• DISSECTING THE ROLE OF YEAST RIM8 IN THEINTERNALIZATION OF THE PLASMA MEMBRANE PROTON PUMPPMA1

      Kane, Patricia; Shoniwa, Makandiwana (2013)
      The vacuolar A-TPase (V-ATPase) is a proton pump that is found ubiquitiously throughout the cells. It uses the hydrolysis of ATP to transport protons across membranes, thereby maintaining homeostatic pH. pH control in the cells of an organism is vital, a disturbance in cellular pH may be lethal. The maintenance of homeostatic pH within the cell appears to be a result of the interplay between V-ATPasesandproton exporters. In yeast and plants, the major proton exporter isthe plasma membrane proton exporter, Pma1. Pma1 is the transporter that is primarily involved in themaintenance of cytosolic pH. In cells in which the function of V-ATPase has been compromised (vma mutants) Pma1 is partially mislocalized.It is known thatmembrane transporterslacking the PY motifare endocytosed via the action of an Arrestin Related Trafficking (ART) protein, which translocates an E3 ligase into close proximity with the transporter, so as to allow for the ubiquitination of the transporter. Rim 8 is the ART protein (adaptor) that has been linked to the endocytosis of Pma1, along with E3 Ubiquitin ligase Rsp5. It is of interest to this project that Rim8 is well studied in its role as an adaptor in the alkaline ambient pH pathway. We thus propose that there may be crosstalk between the ambient pH pathway and the pathway that leads to the internalization of Pma1. Therefore, in this body of work we seek to find other players that may be involved in the Pma1 pathway, as well as to elucidate theareas of interaction between Rim8 and Pma1. Ourfinal goal isbringing a better understanding ofthe pathway that leads to the endocytosis of Pma1. To answer the question posed in this work we monitored the growth phenotype and the localization of Pma1 indouble mutants lacking both V-ATPase subunits and key players in the ambient pH pathway. In addition, we looked to see which cytosolically exposed terminal of Pma1 may be involved in theinteraction with Rim8. In yet another experiment, we mutatedRim8 so as to find which areas of the adaptor werevital for Pma1 internalization.Our results showed that other players tested in the Rim pathway(vma2∆rim20∆and vma2∆vps23∆)werenot required for Pma1 internalization. In addition we observed that mutations in Rim8 that compromise its function in the Rim pathway still allow Pma1 internalization, even though they show synthetic growth phenotypes with vma2∆ mutations. Two-hybrid assay could not detect thesites of interaction between Rim8 and Pma1 and newstrategies will be employed to determine these sites. Changes in electrophoretic mobility of Rim8 suggested that Rim8 undergoes posttranslational modifications, and showed differences in vma2∆mutants and WT mutants.
    • EVALUATION OF THE ROLE OF THE CENTRAL PAIR IN CHLAMYDOMONAS REINHARDTII FLAGELLAR MOTILITY

      Mitchell, David; Smith, Brandon (2013)
      Cilia and flagella are essential for the function of nearly all eukaryotes. This organelle is made up of nine outer doublet microtubules and two central singlet microtubules to form the canonical (9+2) ciliary structure. Cilia and flagella use this structure, as well as several protein complexes, such as the outer and inner dynein arms, the radial spokes, and the proteins that decorate the central pair to propagate the bending that produces motion. Flagellar motion is highly regulated, and each of these structures is necessary to regulate the dynein arms that generate the motile force. The central pair is one of the least understood of these structures. To date there are two major impediments hindering our understanding of the central pair: a lack of understanding as to how distinct central pair structures work in concert, and a general lack of available central pair mutant strains in the model organism Chlamydomonas reinhardtii. In order to further our understanding of how the central pair functions I have used multiple strategies. Firstly I have used previously characterized central pair mutants to study both structural interactions within the central pair and how the double mutant affects motility regulation. Secondly I provide evidence that a potential central pair mutant, H2, is indeed a central pair mutant and affects the C2b projection. Lastly I will attempt to characterize a new Chlamydomonas mutant, 10B5. Together these analyses will demonstrate that double mutants can have an additive effect on the structure of the central pair, and that double central pair mutants do not appear to suppress one another, but are at least ivepistatic to the most severe phenotype. I will also show evidence that 10B5 is not a central pair mutant, but with further study it may offer new insight into motilityregulation.
    • ROLE OF BMP AND ACTIVIN/NODALSIGNALING REPRESSION IN RETINALPROGENITOR CELL SPECIFICATION ANDCONE PHOTORECEPTOR GENESIS

      Viczian, Andrea; Wong, Kimberly (2017)
      Eye formation begins with the specification of the eye field from the anteriorneural plate during the transition from gastrulation to neuralization. The morphogeneticgradients formed by BMP and Activin/Nodal ligands organize the body axis andregulate intracellular signals to bias the cell lineages.Therefore, to understand theextracellular signaling mechanisms governing eye field specification, we looked closerat the mechanisms responsible for neuralization. This project takes advantage of thedevelopmental accessibility of theXenopus laevisembryo to investigate the role ofBMP and Activin/Nodal signaling pathways during early eye development.Nogginis a secreted BMP antagonist and is sufficient to induce formation of ectopic retinaltissue. My results presented in Chapter 2 show that intracellular repression of BMP-Smad1/5/8 and Activin/Nodal-Smad2/3 signaling was sufficient to replicate the retina-promoting activity of Noggin and efficiently drive pluripotentXenopusprimitive ectodermto a retinal progenitor cell fate. Furthermore, my work presented in Chapter 3 suggeststhat repression of BMP and Activin/Nodal in the anterior neural plate allows the anteriorneural patterning transcription factor, Otx2, to directly activate expression of the earliesteye field transcription factor,tbx3. This study elucidates the initial step by which theanterior neural plate is patterned to specify the eye field. We next translated our findings to mouse embryonic stem cell culture (Chapter 4). We observed that mouse embryonic stemcells can be efficiently directed towards retinal progenitor cells by first converting themto an early primitive ectoderm-like state, followed by simultaneous repression of BMPand Activin/Nodal and activation of FGF signaling. After differentiation, we observedthat photoreceptor genesis is synchronized, resulting in 96% of aggregates expressingmarkers for cone photoreceptors after 9 days. This differentiation protocol is more efficientand quicker than previously published mouse stem cell protocols. Optimizing treatmentconditions resulted in up to 62% of cells expressing the cone/rod photoreceptor marker,CRX. This study was the first to show that photoreceptor-like cells can be generated frommouse pluripotent stem cells as efficiently as published human pluripotent stem cell reports,but with a shorter culture period. These studies will provide the basis of future work todetermine the underlying molecular mechanism driving retinal differentiation.
    • THE ROLE OF THE UBIQUITIN E3/DUB DUAL ENZYME A20 IN NEURONS AND SYNAPSES

      Yao, Wei-Dong; Shaolin, Mei (2016)
      Posttranslational ubiquitination and deubiquination protein modifications play an essential role in neuronal development, function, and plasticity. This study investigated the role of the E3/deubiquitinase dual ubiquitin enzyme A20 (also called tumor necrosis factor alpha -induced protein 3, TNFAIP3) on neuronal arborization, dendritic spine morphogenesis, and synaptic transmission. The spatial and temporal expression profiles of A20 were investigated in primary neuronal cultures and rodent brains by western blotting and immunofluorescent staining. Several mammalian cDNA expression and shRNA plasmids with the Myc tag or the DsRed or GFP fluorescent reporters were constructed by subcloning, and their efficiency was validated in HEK293FT cell line, cultured neurons, and yeast. These plasmids were used to upregulate or downregulate the A20 level and investigate the effects on neuronal morphology and synaptic function. Overexpressing A20 diminished spine (mushroom, stubby, and thin subtypes) sizes and reduced dendritic spine densities. Sholl analysis showed that A20 upregulation also decreased neurite arborization numbers at medial-distal branches. Consistently, A20 downregulation significantly enlarged mushroom and stubby spine size and modestly increased spine density, which was rescued by an RNAi-resistant A20 cDNA construct. Moreover, electrophysiological recording of mEPSCs from rat hippocampal neurons showed a slight reduction of the mEPSC frequency but a significantly greater mEPSC amplitude when A20 was knocked down by transducing shA20 lentivirus. Finally, in an attempt to identify potential A20 interacting proteins, we conducted a yeast two-hybrid screening assay using a mouse brain cDNA library. Taken together, this study provided strong evidence that A20 regulates neuronal morphology including dendritic spine size, density, and neurite complexity as well as efficacy of synaptic transmission.
    • THE ROLE OFNORMAL AND ONCOGENICJANUS KINASE 2 IN HEMATOPOIETIC STEM CELLS

      Mohi, Golam; AKADA, HAJIME (2014)
      During my Ph.D. training, I first experimentally proved that the expression of oncogenic Jak2V617F wassufficient to induce MPNs and transformed only HSCs into CSCs for developing MPNs. Thus, it is criticalto understand the role of both normal and oncogenic Jak2 in HSCs to find the mechanism to cureJak2V617F-positive MPNs. Therefore, I have mainly studied two major questions in Jak2:1) The role of normal Jak2 in hematopoietic stem cells for adult hematopoiesis2) The role of oncogenic form of Jak2, Jak2V617F, in cancer stem cells for MPN developmentFirst question has not been addressed since 1998, because conventional Jak2 knock-out mice wereembryonic lethal. Thus, I hypothesized that Jak2 plays a pivotal role in adult hematopoietic stem cellmaintenance. I successfully prove that Jak2 is the one of key regulators of HSCs. Conditional Jak2 deletionin mice caused an irreversible HSCs impairment. My data strongly suggest that Jak2 plays a critical role inthe maintenance of quiescence, survival and self-renewal of adult HSCs.Second question has been studied after the discovery of a somatic point mutation, Jak2V617F, in a majorityof patients with MPNs in 2005. I hypothesized that this oncogenic mutation confers unique properties inCSCs maintenance for MPNs development. Surprisingly, I found that the site of leukemogenesis shiftedfrom BM to spleen, and spleen became the major source of CSCs for Jak2V617F-positive MPNs. The age-associated progressive expansion of CSCs was seen in spleen. Splenic-CSCs were capable to propagateMPN disease and possessed a greater proliferative advantage than BM-CSCs. The Jak2V617F-CSCsestablished a positive-feedback mechanism with CD169+ macrophage progenitors. Depletion of CD169+macrophage progenitors reduced the number of Jak2V617F-CSCs. Gene profiling revealed that splenic-CSCs have distinct gene expression compared to BM-CSCs. Together, I demonstrated that Jak2V617F-CSCs are maintained in spleen for long-term MPN progression.By utilizing gene analysis data from two projects, I discovered a set of unique genes/pathways regulated byonly Jak2V617F but not by wildtype Jak2. All together, my Ph.D. researches provided the potential genetarget a novel therapy for Jak2V617F-positive MPNs.
    • THE ROLE OF DENGUE VIRUS NON-STRUCTURAL PROTEIN 1 IN DISEASE PATHOGENESIS

      King, Christine; Endy, Timothy; Barbachano-Guerrero, Arturo (2020)
      Dengue virus (DENV) causes an estimated 390 million infections worldwide annually, with severe forms of disease marked by vascular leakage and an over reactive inflammatory response. Endothelial cells (EC) are directly responsible for vascular homeostasis and are highly responsive to circulating mediators but are not commonly infected. Mast cells (MC) are potent cells of the innate immune system that play an important role in EC biology and inflammatory responses. DENV encodes 10 proteins; with only one, the non-structural protein 1 (NS1), secreted from infected cells and accumulating in the blood of patients.NS1 has been implicated in the pathogenesis of vascular permeability, but the mechanism is not completely understood. Using a complementary array of in vitroassays and disease relevant ECs and MCs, we described the possible roles for NS1 in dengue disease pathogenesis. Using microscopy and immunoblotting we observed that ECs internalize NS1 into endosomes, where it accumulates and is degraded overtime. Transcriptome and pathway analysis defined changes in global gene expression in ECs that are associated with cell dysfunction. We observed that NS1 induced an increase in multicellular rearrangements and a decrease in barrier function in ECs. We demonstrated that NS1-dependent activation of the p38 MAPK pathway controls the changes in EC permeability in vitro. Further, we discovered iiithat ECs and MCs respond to NS1 by secreting a specific array of proinflammatory cytokines and chemokines that may contribute to the cytokine storm in dengue disease. Finally, we found that NS1 internalization can mediate the uptake of bound antibodies into ECs. Together, these results suggest a vasoactive and proinflammatory role for DENV NS1 that may participate in the development of severe symptoms in dengue disease. The observed functions of NS1 could lead to the discovery of new therapeutic targets in dengue disease.