Thesis (Ph.D.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

<p>The partitioning of translation to the outer membrane of the endoplasmic reticulum is a problem that has been the subject of inquiry since the discovery of the ribosome. The large degree to which ribosomes were found to be tethered to the membrane led to intense investigation of a series of related questions regarding the identity of those mRNAs that are translated on the endoplasmic reticulum, and the functions of that localization in cell stress. In this dissertation, I approach each of these questions in turn and work to reconcile my observations with those models that have been previously proposed. A theme of this work is the application of modern methods, particularly deep sequencing technology, to address problems that had largely been considered solved. The most prominently featured method is ribosome profiling, which is paired with classical biochemical and cell biological techniques. I arrive at several conclusions: 1) a significant fraction of all mRNAs is well represented on the endoplasmic reticulum membrane, 2) the properties of translation diverge substantially between membrane-associated and free ribosomes, and 3) the compartmentalization of translation can serve as an important variable in cell stress.</p> / Dissertation

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX), infamously known as the “red devil,” is considered the most effective antineoplastic drug utilized in oncologic practice today. However, its clinical use is hampered due to cumulative, dose-dependent cardiotoxicity, which can lead to reduced quality of life, irreversible heart failure and death. The mechanisms involved in the pathogenesis of cardiotoxicity have not been fully elucidated, but have previously been demonstrated to involve oxidative stress, calcium dysregulation and mitochondrial dysfunction. Since the mitochondria play a critical role in generation of reactive oxygen species, the maintenance of calcium homeostasis and are the most extensively damaged by DOX, they have become the main focus of novel therapeutic interventions. The morphology and function of these dynamic organelles are regulated in part by mitochondrial fission and fusion events, as well as mitochondrial quality control systems. Since mitochondrial morphology is often associated with crucial cellular functions, this study aimed to investigate the long-term effects of DOX on mitochondrial dynamics and the mitochondrial quality control systems, mitophagy and the ubiquitin-proteasome pathway (UPP). Additionally, since the mitochondria and the endoplasmic reticulum (ER) are two interconnected organelles, and both play a role in maintaining calcium homeostasis, this study further assessed the effects of chronic DOX treatment on ER function and calcium status. Materials and Methods: In order to fully establish the effect of chronic DOX treatment in vitro, two cardiac cell lines were utilized in this study. H9C2 cardiomyoblasts and humanderived Girardi heart cells were cultured under standard culture conditions until ± 70-80% confluency was reached, where after treatment commenced. Cells were treated daily with 0.2 and 1.0 μM of DOX for 96 and 120 hours in order to simulate chronic, cumulative cardiotoxicity. Cell viability and apoptotic cell death were assessed with the MTT assay and Caspase Glo 3/7 assays, respectively. The expression of proteins involved in mitochondrial dynamics, mitochondrial biogenesis, the ubiquitin-proteasome pathway, mitophagy and ER stress were determined with Western blotting. Organelle morphology was visualized with fluorescence microscopy, and flow cytometry was used to assess mitochondrial and ER load. In order to determine the oxidative capacity, stress and status within the cells following treatment, the Oxygen radical absorbance capacity (ORAC), Thiobarbituric acid reactive substances (TBARS) and Glutathione (GSH) assays were employed respectively. Finally, intracellular and mitochondrial calcium was assessed and quantified with superresolution structured illumination microscopy (SR-SIM) and flow cytometry respectively. Results: DOX significantly reduced cell viability and increased apoptosis in both in vitro cardiac cell models. This study further demonstrated that the expression of mitochondrial fusion proteins, Mfn 1 and Mfn 2 were significantly downregulated, whilst the regulators of fission, Drp1 and hFis1, were significantly elevated, therefore shifting the balance of mitochondrial dynamics towards fission. Unopposed and elevated mitochondrial fission was clearly evident from the morphology of these organelles, which displayed short, highly fragmented mitochondria with a dispersed network following treatment. Chronic DOX also downregulated the regulator of mitochondrial biogenesis, PGC-1α, thus inhibiting the formation of new, functional mitochondria. The E3 ligases, MARCH5 and Parkin were highly upregulated following treatment, indicating activation of the UPP and mitophagy. Although chronic DOX stimulated K48 ubiquitination following treatment, it inhibited the catalytic activity of the 26S proteasome, therefore blocking proteasomal degradation. Although the antioxidant capacity (measured as ORAC) was significantly enhanced by both concentrations of DOX, an increase in oxidative stress status was shown following DOX treatment. In this regard lipid peroxidation significantly increased, while redox status of the endogenous antioxidant, glutathione, significantly decreased. Additionally chronic DOX treatment induced ER stress, which lead to an increase in cytosolic and mitochondrial calcium. In response to ER stress, the unfolded protein response (UPR) was then stimulated. Discussion: Results from this study indicate that chronic DOX treatment disrupts the balance of mitochondrial dynamics, favouring mitochondrial fission. Mitochondrial fragmentation is mediated by the downregulation of fusion proteins regulated by the E3 ubiquitin ligase, MARCH5 as well as by the increase in mitochondrial calcium. Mitochondrial fission results in mitophagy, an adaptive response to protect the cardiac cell against damaged mitochondria. This study also indicates that during chronic DOX-induced cardiotoxicity ER stress and the UPR are induced, which is possibly responsible for the disruption in calcium homeostasis. The inhibition of mitochondrial biogenesis coupled with elevated mitophagy as observed in this chronic study, elucidates a plausible mechanism whereby DOX induces mitochondrial dysfunction. Unregulated mitochondrial fragmentation and inhibited mitochondrial biogenesis are known to regulate various cardiomyopathies, therefore since both these effects are induced by chronic DOX treatment suggests a mechanism whereby cardiotoxicity, and ultimately heart failure are produced. This study provides new insight into the role of chronic DOX plays in altering mitochondrial dynamics and mitochondrial quality control systems. Further investigations targeted at limiting mitochondrial fission may reduce the cardiovascular side effects associated with DOX. / AFRIKAANSE OPSOMMING: Inleiding: Doksorubisien (DOX), ook bekend as die “rooiduiwel,” word beskou as die mees effektiewe anti-neoplastiese middel wat tans in onkologie praktyke gebruik word. Die kliniese gebruik hiervan word gerem deur die kumulatiewe dosis-afhanklike kardiotoksisiteit wat tot verlaagde lewenskwaliteit, onomkeerbare hartversaking, en tot die dood kan lei. Die meganismes wat by die kardiotoksiese patogenese betrokke is, is nog onbekend, maar die meganisme het moontlik te doen met oksidatiewe stres, kalsiumwanregulering en mitochondriale wanfunksionering. Omrede die mitochondria ‘n kritieke rol in die vorming van reaktiewe suurstofspesies speel, asook die handhawing van kalsiumhomeostase en die mees beskadigde organelle deur DOX, het die hooffokus na nuwe terapeutiese intervensies verskuif. Die morfologie en funskie van hierdie dinamiese organelle word gereguleer deels deur mitochondriale fragmentering en fussie, asook mitochondriale kwaliteitsbeheersisteme. Omrede mitochondriale morfologie geassosieer is met noodsaaklike sellulêre funksies, het hierdie studie gepoog om die langtermyneffkte van DOX op mitochondriale dinamika en die mitochondriale kwaliteitsbeheersisteme, mitofagie en die ubikwitien-proteosoomweg (UPW) te ondersoek. Siende dat die mitochondria en die endoplasmiese retikulum (ER) twee interverweefde organelle is, en beide ‘n rol speel in die handhawing van kalsiumhomeostase, het hierdie studie verder die effekte van chroniese DOX behandeling op ER funksie en kalsiumstatus ondersoek. Materiaal en Metodes: Om die effek van chroniese DOX behandeling in vitro te verstaan in hierdie studie, is twee hartsellyne gebruik. H9C2 kardiomioblaste en menslike Girardi hartselle is onder standaardtoestande tot ± 70-80% konfluensie bereik is gekweek, waarna behandeling begin is. Selle is daagliks met 0.2 en 1.0 μM DOX vir 96 en 120 uur behandel om chroniese en kumulatiewe kardiotoksisiteit n ate boots. Selvatbaarheid en apoptotiese seldood is onderskeidelik ondersoek deur middel van die MTT en Caspase Glo 3/7 toetse. Die proteïenuitdrukking betrokke by mitochondriale dinamika, mitochondriale biogenese, die ubikwitien-proteosoom weg, mitofagie en ER stres is deur middel van westerse afblatting bepaal. Organelmorfologie is deur middel van fluoresensie mikroskopie gevisualiseer, en vloeisitometrie was gebruik om die aantal mitochondria en ER lading te bepaal. Om die oksidatiewe kapasiteit, stres en status binne die selle na behandeling te bepaal, is die ORAC, TBARS en GSH toetse onderskeidelik gebruik. Laastens was die intrasellulêre en mitochondriale kalsium ondersoek en gekwantifiseer met superresolussie gestruktureerde illuminasie mikroskopie (SR-SIM) en vloeisitomerie. Resultate: DOX het selvatbaarheid betekenisvol verlaag en apoptose in beide in vitro kardiale selmodelle verhoog. Hierdie studie het verder aangetoon dat die uitdrukking van mitochondriale fussie proteïene, Mfn 1 en Mfn 2 betekenisvol afgereguleer is, terwyl die reguleerders van fragmentering, Drp1 en hFis1, betekenisvol verhoog is en daardeur die balans van mitochondriale dinamika na fussie verskuif. Onverhinderde en verhoogde mitochondriale fragmentering is duidelik sigbaar deur die morfologie van die organelle, wat as kort, hoogsgefragmenteerde mitochondria met ‘n verspreide netwerk na behandeling vertoon. Chroniese DOX het ook die mitochondriale biogenese reguleerder, PGC-1α, afgereguleer en daardeur die vorming van nuwe, funksionele mitochondria geinhibeer. Die E3 ligase, MARCH5 en Parkin is hoogs opgereguleer na behandeling, wat aktivering van UPW en mitofagie aantoon. Alhoewel chroniese DOX K48 ubikwitinering na behandeling gestimuleer het, het dit die katalitiese aktiwiteit van die 26S proteasoom geinhibeer en dus die proteosomale degradasie geblokkeer. Antioksidantkapasiteit en oksidatiewe status was betekenisvol na behandeling wat gevolglik tot hoë vlakke oksidatiewe skade binne die selle gelei het. Addisioneel het chroniese DOX behandeling ER stres geïnduseer wat tot ‘n toename in sitosoliese en mitochondriale kalsium gelei het. In reaksie op die ER stres is die UPW gestimuleer. Bespreking: Resultate van hierdie studie het aangetoon dat chroniese DOX behandeling die balans van mitochondriale dinamika onderbreek en sodoende mitochondriale fragmentering bevoordeel. Mitochondriale fragmentering word gemediëer deur die afregulering van fussie proteïene wat deur die E3 ubikwitienligase, MARCH5, gereguleer word, en ook deur die toename in mitochondriale kalsium. Mitochondriale fragmentering induseer mitofagie, ‘n aanpassingsreaksie om die hartselle teen beskadigde mitochondria te beskerm. Hierdie studie toon verder ook dat gedurende chroniese DOX-geïnduseerde ER stres, word die UPW ook geïnduseer, wat moontlik dan verantwoordelik is vir die ontwrigting van kalsiumhomeostase. Die inhibering van mitochondriale biogenese gekoppel met verhoogde mitofagie soos waargeneem in hierdie studie, verklaar ‘n moontlike meganisme waardeur DOX mitochondriale wanfunksionering veroorsaak. Ongereguleerde mitochondriale fragmentering en geinhibeerde mitochondriale biogenese is bekend om verskeie kardiomiopatieë te reguleer. Omrede beide hierdie effekte geinduseer word deur chroniese DOX behandeling kan dit moontlik ‘n meganisme voorstel waarby kardiotokiese en uiteindelik hartversaking ontwikkel. Hierdie studie bied nuwe insig in die rol wat chroniese DOX speel in die wysiging van mitochondriale- dinamika en kwaliteitskontrole sisteme. Verdere ondersoeke wat die mitochondriale fragmentering kan verminder mag moontlik die kardiovaskulêre newe-effekte wat met DOX behandeling geassosieer is, verlaag. / National Research Foundation (NRF)

In mammalian cells newly synthesised proteins are translocated across the ER membrane and their subsequent folding is facilitated by an array of folding factors present in the lumen. These include the lectins calreticulin and calnexin, which form complexes with ERp57 to generate glycoprotein specific molecular chaperones. ERp57 is a member of the protein disulphide isomerase (PDI) family and its binding to ER lectins can be reconstituted in vitro. I have exploited this approach to define the regions of ERp57 that are necessary and sufficient for its specific interaction with calreticulin and calnexin. Truncated forms of ERp57, chimeric proteins containing various domains of ERp57 and PDI (which does not interact with calreticulin) and ERp57 b' domain point mutants have been constructed. By analysing the interactions of ERp57 derivatives with calreticulin using both cross-linking and binding assays I have been able to provide detailed insights into the molecular basis for the specific assembly of these components within the ER lumen. My results indicate that the b and b' domains of ERp57 are necessary, but not sufficient for binding to both calreticulin and calnexin. The more stringent binding assay revealed that the a' domain of ERp57 significantly enhanced binding to biotin-tagged calreticulin. The ERp57 C-terminal extension also increased binding to biotin-tagged calreticulin, perhaps by playing a role in the overall stability of the ERp57. In addition, the ERp57 b' domain point mutants show that certain amino acids in this domain, in particular residues F280, V283 and F299, may be crucial for binding to calreticulin, consistent with the principal lectin-binding site being located in the b' domain. However, the binding region clearly extends into other domains, in particular the b and a' domains.

No description available.

Plant cells contain multiple mobile Golgi bodies. Golgi bodies receive cargo from specialized subdomains of the endoplasmic reticulum (ER), so-called ER export sites (ERES). How ERES operate in plant cells is largely uncharacterized. <p>In mammals and yeast, the commonly recognized ER-to-Golgi transport model asserts that protein transport between these two organelles is mediated by vesicles. Formation of these vesicles is interceded by COPII and COPI coat complexes. COPII coat proteins assemble at ERES. The minimal components of the COPII coat comprise the following proteins: the GTPase Sar1, and two large heterodimeric complexes, Sec23/24 and Sec13/31. COPII vesicles are responsible for forward (anterograde) protein traffic from the ER to the Golgi apparatus. Proteins are constantly recycled from the Golgi back to the ER through a conserved backward (retrograde) pathway mediated by COPI coat proteins. Fusion of the anterograde and retrograde carriers with target membranes is mediated by a subset of specialized proteins called soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Studies conducted in mammalian and yeast systems also concluded that ER-to-Golgi SNARE proteins and membrane cargo proteins are concentrated into COPII vesicles through a direct interaction and binding with the pre-budding complex Sec23/24-Sar1. <p>The COPII component distribution and their biological function in plant cells are largely uncharacterized. Therefore, through the study of the COPII protein Sec24, this work aimed (i) to investigate where and how protein transport between ER and Golgi occurs in plant cells, and (ii) to establish the importance of the anterograde and retrograde transport equilibrium in regulating the ER protein export. To do so, live cell imaging of a fluorescent protein fusion of Sec24 was used and the dynamics of this protein chimaera were followed in tobacco leaf epidermal cells. The imaging investigations were complemented by mutagenesis studies and biochemical analyses. <p>The obtained results indicate that in plant cells Sec24 is localized at specific regions of the ER that represent mobile units continuously joined to the Golgi apparatus. From this study the importance of the balance between the anterograde and retrograde transport in protein ER export has also emerged. I have shown in fact, that blockage of the retrograde pathway using Arf1 mutants and COPI chemical inhibitor determines the collapse of the anterograde protein trafficking from the ER to the Golgi. Moreover, this study has shown that Sec24 is capable of an interaction with the SNAREs Sed5 and Sec22. This is a forward step in our understanding of the role of Sec24 in the mechanism of cargo selection and recruitment.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

Plant cells contain multiple mobile Golgi bodies. Golgi bodies receive cargo from specialized subdomains of the endoplasmic reticulum (ER), so-called ER export sites (ERES). How ERES operate in plant cells is largely uncharacterized. <p>In mammals and yeast, the commonly recognized ER-to-Golgi transport model asserts that protein transport between these two organelles is mediated by vesicles. Formation of these vesicles is interceded by COPII and COPI coat complexes. COPII coat proteins assemble at ERES. The minimal components of the COPII coat comprise the following proteins: the GTPase Sar1, and two large heterodimeric complexes, Sec23/24 and Sec13/31. COPII vesicles are responsible for forward (anterograde) protein traffic from the ER to the Golgi apparatus. Proteins are constantly recycled from the Golgi back to the ER through a conserved backward (retrograde) pathway mediated by COPI coat proteins. Fusion of the anterograde and retrograde carriers with target membranes is mediated by a subset of specialized proteins called soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Studies conducted in mammalian and yeast systems also concluded that ER-to-Golgi SNARE proteins and membrane cargo proteins are concentrated into COPII vesicles through a direct interaction and binding with the pre-budding complex Sec23/24-Sar1. <p>The COPII component distribution and their biological function in plant cells are largely uncharacterized. Therefore, through the study of the COPII protein Sec24, this work aimed (i) to investigate where and how protein transport between ER and Golgi occurs in plant cells, and (ii) to establish the importance of the anterograde and retrograde transport equilibrium in regulating the ER protein export. To do so, live cell imaging of a fluorescent protein fusion of Sec24 was used and the dynamics of this protein chimaera were followed in tobacco leaf epidermal cells. The imaging investigations were complemented by mutagenesis studies and biochemical analyses. <p>The obtained results indicate that in plant cells Sec24 is localized at specific regions of the ER that represent mobile units continuously joined to the Golgi apparatus. From this study the importance of the balance between the anterograde and retrograde transport in protein ER export has also emerged. I have shown in fact, that blockage of the retrograde pathway using Arf1 mutants and COPI chemical inhibitor determines the collapse of the anterograde protein trafficking from the ER to the Golgi. Moreover, this study has shown that Sec24 is capable of an interaction with the SNAREs Sed5 and Sec22. This is a forward step in our understanding of the role of Sec24 in the mechanism of cargo selection and recruitment.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 100-109).