Cell Biology & Molecular Genetics
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Item THE MECHANISMS AND ROLES OF POST-TRANSLATIONAL PROCESSING OF THE DROSOPHILA FIBROBLAST GROWTH FACTOR BRANCHLESS DURING DEVELOPMENT(2019) Sohr, Alexander Ronayne; Roy, Sougata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)During embryonic development, cells communicate with each other to cooperate to form organized tissues. Cells spatiotemporally coordinate with each other by communicating with signaling proteins such as Fibroblast Growth Factor (FGF) that travel from source to target cells to activate various functions. To better understand cell communication during tissue morphogenesis, this study aimed to address a fundamental question: how different cellular and molecular mechanisms in signal-producing cells prepare and release signals at the correct time and location and at an appropriate level. This research focuses on the intercellular communication of the Drosophila FGF Branchless (Bnl) to elucidate this question. Bnl is dynamically produced in restricted groups of cells to induce morphogenesis of tracheal airway epithelial tubes. Tracheal cells receive the signal over distance by extending long receptor-containing filopodia, or cytonemes, to dynamically contact the Bnl-source. This work discovered two post-translational modifications of Bnl that regulate its polarized intracellular trafficking and cytoneme-mediated intercellular dispersal. During intracellular trafficking through the source cell Golgi network, Bnl is endo-proteolytically cleaved at a single site by the protease Furin-1. This cleavage activates polarized intracellular trafficking of the truncated signal exclusively to the surface of the source cells that faces the recipient tracheal cells. Thus, the intracellular cleavage acts as a switch to catalyze the efficient trafficking of the signal to the correct location from where cytonemes can subsequently receive it. Secondly, in the endoplasmic reticulum of source cells, Bnl is modified with a glycosylphosphatidylinositol (GPI) moiety at its C-terminus. This lipid moiety tethers Bnl molecules to the outer leaflet of the cell membrane, inhibiting its free release and ensuring signal exchange solely by direct physical contacts established by cytonemes. Therefore, this study discovered how Bnl is prepared by the source cells to ensure its subsequent target-specific intercellular dispersion through cytonemes. Conserved FGF family proteins are essential for regulating a broad spectrum of biological functions and defects in spatiotemporal levels of FGF signaling leads to severe diseases. Given the conservation of developmental signaling mechanisms in all organisms, the discovery of new regulatory mechanisms of FGF signaling has fundamental implications for understanding development and disease in humans.Item Novel Models for Studying Trophoblast Development and Placental Pathologies(2019) Pence, Laramie; Telugu, Bhanu; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Placental development begins in the mammalian blastocyst, when the first lineage specification event commits one cell population to making extraembryonic tissues, including the placenta, and commits another cell population to making the embryo proper. The mouse is an excellent animal model to study these early events and how the resulting placenta organ supports normal fetal development and a healthy pregnancy in the mother. The studies included in this Dissertation use the mouse to understand the role of long non-coding RNAs during early placental development, and to create a lineage biasing model that takes advantage of what is known about the first lineage specification event in mammals. Using expression analysis and the CRISPR/Cas9 system to create a knockout mouse strain, a placental-specific lncRNA was discovered and shown to be expressed in derivatives of the ectoplacental cone. Additionally, using the line age bias model to cause biased ablation of Hif1α in the placenta has revealed a role for fetal vs. placental contribution of resulting phenotypes.Item NEISSERIA GONORRHOEAE MODULATES INFECTIVITY BASED ON PROPERTIES OF HUMAN CERVICAL EPITHELIA AND PHASE VARIABLE BACTERIAL SURFACE STRUCTURES(2019) Yu, Qian; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) infection in the human female reproductive tract causes various clinical outcomes, from no symptom to severe complications. The major barrier to a better understanding of GC infection in women is the lack of experimental system closely mimicking in vivo infection. Here, I developed a human cervical tissue explant model, which maintains the heterogeneity of the cervical epithelium. Using this model, my thesis research examined the impact of the heterogeneity of the cervical epithelium and the phase variation of GC surface structures on GC infectivity. My research revealed that GC preferentially colonize the ectocervix and the transformation zone (TZ), but exclusively penetrate into the subepithelial tissues of the TZ and endocervix. Pili are essential for GC colonization in all regions of the cervix. Expression of Opa isoforms that bind to the host receptors CEACAM (OpaCEA) enhances GC colonization in the ecto/endocervix but inhibits GC penetration into the endocervix. However, GC infectivity in the TZ does not respond to Opa phase variation, due to the low expression level and intracellular location of CEACAMs in the TZ epithelial cells. OpaCEA enhances GC colonization in the ecto/endocervix by inhibiting epithelial exfoliation and suppresses GC penetration into the endocervical subepithelium by inhibiting GC-induced disassembly of the apical junction. Opa-mediated modulation of GC infectivity depends on the immune receptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and its downstream phosphatase SHP. The effect of epithelial cell polarity on GC invasion was studied using a cell line model. My results show that GC invade more efficiently into non-polarized than polarized epithelial cells without changing the adhesion efficiency. Opa (phase variable) expression enhances both adhesion and invasion in both non-polarized and polarized cells. In non-polarized cells, Opa expression induces F-actin accumulation and microvilli elongation underneath GC microcolonies, suggesting an actin-mediated uptake of GC. In contrast, GC expressing no Opa reduce F-actin and demolish microvilli underneath microcolonies in both polarized epithelial cell line and endocervical epithelial cells potentially by increasing calcium flux, NMII activation and the redistribution of actin nucleation factor Arp2/3 from the apical surface. Taking together, my research demonstrates that both the heterogeneity of the cervical epithelium and the phase variation of bacterial surface structures regulate GC infectivity in the human cervix, either dominated by colonization or penetration, consequently influencing the clinical outcomes of the infection.Item Investigating the role(s) of mammalian heme transport by HRG1(2019) Pek, Rini; Hamza, Iqbal; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The recycling of hemoglobin from damaged or senescent red blood cells (RBCs) contributes almost 90% of daily body iron requirements in humans for bone marrow erythropoiesis. Previously, our cell biological studies have shown that HRG1, a four transmembrane protein first discovered in C. elegans, facilitates the transport of heme within reticuloendothelial system (RES) macrophages during the turnover of RBCs, a process termed erythrophagocytosis. HRG1 transports heme from the phagolysosomes into the cytosol where heme is degraded to liberate iron for erythropoiesis. Here we show that mice deficient for HRG1 are defective in heme- iron recycling by RES macrophages, resulting in over ten-fold excess heme accumulation as visible dark pigments within lysosomal compartments that are 10- 100 times larger than normal. The sequestered heme is tolerated by macrophages through polymerization into crystallized hemozoin, a phenomenon typically observed in blood-feeding parasites as a detoxification method to protect against heme toxicity. HRG1-/- mice display ineffective bone marrow erythropoiesis which results in a reduction in hematocrit and extramedullary erythropoiesis in the spleen. Under iron- deficient conditions HRG1-/- mice are unable to utilize hemozoin as an iron source to sustain erythropoiesis, causing severe anemia and lethality. Our studies establish that polymerizing cytotoxic heme into hemozoin is a previously-unanticipated heme tolerance pathway in mammals.Item Structure-Guided Engineering of a Multimeric Bacteriophage-Encoded Endolysin PlyC(2019) Shang, Xiaoran; Nelson, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Emerging antibiotic resistance has become a global health threat. One alternative to antibiotics is bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage replication cycle resulting in bacterial cell lysis and progeny bacteriophage release. Endolysins are also capable of destroying the Gram-positive bacterial peptidoglycan when applied externally as recombinant proteins. These enzymes typically consist of an enzymatically active domain (EAD) and a separate cell wall binding domain (CBD). Studies have shown therapeutic efficacy of endolysins in vitro and in vivo, with no resistance developed to date. An endolysin from the streptococcal C1 phage, known as PlyC, has the highest activity of any endolysin reported. It also has a unique multimeric structure consisting of one activity subunit (PlyCA) harboring two synergistically acting catalytic domains, GyH and CHAP, and eight identical binding subunits (PlyCB) forming an octameric ring. Groups A, C, and E streptococci as well as Streptococcus uberis are sensitive to the lytic activities of PlyC. In order to harness the potent activity of PlyC for use against other bacteria, we sought to change/extend the host range of PlyC by engineering PlyCB and PlyCA, respectively. We first used a structure-guided mutagenesis method to obtain the single PlyCB monomer subunit, PlyCBK40A E43A (PlyCBm), aiming to study the binding mechanism of PlyCB. Via fluorescence microscopy and binding assays, we determined that PlyCBm retained the host range of the octamer with a much lower binding affinity, which suggests the PlyCB octamer binds concurrently to a specific epitope on the bacterial surface resulting in a tight, stable interaction. Thus, it is not feasible to change/extend the PlyC host range via engineering PlyCB. Next, we proposed a novel design to engineer PlyCA. We successfully created two chimeric endolysins, ClyX-1 and ClyX-2, possessing the synergistic activity of the GyH and CHAP catalytic domains, but extended the host range to include, Streptococcus pneumoniae, Group B streptococci, Streptococcus mutans, and Enterococcus faecalis, all strains previously insensitive to PlyC. Finally, we tested a novel hypothesis that a positively charged catalytic domain could display lytic activity in a CBD-independent manner resulting in a broad host range. Using the PlyC CHAP domain as a model, we converted the net surface charge of the CHAP domain from negative three to positive one through positive seven. Notwithstanding the range of charges, our mutant CHAP domains did not show lytic activity in a CBD-independent manner, suggesting that other factors, like surface charge distribution, need to be considered in such a way of engineering.Item INVESTIGATION OF DEFECTIVE CELL SIGNALING CASCADE INVOLVED IN THE OSTEOGENESIS IN HUTCHINSON-GILFORD PROGERIA SYNDROME(2018) Choi, Ji Young; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human bone homeostasis is maintained through constant bone remodeling, which balances bone formation by osteoblasts and bone resorption by osteoclasts. Patients with Hutchinson-Gilford progeria syndrome (HGPS) have low bone mass that manifests in a high risk of fractures and an atypical skeletal geometry, suggesting impaired bone remodeling. HGPS is a premature aging disease caused by truncated lamin A that is permanently farnesylated. The mutant lamin A is referred as progerin. Several previous clinical reports discussed abnormal skeletal development of the children with HGPS, but the molecular mechanistic study on defective osteogenesis of HGPS stem cells need to be further elucidated. The major aim of my dissertation research is to investigate dysfunction in stem cell differentiation due to aberrant cell signaling in osteoprogenitor cells that express progerin. To achieve this aim, the study demonstrates both in vitro and in vivo models of HGPS to support defective mechanism of the canonical WNT/β-catenin pathway, seemingly at the level of efficiency of nuclear import of β-catenin and impaired osteoblast differentiation. Restoring β-catenin activity rescues osteoblast differentiation and significantly improves bone mass. In particular, HGPS patient-derived induced pluripotent stem cells (iPSCs)-osteoprogenitors and primary mesenchymal stem cells (MSCs) expressing the HGPS mutant progerin display defects in osteoblast differentiation, characterized by deficits in alkaline phosphatase activity and mineralizing capacity. Mechanistic investigation reveals that canonical WNT/β-catenin pathway, a major signaling cascade involved in skeletal homeostasis, is impaired by progerin, causing a reduction in nuclear active β-catenin protein levels and reciprocal aberrant cytoplasmic accumulation which causes reduced transcriptional activity for osteogenesis. Non-farnesylation of progerin in MSCs attains higher level of active β-catenin protein expression and consequently increasing the signaling, enhancing mineralization capacity and ameliorating the defective osteogenesis. Moreover, in vivo analysis of the Zmpste24-/- HGPS mouse model demonstrates that treatment with a sclerostin-neutralizing antibody (SclAb), which targets an antagonist of canonical WNT/β-catenin signaling pathway, fully rescues the low bone mass phenotype to wild-type levels. This study implicates β-catenin signaling cascade as a therapeutic target for restoring defective skeletal microarchitecture in HGPS. Given the fundamental nature of WNT/β-catenin signaling to stem cell renewal and lineage allocation, the findings from this dissertation may provide broader inferences for the treatment options in HGPS.Item THE ROLE OF AURORA KINASE IN THE DEVELOPING MALE GAMETOPHYTE OF MARSILEA VESTITA(2018) Barnes, Alisha Nicole; Cooke, Todd; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The development of the male gametophyte of Marsilea vestita occurs via an ordered sequence of cellular events, which result in the formation of 32 motile sperm. The blepharoplast is a subcellular structure that arises de novo at ~3.5 hours following microspore hydration and functions as a microtubule organizing center during the final mitotic division. I investigated the roles of aurora kinases in the formation and maturation of the blepharoplast. Three unique aurora kinase isoforms were found in the transcriptome of the male gametophyte, each with a unique N-terminal sequence and expression pattern. RNAi knockdowns of each isoform resulted in different stages of developmental arrest, and the absence of blepharoplasts, thereby suggesting that each isoform has a unique function. Centrin phosphomimics acted to stabilize centrin, enabling centrin aggregates to form. My results suggest that each isoform of aurora kinase plays an important role in male gametophyte development in Marsilea.Item THE PHYTOHORMONE ETHYLENE: (I) INVESTIGATING THE MOLECULAR FUNCTION OF RTE1 AND (II) INSIGHTS ON THE EVOLUTION OF THE ETHYLENE BIOSYNTHESIS AND SIGNALING PATHWAYS(2017) Clay, John; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is an important phytohormone that regulates growth, development and stress responses in land plants and charophycean green algae. In Arabidopsis thaliana, ethylene is perceived by a family of five receptors. One of these five receptors, ETR1, is dependent on REVERSION-TO-ETHYLENE1 (RTE1) and Cytochrome B5 (Cb5) while the other four receptors are not. We found that RTE1 and Cb5 interact in planta and used genetic analyses to place Cb5 upstream of RTE1 in the ethylene signaling pathway. After comparing different ethylene receptors we identified an N-terminally localized proline that is important in determining whether a receptor is RTE1-dependent. Our results suggest that Cb5 receives electrons from upstream redox molecules, passes these electrons to RTE1; RTE1 is then able to activate the ETR1 receptor possibly by acting a molecular chaperone that refolds the ETR1 receptor into an active conformation. The ethylene signal transduction pathway is functionally conserved in the charophycean green algae such as Spirogyra pratensis, suggesting that this signaling pathway was present in the common ancestor of charophytes and land plants over 450 million years ago. However, it is unclear whether the central regulator of ethylene response, EIN2, was conserved in charophytes. Furthermore, the details of ethylene biosynthesis in charophytes were unresolved. After examining the genomes and transcriptomes of many green algae we are able to report that EIN2 is conserved in most charophytes and even some of the more distantly related chlorophycean green algae. Moreover, the Spirogyra EIN2 is functionally conserved and able to activate ethylene responses in Arabidopsis. Ethylene is synthesized via a two-step reaction involving the conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase (ACS), followed by oxidation of ACC to ethylene gas by the enzyme ACC oxidase (ACO). We identified S. pratensis ACS homologs and demonstrated that S. pratensis can synthesize ACC. S. pratensis lacks ACO homologs but we find it is still capable of producing low levels of ethylene. From our results we conclude that the ethylene biosynthesis and signaling pathways were established in early charophytes allowing these algae to establish ethylene as an important signalling molecule.Item Meta-Transcriptomic Profiling of Human Cutaneous Leishmaniasis(2018) Christensen, Stephen Michael; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our understanding of the spectral nature of the neglected tropical disease leishmaniasis, and of host-parasite interactions in general, remains incomplete. In this work, we used high throughput RNA-sequencing (RNA-seq) to analyze human host and Leishmania gene expression in cutaneous leishmaniasis patients. Skin biopsies were taken from a total of 25 localized cutaneous leishmaniasis (LCL), 6 diffuse cutaneous leishmaniasis (DCL), and 10 healthy patients. LCL separated into groups that lacked detectable parasite transcripts in lesions (PTNeg) and a group in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection, including B lymphocyte presence, B and T cell activation, and immunoglobulin production. Analysis of DCL lesions revealed distinct differences in host responses relative to LCL, including atypical B lymphocyte accumulation, diminished cytotoxic T lymphocyte responses, and an altered macrophage activation state. Surprisingly, neither localized nor diffuse forms of the disease could be correlated with any indication of a Th2 immune response that had previously been implicated in mouse models of L. major susceptibility. The presence of low levels of parasite transcripts in the majority of LCL patients made it difficult to obtain a comprehensive analysis of the parasite transcriptome in LCL. However, high levels of parasite transcripts in DCL afforded a unique opportunity to examine parasite gene expression in this disease. Despite differences in age, gender, and illness duration, there was a remarkable uniformity in parasite gene expression in all 6 DCL patients. We identified transcripts that were highly expressed by all 6 DCL patients, and then curated a subset of conserved genes highly expressed in multiple Leishmania species. These subsets of genes emerge as targets for further research on host-pathogen interactions and a better understanding of Leishmania infection.. In summary, RNA-seq allowed us to fully examine host and parasite transcriptomes, characterize host responses in localized and diffuse cutaneous leishmaniasis lesions, and determine factors that define the variations in disease manifestation. New approaches to modify host immune responses in this disease and new parasite targets for drug development may emerge from this work.Item CHARACTERIZING THE INHIBITION OF INNATE IMMUNE SIGNALING BY MYCOBACTERIUM TUBERCULOSIS(2017) Ahlbrand, Sarah; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Numerous cytokines are induced as a consequence of infection by Mycobacterium tuberculosis (Mtb), an intracellular pathogen that is responsible for millions of deaths each year. These cytokines play varied roles in eliciting and regulating the innate and adaptive immune responses against Mtb and often determine disease outcome. The pro-inflammatory cytokine interleukin 1β (IL-1β) is required for Mtb maintenance and clearance, as mice lacking the ability to produce IL-1β succumb very quickly to infection compared to infected wildtype mice. In contrast, interferon β (IFNβ) is largely thought to be detrimental to the host during Mtb infection based on collective studies in patients and mouse models. Both IL-1β and IFNβ are induced during Mtb infection, but here we demonstrate that Mtb has also evolved mechanisms to reduce their production and/or signaling. Previously, we’ve shown that Mtb can inhibit IL-1β production induced by the Absent in Melanoma 2 (AIM2) inflammasome. These findings were expanded upon by investigating the mechanisms by which Mtb inhibits AIM2 activation. A gain-of-function screen was also utilized in the non-pathogenic mycobacterial species Msmeg to identify Mtb genomic regions contributing to this phenomenon. In addition, we demonstrate that Mtb inhibits IFNβ signaling by inhibiting IFNβ-induced JAK1 and Tyk2 phosphorylation, leading to changes in the host type I interferon transcriptional profile. These studies provide insight into two previously undescribed mechanisms Mtb utilizes to manipulate the host immune response.