Animal & Avian Sciences Theses and Dissertations
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Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.Item MRP5 AND MRP9 PLAY A CONCERTED ROLE IN MALE REPRODUCTION AND MITOCHONDRIAL FUNCTION(2021) Chambers, Ian George; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels are significantly lower in the DKO testes. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating normal male reproductive functions and mitochondrial sufficiency.Item Investigating Copper Acquisition And Delivery via Transporters and a Pharmacological Chaperone in Copper-Deficient Worms and Mice(2019) Yuan, Sai; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Copper (Cu) is a key micronutrient required for a variety of essential biochemical pathways. Systemic or tissue-specific Cu-deficiencies, caused by insufficient dietary Cu uptake or mutations in Cu transporting genes, result in impaired growth, neuropathy, ataxia, hypopigmentation, osteoporosis and anemia-like symptoms in mammals. How organisms regulate Cu homeostasis at the systemic levels in response to Cu deficiencies remain elusive. In this study, we use Caenorhabditis elegans (C. elegans), a genetically tractable, multi-tissue metazoan to explore Cu homeostasis and investigate these unknowns. The high-affinity Cu transporters encoded by CTR family genes are required for dietary Cu uptake and maintaining systemic Cu balance from yeast to mammals. However, little is known about Cu acquisition mechanisms in C. elegans. We identified ten CTR ortholog genes in C. elegans; of these, chca-1 was functionally characterized. Cu availability regulates transcription of chca-1 in both the intestine and hypodermis, and chca-1 is essential for normal growth, and reproduction in the worm. Additionally, altered Cu balance caused by the loss of CHCA-1 results in defects in Cu-responsive avoidance behavior. Identification of this CTR-like gene in C. elegans, which appears to be essential for normal Cu homeostasis in the worm, illustrates the importance of Cu delivery via CHCA-1 for normal metazoan development and behavioral phenotypes. In addition, we show that a Cu-binding pharmacological chaperone, elesclomol (ES), fully restores the developmental defects and Cu deficiencies in chca-1-depleted worms, as well as the lethality in worms lacking cua-1 expression (Cu exporter ATP7A ortholog), suggesting ES is able to efficiently deliver Cu from dietary sources to peripheral tissues through the intestine in C. elegans. Our study was further expanded to mammalian models such as cardiac-specific Ctr1-depleted (Ctr1hrt/hrt) mice. We found that ES administration fully restores the postnatal lethality, developmental defects and cardiac hypertrophy found in Ctr1hrt/hrt mice, as well as rescuing the secondary systemic Cu homeostasis responses, including aberrant ATP7A protein levels in the liver and intestine. Moreover, ES shows the potential ability to transport Cu across the blood-brain-barrier in in vitro studies. These results illustrate the capability of ES to rescue systemic Cu deficiency in worms and mice, independent of the presence of functional Cu transporters, and shed light on the therapeutic usage of ES in Cu-deficient human diseases.Item FUNCTIONAL CHARACTERIZATION OF HEME TRANSPORTERS IN ZEBRAFISH(2017) Zhang, Jianbing; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hrg1 and Mrp5 are identified as eukaryotic heme importer and exporter, respectively. Two Hrg1 paralogs have been annotated in zebrafish genome, Hrg1a (Slc48a1b) and Hrg1b (Slc48a1a) with 84% homology in protein sequences. Hrg1a and hrg1b are widely expressed in embryonic and adult zebrafish. Yeast growth assays reveal that zebrafish Hrg1a and Hrg1b are both capable of heme import. However, hrg1a and hrg1b double knockout (hrg1 DKO) zebrafish generated by CRISPR/Cas9 has no overt defects in differentiation and maturation of erythroid cells. Knockdown of hrg1a in hrg1b mutants or vice versa does not impair erythroid lineage in zebrafish embryos. These genetic results suggest that Hrg1 is not required for maturation and hemoglobinization of primitive erythroid cells. Hrg1a and hrg1b mRNA are upregulated in adult kidneys and spleens upon PHZ-induced hemolysis, together with hmox1, a downstream heme degrading enzyme, suggesting that Hrg1 is involved in adult heme-iron recycling during erythrophagcytosis in kidney and spleen of adult zebrafish. DAB-enhanced Perl’s iron staining reveals that iron is accumulated in macrophages in the kidney and spleen in adult wild-type zebrafish. However, macrophages with positive Perl’s staining are rarely found in the kidney of hrg1 DKO and instead large amount of iron is deposited in renal tubules, suggesting defects in heme-iron recycling by kidney macrophages in hrg1 DKO under PHZ-induced hemolysis. Whole transcriptome sequencing of mRNA extracted from spleens and kidneys reveals massive differentially expressed genes in hrg1 DKO involved in immune response, lipid transport, oxidation-reduction process and proteolysis. These indicate that hrg1 DKO are deficient in recycling heme-iron derived from damaged RBCs in the absence of functional Hrg1. Phylogenetic analysis reveals that Mrp5 and Mrp9 are closed homologs in the zebrafish genome. Yeast growth assays reveal that both zebrafish Mrp5 and Mrp9 are capable of heme export. Morpholino knockdown of mrp5 and mrp9 in zebrafish showed severe anemia in developing embryos indicating their involvements in erythropoietic development. Subsequent generation and characterization of mrp5 and mrp9 mutants by CRISPR/Cas9 will further define the function of Mrp5 and Mrp9 during zebrafish development.Item THE REGULATION OF THE INTESTINAL COPPER EXPORTER IS COORDINATED WITH SYSTEMIC COPPER HOMEOSTASIS(2017) Chun, Haarin; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Copper (Cu) plays key catalytic and regulatory roles in biochemical reactions essential for normal growth, development, and health. Defects in Cu metabolism cause Menkes and Wilson’s disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of Cu. While it is well established that the enterocyte is a key regulatory point for Cu absorption into the body, how the intestine responds to systemic Cu requirements is poorly understood. Here, we demonstrate that fine-tuned Cu homeostasis is required for normal growth and development in C. elegans. Moreover, we show that CUA-1, the ATP7A/B homolog in worms, localizes to lysosome-like organelles (gut granules) in the intestine under Cu-overload conditions for Cu detoxification, while Cu-deficiency results in a redistribution of CUA-1 to basolateral membranes for Cu efflux to peripheral tissues. Defects in gut granule biogenesis exhibit result in abnormal Cu sequestration and increased susceptibility to toxic Cu levels. Our studies establish that CUA-1 is a key intestinal Cu exporter, and that its trafficking is regulated in response to systemic Cu status in worms. In addition, while the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation in mammals. Here we show, using Cu-deficient mouse models, that steady-state levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Importantly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestine-specific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Altogether, our results implicate CUA-1/ATP7A Cu exporter in the intestine as a key modulator for organismal Cu homeostasis in metazoans.Item Investigation of spermatozoal metabolism and substrate utilization in domestic and non-domestic felids(2015) Weiner, Halli Sigal; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cryopreservation of spermatozoa is a critical aspect of assisted reproduction. However in most cases, this process causes diminished spermatozoal function and low cellular survival. The effects of cryopreservation are further compounded in spermatozoa from individuals with disorders known to affect fertility. Teratospermia, characterized by the ejaculation of >60% morphologically abnormal spermatozoa, is one such disorder. Sperm from individuals with this condition exhibit reduced motility, impaired mitochondrial function, and a reduced ability to bind, penetrate, and fertilize an oocyte compared to sperm from normospermic males. Interestingly, even spermatozoa from teratospermic males that appear normal exhibit reduced functional capacity and following cryopreservation all cells are further impaired. More than 90% of felid species are affected by this condition, and it is prevalent among humans. Previous research suggested that impaired sperm metabolism in cheetahs – a ubiquitously teratospermic species – contributes to their poor cryosurvival and function, but the specific mechanisms are unknown. In this research, we hypothesized that inherent differences exist between the substrates and metabolic pathways utilized by sperm from normospermic and teratospermic individuals and on a species-specific basis. Gas chromatography - mass spectrometry (GC-MS) was used to investigate felid sperm metabolism using semen collected from domestic cats (Felis catus), cheetahs (Acinonyx jubatus), and clouded leopards (Neofelis nebulosa). The main objectives of this research were to: 1) characterize the metabolome of spermatozoa and seminal fluid; 2) investigate the role of β-oxidation in sperm motility and metabolism; and 3) interrogate the significance of specific metabolic pathways using metabolic activity profiling and heavy isotope tracers. Using GC-MS enabled the identification of metabolites which were unique to each species and sperm status (normospermic vs. teratospermic). Additionally, treatment of sperm with a β-oxidation inhibitor caused impaired motility in cheetah - but not domestic cat or clouded leopard - spermatozoa. Finally, fluxomic analysis demonstrated that glucose, fructose, and pyruvate are metabolized by felid sperm, but pathway utilization is species-specific. This is the first study to utilize a metabolomic and fluxomic approach to studying felid sperm, and the results of these studies illustrate the complexity of sperm metabolism on a species-specific basis. Understanding metabolic pathway activity in these cells will aid in the development of improved assisted reproduction techniques that may better facilitate sperm function and survival.Item MOLECULAR MECHANISMS UNDERLYING CADHERIN-6B INTERNALIZATION IN PREMIGRATORY CRANIAL NEURAL CREST CELLS DURING THEIR EPITHELIAL-TO-MESENCHYMAL TRANSITION(2015) Padmanabhan, Rangarajan; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The generation of migratory cells from immotile precursors occurs frequently throughout development and is crucial to the formation and maintenance of a functioning organism. This phenomenon, called an epithelial-to-mesenchymal transition (EMT), involves the disassembly of intercellular adhesions and cytoskeletal rearrangements in order to promote migration. Importantly, aberrant EMTs and cell migration can lead to devastating human conditions including cancer metastasis and fibrosis. How cells accomplish EMT to become migratory is still an unanswered question in the biomedical field. To this end, we use chick neural crest cells as an in vivo model to elucidate the molecules and pathways that regulate EMT and migration. Neural crest cells are a population of embryonic cells that are originally stationary within the dorsal neural tube but later migrate to form a variety of adult derivatives, such as the craniofacial skeleton, skin pigment cells and portions of the heart. To facilitate EMT, chick premigratory neural crest cells lose intercellular contacts mediated, in part, by the transmembrane cell adhesion protein Cadherin-6B (Cad6B). While Cad6B mRNA is transcriptionally repressed in premigratory neural crest cells, loss of Cad6B protein does not directly follow and instead occurs ~90 minutes later, just prior to migration. This rapid depletion of Cad6B is all the more striking given that the half-life of most cadherins, including Cad6B, is ~6-8 hours in vitro. As such, unique post- translational mechanisms must exist to remove Cad6B from premigratory neural crest cell plasma membranes to facilitate neural crest EMT. Since cadherins are known to be downregulated through internalization mechanisms (e.g., endocytosis, macropinocytosis) in other in vitro systems, the hypothesis of this dissertation is that Cad6B is internalized, and that this process plays a critical function to enable neural crest EMT. To this end, we document the existence of Cad6B cytoplasmic puncta in cultured cells, cultured neural crest cells and transverse sections of chick embryos. We subsequently identified a p120-catenin binding motif in the Cad6B cytoplasmic tail and demonstrated its functionality through site-directed mutagenesis, revealing a role in enhancing Cad6B internalization and reducing the stability of membrane-bound Cad6B. Furthermore, we uncover for the first time that Cad6B is removed from premigratory cranial neural crest cells through cell surface internalization events that include clathrin-mediated endocytosis and macropinocytosis. Both of these processes are dependent upon the function of dynamin, and inhibition of Cad6B internalization abrogates neural crest cell EMT and migration. Collectively, our findings provide a molecular blueprint for how cadherins are dynamically regulated during the formation of migratory cell types required for normal embryonic development and tissue repair as well as those generated during human diseases and cancers. Importantly, our research is multi-disciplinary, integrating cell biology and physiology to reveal how a cellular event, the active downregulation of a membrane protein, results in a physiological event, neural crest EMT and migration.Item LONG-RANGE SIGNALING AT THE INTESTINAL-NEURAL AXIS PROMOTES ORGANISMAL HEME HOMEOSTASIS IN C. ELEGANS(2014) Sinclair, Jason Wallace; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metazoans synthesize and regulate intracellular heme in a cell autonomous manner although genetic evidence in vertebrates suggests that cell non-autonomous mechanisms may exist at the organismal level. In C. elegans, a heme auxotroph, extraintestinal tissues are intrinsically dependent on the intestine, which acquires dietary heme for sustenance, supporting the concept that intestinal heme status must be coordinated at the systemic level to regulate whole-organism heme homeostasis. Here we show, by conducting a functional genome-wide RNAi screen in an intestinal-restricted heme sensor worm, that an interorgan heme signaling pathway exists and that >30% of the genes identified from the RNAi screen altered heme homeostasis in the intestine even though these genes are not expressed in the intestine. The biological basis for this signaling is underscored by HRG-7, a cathepsin protease-like protein secreted by the intestine and internalized by distally-located neurons. HRG-7 is specifically secreted from the intestine during heme limitation and hrg-7 depletion causes embryonic lethality concomitant with a heme deficiency response. Reciprocally, neuron-to-intestine heme signaling is mediated by the bone morphogenic protein homolog DBL-1, which recapitulates hrg-7 deficiency when depleted. Remarkably, depletion of both genes simultaneously results in markedly enhanced growth and heme deficiency phenotypes, suggesting that bidirectional signaling between the intestine and neurons mediates systemic heme homeostasis. Our results have uncovered an unexpected role for a protease family member in long-range communication between organs at the intestinal-neural axis to regulate systemic heme homeostasis in metazoa. As humans have over thirty cathepsin and cathepsin-like proteases, several of which are secreted, we anticipate that these proteins may play analogous roles in mammalian biology.Item CELLULAR PATHWAYS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITIONS IN NEURAL CREST CELLS(2013) Li, Shen; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neural crest cells are a population of multi-potent progenitor cells in the developing vertebrate embryo that undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively to generate diverse derivatives. As such, abnormal development of neural crest cells can lead to human congenital and hereditary malformations, diseases and cancers. Both internal molecular signals and external mechanical factors play essential roles in facilitating neural crest cell EMT. How cells modulate their adhesion machinery and dynamically reorganize their actin cytoskeleton to respond to the mechanical features of their external environment during EMT is not well understood. To evaluate the role of the actomyosin cytoskeleton during neural crest cell EMT and migration, midbrain neural folds that contain premigratory neural crest cells were dissected out from chick embryos, explanted into chamber slides, and incubated to allow for the formation of migratory neural crest cells. Time-lapse imaging technique was used to record cell behaviors. To elucidate cellular pathways controlling EMT and migration, chemical inhibitors (blebbistatin, Y-27632, latrunculin-A, and nocodazole) that perturb molecular cascades regulating cellular structures were employed. Effects of these perturbations on neural crest cell EMT and migration were quantified in terms of the spreading rate of the explants, and vorticity of collectively moving cell groups. We observed that blebbistatin treatment reduced the overall velocity of migratory neural crest cells to negligible levels. Moreover, migratory neural cells developed rounder cell bodies, and lamellipodia were transformed into filopodia at the periphery of the extract. Y-27632 treatment led to more neural crest cells coming out from these explants within a shorter time period compared to control. Nocodazole treatment blocked neural crest cell EMT and the resumption was dose-dependent. Latrunculin-A caused cell death at a very low concentration. These results implicate roles for non-muscle myosin II, the target of blebbistatin, and ROCK, the target of Y-27632, as well as microtubules and actin filaments, in chick midbrain neural crest cell EMT and migration. Actin crosslinkers such as α-actinin and actin-associated proteins like palladin also participate in pathways affected by these cytoskeletal inhibitors through their regulation of focal adhesion formation and cytoskeletal organization, thereby modulating cell stiffness and migration. We are also documented the distribution of α-actinin and palladin in migratory neural crest cells in vivo. Collectively, our studies have provided insight into specific cellular pathways regulating neural crest cell EMT and migration and the impact on various biophysical parameters upon perturbation of these pathways.