathrin binding was important and how this interaction contributes mechanistically to endocytic progression. By imaging Sla1 and clathrin, our work demonstrates that Sla1 contributes to proper clathrin recruitment to endocytic sites. A loss of proper recruitment of clathrin to endocytic sites by mutation of the Sla1 variable clathrin box also resulted in significant accumulation of other endocytic proteins, including those involved in actin polymerization. The lifetime of these additional endocytic components lasted for significantly longer at endocytic sites, some having a disruption in normal recruitment dynamics. Despite this accumulation of the actin polymerization machinery, there is a significant delay in actin polymerization and an increase in actin polymerization time and levels at endocytic sites. Our results also demonstrate defects in the formation of the endocytic invagination and delays in scission. Thus, the Sla1-clathrin interaction is needed for normal progression through different stages of the endocytic process. A second question in the endocytic field that has received little attention is the role cargo plays in the recruitment of the endocytic machinery. The conventional view is that first the endocytic machinery forms an endocytic site and then cargo is concentrated by binding adaptor proteins. Sla1 has previously been shown to bind to endocytic protein cargo that contains the amino acid sequence NPFxD through its SHD1 domain. It has also been shown through biochemical experiments that Sla1 binds Ubiquitin via its third SH3 domain. Both NPFxD and Ubiquitin have been shown to be important signals of cargo for entry into the endocytic pathway. The question, however, remained as to whether cargo binding via these signals contributes to recruitment of the adaptor Sla1 to endocytic sites. The work described in this dissertation will present evidence that this is indeed the case.
Open Access
Cholinergic synaptic homeostasis is regulated by Drosophila α7 nicotinic acetylcholine receptors and Kv4 potassium channels
(Colorado State University. Libraries, 2021) Eadaim, Abdunaser Omar, author; Tsunoda, Susan, advisor; Tamkun, Michael, committee member; Amberg, Gergory, committee member; Bouma, Gerrit, committee member; Clay, Colin, committee member; DeLuca, Jennifer, committee member
Homeostatic synaptic plasticity (HSP) is an important mechanism that stabilizes neural activity during changes that occur during development and learning and memory formation, and some pathological conditions. HSP in cholinergic neurons has been implicated in pathological conditions, such as Alzheimer's disease and nicotine addiction. In a previous study in primary Drosophila neuron culture, cholinergic activity was blocked using pharmacological tools and this induced a homeostatic response that was mediated by an increase in the Drosophila α7 (Dα7) nAChR, which was subsequently tuned by an increase in the voltage-dependent potassium channel, Kv4/Shal. In this study, we inhibit cholinergic activity in live flies using temperature-sensitive mutant alleles of the choline acetyltransferase gene (Chats2 mutants). We show that this in vivo activity inhibition induces HSP similarly mediated by Dα7 nAChRs followed by an up-regulation of Kv4/Shal. We show that the up-regulation of Dα7 nAChRs alone is sufficient to induce an increase in Kv4/Shal protein, as well as mRNA. Finally, we test the involvement of transcription factors, dCREB2 and nuclear factor of activated T cells (NFAT) in the up-regulation of Kv4/Shal. In particular, we find that NFAT is required for the inactivity-induced up-regulation of Kv4/Shal channels. Our studies reveal a novel receptor-ion channel system transcriptionally coupled to prevent over-excitation.
Open Access
Determination of the functions of Rab32, Rab38, and their effector Myosin Vc in the biogenesis of melanosomes
(Colorado State University. Libraries, 2013) Bultema, Jarred, author; Di Pietro, Santiago, advisor; Ross, Eric, committee member; DeLuca, Jennifer, committee member; Chen, Chaoping, committee member; Reddy, Anireddy, committee member
In mammals, pigment produced within specialized cells is responsible for skin, hair, and eye coloration. Melanocytes are specialized cells that produce pigment within an organelle known as the melanosome. Melanosomes are a member of a specialized class of organelles, known as Lysosome-related organelles (LRO), which are responsible for a number of critical functions in mammals such as pigmentation, blood clotting, lung function, and immune function. LROs are related to the ubiquitous lysosome, and are formed using the same molecular mechanisms as lysosomes that rely upon the Adaptor Protein complexes -1 (AP-1) and -3 (AP-3), and the Biogenesis of Lysosome-related Organelles Complex (BLOC)-2 (BLOC-2). These protein complexes are critical for the trafficking of specialized cargoes to melanosomes required for proper melanin synthesis. But, these complexes are also used for the formation of lysosomes, and no mechanism is known to distinguish between trafficking to lysosomes and melanosomes. The melanosome serves as a model system to study the formation of LROs, and insights from the study of melanosomes help explain the biogenesis of other LROs. In this dissertation, I present the finding that Rab32 and Rab38 function as melanosome-specific trafficking factors that allow for the use of AP-3, AP-1, and BLOC-2 in melanosome biogenesis. Using biochemical approaches, I show that Rab32 and Rab38 bind directly to AP-3, AP-1, and BLOC-2 on membranes. In microscopy experiments, I demonstrate that Rab32 and Rab38 localize to early endosomal subdomains where AP-3, AP-1, and BLOC-2 function. Using a combination of biochemical and microscopic approaches, I show that Rab32 and Rab38 serve partially redundant functions in trafficking of specialized cargoes to melanosomes. I report the discovery that Myosin Vc, a class V myosin motor, interacts with Rab32 and Rab38 and serves novel functions in melanosomes trafficking. I show, using biochemical approaches, that Myosin Vc directly binds to several melanosomal Rab proteins and serves as an effector of these proteins in melanosome biogenesis. Using a combination of approaches, I demonstrate that depletion of Myosin Vc from melanocyte cells causes defects in the trafficking of cargoes to melanosomes, but also causes severe defects in the secretion of mature melanosomes. With biochemical and microscopic approaches, I compare the function and localization of Myosin Vc in melanocytes to related proteins Myosin Va and Myosin Vb, and provide evidence to suggest that all three of these proteins function in distinct steps of melanosome trafficking. My results answer outstanding questions about the use of ubiquitous trafficking machinery (AP-3, AP-1, and BLOC-2) in trafficking to a specialized organelle. I provide evidence to answer outstanding questions about the mechanism of action of Rab32 and Rab38 in melanosome trafficking through my studies with Myosin Vc. I also establish new areas of research in the comparison of Myosin Va, Myosin Vb, and Myosin Vc in melanosome trafficking. My results address numerous unknown areas in melanosome biogenesis, expand the knowledge of melanosome biogenesis, and provide numerous new avenues of research to explore to understand specialized trafficking to LROs.
Open Access
Development of single cell shape measures and quantification of shape changes with cancer progression
(Colorado State University. Libraries, 2018) Alizadeh, Elaheh, author; Prasad, Ashok, advisor; DeLuca, Jennifer, committee member; Munsky, Brian, committee member; Snow, Christopher D., committee member
In spite of significant recent progress in cancer diagnostics and treatment, it is still the second leading cause of death in the United States. Some of the complexity of cancer arises from its heterogeneity. Cancer tumors in each patient are different than other patients. Even different tumors from one patient could differ from each other. Such a high diversity of tumors makes it challenging to correctly characterize cancer and come up with the best treatment plan for each patient. In order to do that, a complex combination of clinical and histopathological data need to be collected. This dissertation provides the evidence that the shape of the cells can be used in conjunction with other methods for a more reliable cancer characterization. In this study, experimental studies, numerical representation of the cell shape, big data analysis methods, and machine learning techniques are combined to provide a tool to better characterize cancer cells using their shape information. It provides evidence that cell shape encodes information about the cell phenotype, and demonstrates that the former can be used to predict the latter. This dissertation proposes detailed quantitative methods for quantifying the shape and structure of a cell and its nucleus. These features are classified into three main categories of textural, spreading and irregularity measures, which are then sub-categorized into nine different shape categories. Textural measures are used to quantify changes in actin organization for the cells perturbed with cytoskeletal drugs. Using the spreading and irregularity measures, it is shown that the changes in actin structure lead to significant changes in irregularity of the boundary of a cell and spreading of the cell and nuclei. Using these methods, the shape of retina, breast, and osteosarcoma cancer cells are quantified and it is shown that the majority of cells have similar changes in their shape once they become cancerous. Then, a neural network is trained on the shape of the cells which leads to an excellent prediction of class of cancer cells. This study shows that even though cancer cells have different characteristics, they can be categorized into clinically relevant subgroups using their shape information alone.
Open Access
Dynamics of H3 and H2B octamer variants
(Colorado State University. Libraries, 2020) McVay, Abigail Lea, author; Hansen, Jeffrey C., advisor; Quackenbush, Sandra, committee member; DeLuca, Jennifer, committee member
Introductions of alterations within a nucleosome can lead to drastic changes to the accessibility and stability of genetic material during various phases of the cell cycle. To understand these changes, three octamer mutants were recombined, reconstituted with a high affinity tandem repeat sequence, and tested using a combination of in vitro experimental methods. All Tailless, H2BTL and an H2BTL & H3TL nucleosomal array mutants were sedimented, digested, re-associated, and visualized in order to determine the role of histone tails and their influence on chromatin condensate structure. The All Tailless octamer mutant expressed an inability to form complex molecular structures, suggesting that histone tails are necessary to further the process of chromatin condensate association and subsequent folding. The H2BTL mutant expressed high levels of concentration and an increased level of association compared to the other mutants. This lead to the assumption that the exclusion of only one histone tail lead to a greater ability to associate compared to mutants lacking two or more tails. The H2BTL & H3TL mutant had a possibility of two distinct populations within solution, suggesting that the exclusion of at least two tails led to a loosely compacted and easily accessible chromatin condensate structure. In summary, this data suggests that as histone tails are excluded from octamer mutants, the chromatin condensates expresses a decreased ability to form higher degrees of compaction. Exclusion of histone tails from octamer mutants also resulted in a more accessible 601-12 tandem repeat sequence susceptible to various changes.
Open Access
Dynein mutagenesis reveals the molecular basis for dynein regulation in broad spectrum neurological diseases
(Colorado State University. Libraries, 2020) Marzo, Matthew G., author; Markus, Steven M., advisor; Bamburg, James R., committee member; DeLuca, Jennifer, committee member; Prasad, Ashok, committee member
Eukaryotic cells rely on cytoskeletal networks to organize materials, transport organelles, give cells shape, and provide locomotion. The cytoskeleton is comprised of many diverse proteins, and three classes of polymeric protein structures are the actin, microtubule, and intermediate filament networks. The microtubule network, and its associated motors, dynein and kinesin, is of interest to the field of neurological disease, due to the prevalence of mutations in the microtubule network in human disease. To better understand the molecular basis for the diseases caused by de novo dynein mutations, we performed a screen of mutants using budding yeast dynein. The results from our experiments present a platform for the molecular dissection of dynein mutations which can be readily applied to new mutations or precisely explore known mutations. The screen-based approach allowed us to identify a new mechanism of yeast dynein regulation, which is autoinhibition of the dynein motor. We demonstrate that this mechanism regulates dynein activity in cells and functions to limit in vivo motor activity in the cytoplasm. Autoinhibition is regulated by Pac1 in yeast, a Lissencephaly-1 homolog, and we demonstrate that Pac1 operates in the dynein autoinhibition pathway by preventing the "closed" autoinhibited state, thereby promoting "open" dynein. This represents an entirely novel function of Pac1/LIS1, and allows us to further refine our model for cortical offloading.
Open Access
Force spectroscopy and dynamics in biological systems
(Colorado State University. Libraries, 2019) Schroder, Bryce William, author; Krapf, Diego, advisor; Bark, David, committee member; Popat, Ketul, committee member; DeLuca, Jennifer, committee member
Communication is key to any process involving the transmission of information or some sort of signal. For communication to occur, a signal must be created that can be detected. Cells communicate through cues transmitted in the forms of chemical and mechanical signals. The most fundamental means for transmitting chemical cues is through the process of diffusion. A single particle undergoing diffusion is considered to undergo Brownian motion, which can be modelled as a random walk. The random walk behavior is characteristic of both the particles properties and the fields in which it is occurring. An unbiased walk will be completely random without outside influence. A biased walk will be random within the confines of a potential influencing its direction. Both are Stochastic processes characterized through probabilistic models with known solutions. The work herein presents the development of single molecule experiments and the associated particle tracking tools targeting particles undergoing biased random walks within a trapping potential on or near a cellular membrane. In the first set of experiments, the trapping potential, an optical tweezers setup, has been developed and employed in measuring cellular membrane biophysical properties as well as blebbing forces. The optical trap was also used to directly measure flow driven forces in live embryonic zebrafish, the first known measurements of this kind. In the second set of experiments, synthetic lipid bilayers provided a trapping potential in a single dimension for protein binding experiments leading to exchanges between free, 3-dimensional diffusion and bound, or biased, 2-dimensional diffusion. In all cases, stochastic models have been used in conjunction with image-based particle tracking tools to better characterize the biophysical properties and forces associated with the cellular membrane and its means of signal transduction. These measurements are key to understanding both the chemical and mechanical signaling means by which the cellular membrane transduces an external signal into an internal response.
Open Access
Genome instability: a pre-existing condition
(Colorado State University. Libraries, 2018) Sedam, Hailey Nicole Conover, author; Argueso, Lucas, advisor; DeLuca, Jennifer, committee member; Nickoloff, Jac, committee member; Wiese, Claudia, committee member
Copy number variations (CNV), or large amplifications or deletions in the genome, account for about 50% of human genetic diversity. CNVs across genomic regions essential for development and function can lead to disease. The underlying mechanisms of CNV formation are typically traced to a combination of endogenous or environmental sources of DNA damage coupled with defects in DNA repair, replication, and recombination. This dissertation describes two endogenous sources of genome instability involved in both mitotic and meiotic CNVs. Each chapter of this dissertation focuses on one endogenous contribution to genome instability, using the budding yeast Saccharomyces cerevisiae as a model system to investigate the conserved cellular processes that, when gone awry, can lead to CNVs. In the first phase of my research, I focused on the mitotic mutagenic effects of ribonucleotide incorporation into DNA. In the absence of RNase H2, RNA-DNA hybrids (R-loops) accumulate in the genome and ribonucleotides that are misincorporated into the DNA are not efficiently excised. Instead, the latter function is taken over by topoisomerase 1 which inappropriately removes ribonucleotides in a way that leads to accidental/unscheduled DNA double strand breaks (DSBs). My work showed that the accumulation of these lesions in RNase H deficient mutants was sufficient to increase the rate of genome rearrangements through both Loss of Heterozygosity (LOH) and Non Allelic Homologous Recombination (NAHR). Modulating the number of ribonucleotides incorporated into the leading DNA strand during replication through the use of DNA polymerase epsilon mutants affected the rate of LOH and NAHR. Additionally, the RNase H2-Ribonucleotide Excision Repair Deficient (RNase H2-RED) separation of function allele allowed further investigation of genomic instability when R-loops are properly processed but misincorporated ribonucleotides are not. The RNaseH2-RED study revealed that both ribonucleotide excision repair and R-loop removal contribute roughly equally to chromosomal stability under normal conditions. Together, the results of these studies indicated that the effects of ribonucleotides and R-loops on chromosomal instability may vary under different genomic contexts of variable R-loop formation and ribonucleotide density. Next, I designed, constructed, optimized and validated a new yeast assay system to study meiotic NAHR leading to de novo recurrent CNVs. The chromosomal rearrangements analyzed through this system are directly analogous to human pathogenic CNVs that are formed in germ cells through recombination between Low Copy Repeat elements (LCRs). While there are assays available to investigate factors involved in mitotic CNV formation, few assays have been developed to experimentally test factors involved in meiotic recurrent CNVs. Previous studies of human patient cohorts have shown that the size and distance between LCRs is strongly correlated with the frequency of recurrent CNV formation. We used this basic observation to validate our experimental system and ask whether it could faithfully recapitulate the phenomenon in our yeast model system. I constructed four diploid strains containing LCRs engineered to range in size from 5-35 Kb and determined the meiotic NAHR frequency in each construct. We detected a very clear linear correlation between LCR size and CNV frequency, and thus established our system as a pertinent assay for interrogation of factors involved in meiotic recurrent CNV formation. The results described within this dissertation have deepened our understanding of the endogenous causes of genome instability leading to CNVs, and provide perspective into the ability of normal cellular processes to trigger both mitotic and meiotic CNV formation. Additionally, I describe a unique method for future screens of both endogenous and exogenous stimulants of meiotic CNV.
Open Access
Identification of therapeutic targets in canine bladder cancer: a translational model for MAPK pathway-targeted and immune-based therapies
(Colorado State University. Libraries, 2021) Cronise, Kathryn Elizabeth, author; Duval, Dawn, advisor; Gustafson, Daniel, advisor; DeLuca, Jennifer, committee member; Page, Rodney, committee member; Thamm, Douglas, committee member
Activating mutations in the proto-oncogene BRAF are drivers of oncogenesis in several human cancers, including melanoma, thyroid and colorectal carcinomas, and hairy-cell leukemia. Small molecule inhibitors targeting oncogenic BRAF demonstrate initial efficacy in approximately 50% of BRAF mutant melanoma patients; however, acquired resistance invariably develops. Other individuals, including the majority of colorectal cancer patients, exhibit intrinsic resistance to BRAF inhibitors. Combined inhibition of BRAF and its downstream target MEK improves the rate and duration of patient response, but resistance remains an issue. Thus, more effective and robust therapies are necessary. Transitional cell carcinoma (TCC) is the most common bladder cancer in dogs and humans. In this study, we provide a molecular characterization of 11 canine TCC (cTCC) tumors and identified BRAF mutations in 8 out of 11 samples. All BRAF mutations were valine-to-glutamic acid missense substitutions at amino acid residue 596 of canine BRAF (V596E), analogous to the V600E driving variant in human cancer. Additionally, 22 out of 32 formalin-fixed paraffin embedded samples expressed mutant BRAF, indicating an overall prevalence of 70%. Further analysis identified four tumors, three being BRAF mutant, that exhibited increased expression of immune gene markers and gene signatures associated with complete clinical response to checkpoint inhibition in human bladder cancer. We also found that all TCC tumors overexpress cell cycle, DNA repair, and immune-related genes. The high prevalence of BRAF mutations in cTCC makes targeting BRAF with small molecule inhibitors an attractive therapeutic option. We explored this possibility in vitro and determined that BRAF mutant cTCC cell lines are insensitive to the BRAF inhibitor vemurafenib but are sensitive to the newer, "paradox-breaking" BRAF inhibitor PLX7904. All tested cTCC cell lines were sensitive to the MEK1/2 inhibitors trametinib and selumetinib. A phenomenon observed with single-agent BRAF or MEK inhibition was the reactivation of ERK1/2 within 24 hours post-treatment, suggesting built-in mechanisms of bypassing BRAF and MEK inhibition. We also observed upregulation of genes encoding the ErbB family receptors, EGFR and ERBB2, and the EGFR ligand, EREG, in cTCC cell lines compared to other canine cancer cell lines. Treatment with the pan-ErbB inhibitor sapitinib synergized with BRAF or MEK inhibition in the BRAF mutant Bliley cell line and in the BRAF wild-type Kinsey cell line. Next, we generated trametinib-resistant clonal derivatives of the BRAF mutant Tyler1 cTCC cell line (Tyler1-TramR). Tyler1-TramR cells exhibited trametinib IC50 values over 500 nM and maintained suppression of ERK1/2 phosphorylation for 24 hours following trametinib treatment. This response, combined with the insensitivity of Tyler1-TramR cell lines to the ERK1/2 inhibitor ravoxertinib, suggests that resistance to trametinib is independent of ERK1/2 reactivation. Further analysis of two Tyler1-TramR clones using RNA-Seq identified a loss of epithelial gene markers, while mesenchymal genes and transcription factors controlling the epithelial-to-mesenchymal transition were upregulated. Analysis of basal cellular metabolism using a Seahorse XF analyzer revealed that one of the Tyler1-TramR clones exhibited altered metabolism compared to the parental Tyler1 characterized by decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and decreased glycolytic reserve. Collectively, these results demonstrate that spontaneous, BRAF mutant cTCC can be utilized as a translational model for investigating novel targeted and immune-based therapies that may improve treatment in both canine and human MAPK-driven cancers.
Open Access
Investigating the regulators of cytoplasmic dynein
(Colorado State University. Libraries, 2019) Dilsaver, Matthew, author; Markus, Steven, advisor; Di Pietro, Santiago, committee member; DeLuca, Jennifer, committee member; Argueso, Juan Lucas, committee member
Organization of the cell is a dynamic and complex process that is often underappreciated. To accomplish this, cells use motor proteins to move different cargo to their destination. Cytoplasmic dynein is one such motor protein that uses filaments called microtubules as tracks. However, there is only one cytoplasmic dynein to accomplish over forty tasks. To achieve this, the cell uses a complex array of cofactors and regulators to specifically control dynein. But the role of each of these cofactors and regulators in poorly understood. To better understand how dynein is regulated we turn to budding yeast that provides a simplified system where dynein only has one known function, this is to position the spindle in the division plane between two dividing cells. Localizing dynein is extremely important. One regulator of dynein is Pac1 which was recently found to also activate dynein motility in vitro. Pac1 works to localize dynein to microtubule plus ends where it can interact with dynactin and Num1. Ndl1 is known to interact with Pac1, knockouts of Ndl1 led to a mild phenotype mimicking a dynein knockout. But how Ndl1 functions is poorly understood. Dynactin is an essential regulator of almost all dynein's tasks in humans and dynein's only role in yeast. Without dynactin, dynein cannot reach Num1 patches at the cell periphery and pull the spindle. In this study we sought to better understand dynactin and Ndl1's role in dynein regulation using in vitro single molecule assays where the activity of dynein can be recorded. Initial attempts to purify dynactin for these assays failed. We then developed a cell lysis assay to study dynactin and other proteins role in dynein regulation. We found in preliminary results that dynactin increased dynein activity. We also attempted to use a protein known as Num1, that is essential to dynein localization and interacts with dynactin, to purify the dynein-dynactin complex. Preliminary results showed that this complex was motile, indicating an intact complex. We also found that Ndl1 can bind motile dynein and increase run length using in vitro assays. We also were able to determine that Pac1 cannot bind dynein and Ndl1 at the same time indicating that there is a release mechanism for Pac1 from Ndl1 to bind dynein. We were able to map Ndl1's binding site to the N terminus of the dynein accessory chain Pac11. Then we tested to see if Ndl1 influence on Pac1-dynein interaction and found that Ndl1 was able to increase Pac1 comigrating with dynein in these assays. This work has opened new strategies for studying the regulators of dynein as well as better determined the interaction between Ndl1, dynein and Pac1. Further work will determine how each of these proteins affect dynein activity.
Embargo
IQGAP1 is a novel effector of gonadotropin-releasing hormone receptor signaling
(Colorado State University. Libraries, 2023) Alqahtani, Huda A., author; Amberg, Gregory, advisor; Clay, Colin, committee member; Tamkun, Michael, committee member; DeLuca, Jennifer, committee member
Stimulation of gonadotropin-releasing hormone (GnRH) receptors on the surface of anterior pituitary gonadotrope cells is a key signaling event for the hypothalamic-pituitary-gonadal axis. One important downstream component of GnRH receptor signaling is activation of the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase), which is essential for the production of the gonadotropin luteinizing hormone. Evidence suggests that GnRH receptors reside in low-density plasma membrane domains where they participate in multiprotein signaling complexes. Here we used quantitative proteomics to identify proteins associated with low-density plasma membrane domains and to measure changes in their relative abundance in these domains in response to GnRH. Using αT3-1 gonadotropes, we identified 537 proteins in detergent-free subcellular fractions containing low-density plasma membranes. SILAC (stable isotope labeling by amino acids in cell culture) in combination with mass spectrometry demonstrated that GnRH, within 10 min, altered the association of 87 proteins with this plasma membrane fraction. Ontology analysis revealed that GnRH promoted an enrichment of actin cytoskeletal and adherens junction-related proteins including the molecular scaffold IQGAP1 and the small GTPase Rac1. Subsequent investigation revealed that the association between Rac1 and IQGAP1 increased with GnRH receptor stimulation and that GnRH increased Rac1 activity. Demonstrating functional relevance, inhibiting Rac1 reduced GnRH-dependent ERK activation. Our data reveals an upstream activation of signaling and structural molecules, including Ca2+, CDC42 and Rac1, E-cadherin, N-cadherin, and β-catenin. We also identified interactions between the scaffold protein IQGAP1 and these molecules, indicating that IQGAP1 is a fundamental regulator of GnRH-dependent signaling in gonadotropes. Furthermore, our data shows that IQGAP1 has a transcriptional regulatory role in gonadotropes treated with GnRH. In sum, these data indicate that IQGAP1 complexed with Rac1 modulates ERK activity and as such serves as an essential effector in modulating cell polarity and cell-cell contacts in gonadotropes. Altogether, our proteomics data show that acute stimulation of GnRH receptors (3 nM for 10 min) alters the PAM fraction abundance of proteins, such as IQGAP1, mechanistically linked to gonadotrope activation.
Open Access
Modeling human trophoblast development during the peri-implantation period using extended embryo culture
(Colorado State University. Libraries, 2023) Logsdon, Deirdre Maria, author; Winger, Quinton, advisor; Krisher, Rebecca, advisor; Yuan, Ye, committee member; Tesfaye, Dawit, committee member; DeLuca, Jennifer, committee member
During the peri-implantation period, a human embryo must transition from a pre-implantation stage blastocyst to a gastrulating embryonic disc surrounding by the primitive placenta. The primitive placenta at this time establishes contact, proliferates, invades, modulates the maternal immune system, and provides a primitive form of nutrients to the implanting embryo proper. Insights into this period have been largely stunted due to the ethical and technical challenges that accompany human embryo research. Studies using donated human embryos following fertility treatment are complicated by confounding infertility diagnoses and limited sample sizes. The development of the extended culture system has provided an avenue to functionally study the peri-implantation period. Further, by using a variety of models including mouse embryos, human embryos, and stem cell-derived blastoids in the extended culture system, researchers are finally able to begin to piece together the puzzle of the peri- implantation period. Here, our objectives were to demonstrate the utility of mouse models in modeling human trophoblast during peri-implantation extended culture, examine and summarize human development during peri-implantation in the context of confounding fertility diagnoses, compare human trophoblast in extended culture to other widely available regenerative trophoblast models, and determine to what extent blastoids are able to reflect human peri-implantation development and maternal-fetal crosstalk in extended culture. Further, we show that estrogen signaling in trophectoderm may be conserved between mouse and human embryos, aged embryos exhibit hindered growth in extended culture, peri-implantation trophoblast cells have unique transcriptional priorities, and the presence of endometrial stromal cells encourage fusion of syncytiotrophoblasts. Our studies both reinforce the significance of the extended culture system and lay the groundwork for future studies on early trophoblast and embryo development during peri-implantation.
Open Access
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.
Open Access
Oxidant-dependent regulation of L-type calcium channel activity by angiotensin in vascular smooth muscle
(Colorado State University. Libraries, 2015) Chaplin, Nathan L., author; Amberg, Gregory, advisor; DeLuca, Jennifer, committee member; Tamkun, Michael, committee member; Tsunoda, Susan, committee member
Resistance arteries are a major point of physiological regulation of blood flow. Increases in vessel wall stress or sympathetic activity stimulate vascular wall angiotensin signaling, resulting in smooth muscle contraction which directly increases peripheral resistance. Calcium influx through voltage-gated L-type calcium channels underlies vascular smooth muscle contraction. Roughly half of calcium influx in these cells occurs through a small number of persistently active channels, whose activity increases with membrane depolarization. The number of channels gating in this manner is increased by activation of angiotensin receptors on the cell membrane, and basal L-type channel activity is increased during hypertension. Reactive oxygen species are also generated by vascular smooth muscle in response to vessel stretch and by several paracrine signaling pathways including angiotensin signaling. Oxidative stress and augmented calcium handling resulting from chronic angiotensin signaling in the vasculature each contribute to enhanced vessel reactivity, pathological inflammation and vessel remodeling associated with hypertension. This study uses a multidisciplinary approach to investigate the role of hydrogen peroxide in angiotensin signaling in vascular smooth muscle. Using calcium- and redox-sensitive fluorescent indicators, local generation of hydrogen peroxide by NAD(P)H oxidase and mitochondria are shown to synergistically promote PKC-dependent persistent gating of plasma membrane L- type calcium channels in response to angiotensin II. We show that broad inhibition of hydrogen peroxide signaling by catalase and targeted inhibition of mitochondrial reactive oxygen species production attenuates cerebral resistance artery constriction to angiotensin. We further demonstrate the role of endothelium-independent mitochondrial reactive oxygen species in development of enhanced vessel tone and smooth muscle calcium in a murine model of hypertension. Together, these findings contribute to the understanding of intracellular calcium and oxidative signaling in vascular physiology and disease and may provide insight into local signaling dynamics involving these second messengers in various other systems.
Open Access
Physicochemical characterization of self-associated chromatin oligomers
(Colorado State University. Libraries, 2016) Rogge, Ryan, author; Hansen, Jeffrey, advisor; Luger, Karoline, committee member; DeLuca, Jennifer, committee member; Mykles, Donald, committee member
The DNA of chromosomes is extensively compacted within the nuclei of eukaryotic nuclei. Chromosomes are composed of chromatin which is a repeating polymer of nucleosomes bound by additional chromatin proteins. Chromatin can be reconstituted in vitro using purified DNA and histone proteins to form nucleosomal arrays. Reconstituted chromatin fibers are structurally dynamic and the structures formed are highly dependent on the buffer conditions, particularly polyvalent cations. The addition of Mg2+ favors nucleosome-nucleosome interactions. At low concentrations nucleosomes on the same fiber interact resulting in folding, while at higher concentrations inter-fiber interactions result in chromatin self-association. Unlike folded chromatin, the oligomeric structure of chromatin is unkown, to address this deficiency, in this dissertation the oligomeric structures formed by 12-mer nucleosomal arrays were characterized by microscopy, sedimentation velocity, and SAXS experiments. The oligomeric chromatin complexes were globular throughout all stages of the cooperative assembly process, and ranged in size from ~50 nm to a diameter of ~1000 nm. The oligomer sedimentation coefficients under these conditions ranged from 5000-350,000S, corresponding to ~1-400 Mb DNA/oligomer. The nucleosomal arrays were packaged within the oligomers as interdigitated 10-nm fibers, rather than folded 30-nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in 0 mM MgCl2, conditions that also dissociated the oligomers in vitro. These results indicate that a 10-nm array of nucleosomes has the intrinsic ability to self-assemble into large chromatin globules stabilized by nucleosome-nucleosome interactions, and suggest that the oligomers are good in vitro model for investigating the structure and organization of interphase chromosomes.
Open Access
Regulation of dynein activity during spindle positioning in budding yeast
(Colorado State University. Libraries, 2020) Lammers, Lindsay, author; Markus, Steven, advisor; DeLuca, Jennifer, committee member; Tao, Tingting, committee member; Reddy, Anireddy, committee member
Cytoplasmic dynein is a minus-end directed, microtubule motor that is highly regulated to ensure it is targeted to the correct location at a specific time for its function in cells. This is particularly important for the process of spindle positioning during mitosis. Dynein is targeted to the cell cortex and activated to pull on astral microtubules attached to spindle poles to move the spindle into position at the site of cytokinesis. The position of the spindle dictates the plane of division and influences whether a cell divides asymmetrically or symmetrically- an important distinction during embryonic development and homeostasis. Using the model organism budding yeast, we confirmed that dynein is held in an inactive state before reaching its destination at the cell cortex by identifying a key factor in dynein activation- the cortical receptor Num1. We determined that the mechanism of activation involves enhancing dynein-dynactin interaction and releasing the recruitment factor, Pac1/Lis1. Additionally, I determined the role of another regulator in the dynein pathway, Ndl1/NudE. Ndl1/NudE aids the recruitment factor, Pac1/Lis1 in targeting dynein to astral microtubule plus ends that then deliver the motor to the cortex. Interestingly, it appears Ndl1/NudE may have another function that competes Pac1/Lis1 off dynein in a specific context. Next, I explored the two possible mechanisms of Num1-mediated dynein activation. First, I established an in vitro motility assay to observe how the regulators dynactin, Num1 and Pac1/Lis1 may coordinate to affect dynein activity. I determined the purification conditions for complete dynactin complexes as well as Num1 constructs to test whether Num1 acts as an adapter to activate the dynein-dynactin complex. Finally, I examined the second mechanism of Num1-mediated activation by initiating the release of Pac1/Lis1 from dynein complexes. I predicted that Num1 may influence the conformational changes of dynein during its mechanochemical cycle in conjunction with dynein engaging the microtubule that could induce Pac1/Lis1 release. To test this, I mutated dynein in a way that restricted conformation changes and observed how this affected Pac1/Lis1 interaction. The results show that Pac1/Lis1 binding is profoundly affected by dynein structure. Further, Num1 can still initiate Pac1/Lis1 release despite restriction in conformational changes, which suggests Num1 may initiate Pac1/Lis1 disassociation in another way. Together these data reveal important details of how regulatory proteins coordinate to spatially and temporally regulate dynein during spindle positioning.
Open Access
The biophysical, biochemical and structural characterization of Poly(ADP-ribose) Polymerase-1 (PARP-1) and its complexes with DNA-damage models and chromatin substrates
(Colorado State University. Libraries, 2013) Clark, Nicholas James, author; Luger, Karolin, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey C., committee member; Woody, Robert, committee member
Eukaryotic DNA is highly dynamic and must be compacted and organized with the help of cellular machines, proteins, into 'heterochromatin' state. At its basic level, chromatin is comprised of spool-like structures of protein complexes termed histones, which bind and organize DNA into larger fibrous structures. Cellular processes like transcription and DNA-damage repair require that chromatin be at least partially stripped of its protein components, which in turn allows for complete accessibility by DNA-repair or transcription machinery. A number of protein factors contribute to chromatin structure regulation. Poly(ADP-ribose) Polymerase-1 (PARP-1) is one of these proteins that exists in all eukaryotic organisms except for yeast. In its inactive form, it compacts chromatin, but performs its chromatin-opening function by covalently modifying itself and other nuclear proteins with long polymers of ADP-ribose in response to DNA damage. Thus, it also serves as a first responder to many types of DNA damage. The highly anionic polymers serve to disrupt protein-DNA interactions and thus allow for the creation of a temporary euchromatin structure. Contained herein are investigations aimed at addressing key questions regarding key differences between the interactions of PARP-1 and chromatin and its DNA-damage substrates. Our experiments show that human PARP-1 interacts with and is enzymatically activated to a similar level by a variety of different DNA substrates. In terms of chromatin, it appears that PARP-1 fails to interact with nucleosomes that do not have linker DNA. PARP-1 most effectively interacts with chromatin by simultaneously binding two DNA strands through contacts made by its two N-terminal Zn-finger domains. Small-Angle X-ray (SAXS) and Neutron Scattering (SANS) and molecular dynamics (MD) experiments were combined with biophysical and biochemical studies to better describe the structural effects of DNA binding on PARP-1. The average solution structure of PARP-1 indicates that the enzyme is a monomeric, non-spherical, elongated molecule with a radius of gyration (Rg) of ~55Å. The DNA-bound form of PARP-1 is also monomeric and binding DNA causes the molecule to become more elongated with an average Rg of ~80Å.
Open Access
The chromatin binding factor Spn1 contributes to genome instability in Saccharomyces cerevisiae
(Colorado State University. Libraries, 2018) Thurston, Alison K., author; Stargell, Laurie, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey, committee member; Luger, Karolin, committee member
Maintaining the genetic information is the most important role of a cell. Alteration to the DNA sequence is generally thought of as harmful, as it is linked with many forms of cancer and hereditary diseases. Contrarily, some level of genome instability (mutations, deletions, amplifications) is beneficial to an organism by allowing for adaptation to stress and survival. Thus, the maintenance of a "healthy level" of genome stability/instability is a highly regulated process. In addition to directly processing the DNA, the cell can regulate genome stability through chromatin architecture. The accessibility of DNA for cellular machinery, damaging agents and spontaneous recombination events is limited by level of chromatin compaction. Remodeling of the chromatin for transcription, repair and replication occurs through the actions of ATP remodelers, histone chaperones, and histone modifiers. These complexes work together to create access for DNA processing and to restore the chromatin to its pre-processed state. As such, many of the chromatin architecture factors have been implicated in genome stability. In this study, we have examined the role of the yeast protein Spn1 in maintaining the genome. Spn1 is an essential and conserved transcription elongation factor and chromatin binding factor. As anticipated, we observed that Spn1 contributes to the maintenance of the genome. Unexpectedly, our data revealed that Spn1 contributes to promoting genome instability. Investigation into a unique growth phenotype in which cells expressing a mutant form of Spn1 displayed resistance to the damaging agent, methyl methanesulfonate revealed Spn1 influences pathway selection during DNA damage tolerance. DNA damage tolerance is utilized during replication and G2 to bypass lesions, which could permanently stall replication machinery. This pathway congruently promotes and prevents genome instability. We theorize that these outcomes are due to the ability of Spn1 to influence chromatin structure throughout the cell cycle.
Open Access
The microtubule-associated protein She1 regulates dynein-mediated spindle positioning in budding yeast
(Colorado State University. Libraries, 2020) Ecklund, Kari, author; Markus, Steven, advisor; DeLuca, Jennifer, committee member; Peersen, Olve, committee member; Krapf, Diego, committee member
Microtubules are polar filamentous proteins part of a complex cytoskeletal network within cells that provides an organized interface with which motors use to transport vesicular cargoes and organelles, and mediate positioning of the mitotic spindle during cell division. There are two groups of molecular motor proteins that use microtubules as a track: (1) kinesins, the predominant anterograde motors and which are represented by six distinct different motors in budding yeast and (2) dynein, the predominant retrograde motor to which there is only one, cytoplasmic dynein, in budding yeast. Regulation of motor proteins is paramount to ensure that these various functions are achieved efficiently in a time and space-sensitive manner. There are many ways microtubules regulate their track, including through a class of highly diverse proteins called microtubule-associated proteins (MAPs), one of which in budding yeast is She1. In budding yeast, the only currently known role of cytoplasmic dynein is positioning the mitotic spindle during cell division. To direct the polarized movement of the spindle towards the daughter-cell, dynein relies on the MAP She1. To understand the mechanism by which She1 may regulate dynein-mediated spindle positioning, we first characterized the effects of She1 on dynein motility using recombinant protein. Our results demonstrated that She1 affects dynein motility by enhancing dynein-microtubule binding through simultaneous interactions with the dynein microtubule binding domain (MTBD) and the microtubule. From our in vitro data, we suggested a model where She1 assists dynein force generation to pull the large nucleus into the narrower bud neck connecting mother and daughter cells. However, we tested this model in vivo and found no such effects on nuclear translocation success, leaving us to investigate an alternative model where She1 polarizes spindle movements towards the daughter cell through inhibiting dynein activity in the mother cell. We explored this model in vivo using a comprehensive analysis of dynein-mediated spindle movements which revealed She1 ensures dynein in the daughter cell maintains bud neck proximity by inhibiting dynein activity and the initiation of dynein-mediated spindle movements in the mother cell. Moreover, we find that this process depends on She1 binding to aMTs in the mother cell and not spindle microtubules where She1 also localizes. Finally, we provide evidence that She1 requires the MTBD of dynein for some aspects of this inhibition, reconciling, in part, our in vitro and in vivo data. Our data provides a fascinating new mechanism of regulation by a MAP and suggests a new angle to approach future exploration of MAP-mediated regulation in higher eukaryotes.