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Wong, Mei Wai Mie. "Functions of the golgin coiled-coil proteins of the Golgi apparatus." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708308.
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Au, Catherine. "Organellar proteomics of the Golgi apparatus and Golgi derived COPI vesicles." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18742.
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Studying an organelle with traditional biochemistry, histology, or microscopy techniques allows the determination of the presence of up to three proteins simultaneously. Mass spectrometry based proteomics has changed the study of organelles; for the first time it is possible to investigate the whole protein complement of a subcellular compartment. In this work I demonstrate our ability to use redundant peptide counting as a quantitative technique to compare the relative abundance of different proteins in a complex sample, specifically, enriched organellar preparations. I present the pipeline that we use to isolate, characterize, and prepare samples for mass spectrometric analysis, followed by the automatic mass spectrometric data acquisition and data processing that result in the output of a list of protein identifications. A highly involved and time consuming manual annotation effort is applied to this preliminary list in order to generate a final set of tables where standardized functional categories and assigned names are applied to every protein identified in order facilitate the application of redundant peptide counting. The organelles of the early secretory pathway are processed by the pipeline, and after rigorous manual verification of the data, the proteomes of the rough microsomes, smooth microsomes, Golgi apparatus, and Golgi derived COPI GTP and COPI GTP?S vesicles are determined. The focus of this thesis is on the proteomes of the Golgi and Golgi derived vesicles. The characteristics and most abundant proteins of the proteomes of the Golgi apparatus, COPI GTP, and COPI GTP?S vesicles are described in detail. The hypothesis of cisternal maturation, a theory describing secretory cargo progress through the Golgi apparatus, is tested and eventually supported by our proteomics data. Finally, outlines of the abundant proteins of unknown function of the Golgi, COPI GTP and COPI GTP?S vesicles are presented.Les techniques traditionnelles utilisées en biochimie, en histologie ainsi qu'en microscopie permettent la détermination d'un maximum de trois protéines à la fois dans l'étude d'une organelle. La mise en œuvre de la spectrométrie de masse en protéomique a complètement changé le panorama d'investigation des organelles. Pour la première fois, il est possible d'étudier le panel entier de protéines présent dans un compartiment sub-cellulaire. Dans cette étude, je démontre dans un premier temps que l'utilisation du dénombrement de peptides redondants permet la quantification des protéines et donc la capacité de comparer l'abondance relative de différentes protéines dans un échantillon complexe tel qu'une préparation d'organelle. Je présenterai par la suite le pipeline que nous utilisons pour isoler, caractériser et préparer les échantillons avant leur analyse par acquisition automatique par spectrométrie de masse laquelle est suivie par le traitement des données dont le résultat consiste à l'identification d'une liste de protéines. Un effort manuel important d'annotation est appliqué à cette liste préliminaire afin de générer un tableau final où sont assignées à la fois la fonction dans laquelle chaque protéine identifiée est impliquée, ainsi que l'attribution de la nomenclature la plus appropriée. Ce travail laborieux facilite par conséquent le dénombrement et l'attribution des peptides redondants aux protéines. Les organelles de la voie précoce de sécrétion sont analysées par le pipeline et après une vérification manuelle rigoureuse des données, les protéomes des microsomes rugueux, des microsomes lisses, de l'appareil de Golgi, et des vésicules dérivées du Golgi COPI GTP et COPI GTP?S sont déterminés. L'objectif de cette étude porte sur les protéomes du Golgi et des vésicules dérivées du Golgi dont les protéines les plus abondantes ainsi que leurs caractéristiques sont décrites en détail. L'hypoth
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Dworkin, Joel. "Cell-free reconstitution of the Golgi apparatus." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59884.
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The Golgi apparatus is organized into a characteristic differentiated stack in all eukaryotic cells. During mitosis, the Golgi apparatus disassembles into smaller clustered vesicles lacking recognizable cisternae whereupon they recombine to form typical stacks in each of the daughter cells (Lucocq et al., 1987). Paiment et al. (1989) have demonstrated that dispersed (unstacked) Golgi fragments will reconstitute into a functional stacked Golgi apparatus when microinjected into Xenopus laevis oocytes. Disrupted hepatic Golgi fractions (Gi) were isolated on discontinuous sucrose gradients and incubated at 37$ sp circ$C in the presence or absence of calf brain cytosol, ATP, and GTP$ gamma$S with or without pretreatment by N-ethylmaleimide. The reconstitution of saccule stacking was assayed using transmission electron microscopy on pelleted and filtered fractions. The role of cytosolically exposed Golgi membrane proteins in maintaining saccule was also addressed using controlled protease digests.
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Hui, Hu. "Targeting and retention in the Golgi apparatus." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263648.
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Gilchrist, Annalyn. "Proteomics analysis of the endoplasmic reticulum and Golgi apparatus." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18715.
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Isolated rough and smooth microsomes of the endoplasmic reticulum and isolated Golgi apparatus from rat liver were analyzed by proteomics using mass spectrometry, identifying 1064 proteins among the three fractions. An additional 598 proteins were identified by biochemically subfractionating the rough and smooth microsomes by treatment with a high salt wash and the detergent Triton-X 114. Proteins were quantified by redundant peptide counts which enabled an assessment of the extent of cross-contamination between the endoplasmic reticulum and Golgi fractions and other organellar contamination. Results of this analysis revealed that the Golgi fraction was contaminated up to 30% by proteins of the endoplasmic reticulum and that the mitochondria constitute the largest source of organellar contamination for all three fractions. Hierarchical clustering of the distribution profiles of proteins among the three fractions assigned proteins to either the rough and/or smooth endoplasmic reticulum or the Golgi apparatus. In doing so, the protein disulphide isomerase, ERp44, was localized to the Golgi. This result was verified by immunolocalization with an ERp44 antibody. Furthermore, hierarchical clustering assigned a location for 176 previously uncharacterized proteins in the endoplasmic reticulum providing a subcellular context to their putative functions predicted by bioinformatics. Additionally, the biochemical subfractionation of the rough and smooth microsomes assigned proteins to the cytosolic, membrane or luminal subcompartments of the endoplasmic reticulum. These results guided the selection of uncharacterized membrane proteins for further characterization leading to the identification of 7 proteins upregulated by ER stress, which included 4 new molecular chaperones. Finally, a comparison of this work with previous proteomics analyses of these organelles showed that the proteomes of the endoplasmic reticulum and Golgi apparatus presented here may be the moL'analyse par protéomique des microsomes du Réticulum Endoplasmique rugueux, des microsomes du Réticulum Endoplasmique lisse et de l'appareil de Golgi a permis l'identification de 1064 protéines par spectrométrie de masse. Par ailleurs, le fractionnement biochimique des microsomes lisses et rugueux par lavage avec une solution saline concentrée suivi d'un traitement au détergent Triton X-114 a permis l'identification de 598 nouvelles protéines. Les protéines furent quantifiées en fonction du nombre de peptides identifiés par spectrométrie de masse. La quantification des protéines a permis d'évaluer le degré de contamination croisée présent dans les fractions du Réticulum Endoplasmique, de l'appareil de Golgi et celui provenant des autres organelles. Les résultats de cette analyse ont révélé que la fraction de Golgi était contaminée jusqu'à un maximum de 20% par les protéines provenant du Réticulum Endoplasmique et que les mitochondries constituaient la source essentielle de contamination dans ces trois fractions. La clustérisation hiérarchique des protéines quantifiées a permis de dresser le profile de distribution des différentes protéines et ainsi de les assigner au sein des différents compartiments, à savoir aux microsomes du Réticulum Endoplasmique rugueux et/ou lisses, ou alors à l'appareil de Golgi. De ce fait, la protéine disulphide isomerase, ERp44, a été localisée dans l'appareil de Golgi. Ce résultat a été confirmé par immunolocalisation avec l'anticorps ERp44. Par ailleurs, cette même clustérisation hiérarchique a permis de localiser pour la première fois 176 protéines dans le Réticulum Endoplasmique correspondant ainsi à leur fonction putative prédite par bioinformatique. De plus, le fractionnement biochimique des microsomes lisses et rugueux a permis d'assigner les protéines dans les compartiments subcellulaires du Réticulum Endoplasmique : cytosol, membrane ou lumière. Ces résultats ont é
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Rocchetti, Alessandra. "Interactions between the plant Golgi apparatus and the cytoskeleton." Thesis, Oxford Brookes University, 2016. https://radar.brookes.ac.uk/radar/items/e035b419-1acc-4031-aadd-2cfc1f9ed3c8/1/.
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In animal cells, the relationship between the Golgi apparatus and cytoskeleton has been well characterised but not much is known in plants. The functions of the Golgi apparatus are conserved amongst eukaryotes. It is one of the main stations in the secretory pathway and is involved in protein processing and sorting to different destinations. In plants, it is also involved in trafficking and positioning of cell wall components. In tobacco epidermal cells, fluorescent labelling with Golgi marker proteins has shown that the Golgi apparatus is made of hundreds of individual units scattered in the cortical cytoplasm and moving on the actin cytoskeleton. The contribution of actin filaments to Golgi body motility in plant has been extensively described, but this actin-centric view has recently been challenged. Emerging evidence suggests that microtubules may contribute to short distance movement and 'fine tuning' of Golgi body displacement. Moreover, proteomic studies linking the actin- cytoskeleton to microtubules have demonstrated that these two components of the cytoskeleton are closely related and a role of the microtubules in Golgi movement cannot be excluded. In this thesis, automated tracking of Golgi bodies was used to understand and quantify the contribution of actin filaments and microtubules to the organelle dynamics. The tracking technique is also used to assess how the labelling of the cytoskeleton, with a novel fluorescent nanoprobe, affects the dynamics and stability of the actin filaments and the movement of Golgi bodies; FRAP analysis (fluorescent recovery after photo-bleaching) was also used to investigate the binding properties of the fluorescent nanoprobe to the actin filaments. The nanoprobe was compared with another cytoskeletal marker, Lifeact-GFP, to evaluate their suitability for studying the organelle's motility in relation to the actin-cytoskeleton. Micromanipulation of Golgi bodies with optical tweezers was used to test if there are physical links between the organelles and the cytoskeleton. The widely accepted model is that organelles move on actin filaments and movement is powered by myosins. The hypothesis that actin filaments slide one of top of the other, and drag the organelles along, was tested using the FRAP technique. Kinesin-13a is the only microtubule motor protein localized on Golgi bodies by immunochemical studies. Its localization was investigated in vivo to evaluate if it is involved in linking Golgi bodies to microtubules.
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Short, Benjamin. "Characterisation of rab-effector complexes at the Golgi apparatus." Thesis, University of Glasgow, 2003. http://theses.gla.ac.uk/31014/.
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The rab family of small, ras-like GTPases regulate membrane trafficking events in the secretory and endocytic pathways. They appear to be involved in all stages of vesicular transport, from vesicle budding and cargo selection to motility, docking and membrane fusion. Through a cycle of GTP binding and hydrolysis, rab-effector proteins are recruited to membrane sub-domains in a temporally and spatially specific manner. Several rab proteins localise to the Golgi apparatus, the organelle consisting of stacked, flattened, membrane-bound cisternae through which newly-synthesised proteins are transited and modified, and where proteins and lipids are sorted and packaged for transport to other subcellular destinations. The structure of the Golgi is maintained by a matrix of proteins, many of which have now been shown to be rab-effector proteins. This thesis focuses on Golgi-localised rab proteins and their effector proteins. The cis-Golgi-localised rab protein, rabl, is shown to interact with the Golgi matrix/golgins GM130 and p115 while rab2 binds GM130 as well as a novel tethering factor named golgin-45. siRNA-mediated depletion of these rabs and golgins revealed them to be important for the maintenance of Golgi structure and suggested that p115 is primarily recruited to Golgi membranes by its interaction with rab1 rather than its association with GM130. A search for additional rab1 effectors revealed potential interactions between rab1 and the phosphoinositide- binding/metabolising proteins centaurin?2 and MTMR6. A search for novel effectors of the trans-Golgi-localised rab protein, rab6, was also made, revealing specific interactions with the dynactin subunit p150glued and the dynactin/dynein accessory proteins BicD1 and BicD2. These interactions are proposed to mediate the recruitment of dynactin/dynein to membranous cargo and control minus-end directed, microtubule-dependent vesicle motility. An additional rab6 effector, GOPC, was also identified, which may be responsible for cargo recognition and sorting. Finally, the cis-Golgi rab-effector and matrix protein, GM130, is shown to have a hitherto unsuspected role in the activation of a family of Golgi-localised Ste20 kinases. The implications of all these interactions for Golgi structure and function is discussed.
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Rivinoja, A. (Antti). "Golgi pH and glycosylation." Doctoral thesis, University of Oulu, 2009. http://urn.fi/urn:isbn:9789514292699.
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Abstract
Glycans, as a part of glycoproteins, glycolipids and other glycoconjugates, are involved in many vital intra- and inter-cellular tasks, such as protein folding and sorting, protein quality
control, vesicular trafficking, cell signalling, immunological defence, cell motility and adhesion. Therefore, their correct construction is crucial for the normal functioning of eukaryotic cells and
organisms they form. Most cellular glycans are constructed in the Golgi, and abnormalities in their structure may derive, for instance, from alkalinization of the Golgi lumen.
In this work we show that Golgi pH is generally higher and more variable in abnormally glycosylating, i.e. strongly T-antigen (Gal-β1,3-GalNAc-ser/thr) expressing cancer cells, than in
non-T-antigen expressing cells. We also confirmed that the Golgi pH alterations detected in cancer cells have the potential to induce glycosylation changes. A mere 0.2 pH unit increase in Golgi pH is
able to induce T-antigen expression and inhibit terminal N-glycosylation in normally glycosylating cells. The mechanism of inhibition involves mislocalization of the corresponding
glycosyltransferases.
We also studied potential factors that can promote Golgi pH misregulation in health and disease, and found that cultured cancer cells, despite variation and elevation in Golgi pH, are fully
capable of acidifying the Golgi lumen under the normal Golgi pH. Moreover, we introduce a Golgi localized Cl-/HCO3- exchanger, AE2a, that participates in Golgi pH regulation by
altering luminal bicarbonate concentration and thus also buffering capacity. Participation of AE2a in Golgi pH regulation is especially intriguing, because it also provides a novel mechanism for
expelling protons from the Golgi lumen.
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Albariri, Areej [Verfasser], and Thomas [Akademischer Betreuer] Kuner. "Golgi apparatus and Golgi outposts in neurons studied by correlative microscopy / Areej Albariri ; Betreuer: Thomas Kuner." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1220608793/34.
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Lock, John George. "Dynamic imaging of post-Golgi protein transport /." [St. Lucia, Qld.], 2005. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe19397.pdf.
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Kam, Chuen. "Functional study of PICK1-ICA69 complex in the golgi apparatus /." View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?NSNT%202008%20KAM.
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Lian, Yen-Ling. "Mechanisms of retrograde transport from Golgi apparatus to endoplasmic reticulum." Electronic Thesis or Diss., Université Paris sciences et lettres, 2022. http://www.theses.fr/2022UPSLS067.
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Les cellules de mammifères sont caractérisées par la coexistence de plusieurs voies de transport, dont le transport antérograde et rétrograde. L'appareil de Golgi joue un rôle central dans le traitement et le tri des protéines dans le trafic bidirectionnel. Le système Retention Using Selective Hooks (RUSH) permet de synchroniser le transport des protéines depuis le RE et d'analyser systématiquement les voies sécrétoires. Grâce à l'interaction entre la streptavidine (Str) et un peptide de liaison à la streptavidine (Streptatividin Bioding Peptide, SBP), la protéine rapporteur peut être retenue dans le RE et ensuite libérée par l'ajout de biotine. Cependant, la biotine a une grande affinité pour la streptavidine, ce qui nuit à la réversibilité du test RUSH. Grâce aux ligands artificiels de la streptavidine (ALiS), le transport rétrograde du Golgi vers le RE peut être analysé à l'aide de l’outil RUSH après lab-vage de ALiS.Le test RUSH réversible a d'abord été mis en place pour étudier deux mécanismes de transport rétrograde depuis Golgi vers le RE, notamment la voie médiée par le motif KDEL et la voie de recyclage des enzymes de glycosylation. Str-KDEL ou Ii-Str ont été utilisées comme protéines d’ancrage et les enzymes golgiennes ManII* (Mannosidase II)-SBP-EGFP ou ST* (Sialyltransférase)-SBP-GFP ont été utilisées comme rapporteurs RUSH. Notre analyse cinétique a montré que le transport de l’appareil de Golgi vers le RE de ManII* et de ST* est plus lent par la voie de recyclage des enzymes de glycosylation que par la voie induite par le motif KDEL. Pour caractériser le rôle des facteurs de régulation dans le transport rétrograde médié par le motif KDEL, nous avons réalisé des expériences d'ARNi ciblant COG3 et Rab6. Nos données montrent que la déplétion de COG3 entraîne un retard dans le transport rétrograde de ManII* et ST* et que la déplétion de Rab6 entraîne une altération du trafic de ST*, ce qui est cohérent avec les études précédentes. Nos données confirment l’existence et la différence de cinétique du transport rétrograde Golgi-RE dépendant du motif KDEL et le recyclage des enzymes golgiennes de glycosylation.Enfin, nous avons synchronisé le transport des enzymes golgiennes de glycosylation endogènes en utilisant des approches de knock-in CRISPR-Cas9 ou CRISPaint et l’outil RUSH. La distribution subcellulaire de ManIIEN-SBP-mNeonGreen, GalNAc-T1EN-SBP-mNeonGreen et B4GalT1EN-SBP-EGFP a été détectée dans le Golgi, et nous avons pu synchroniser leur transport bidirectionnel grâce à l'expression de Str-KDELMammalian cells are characterized by the co-existence of multiple pathways, including anterograde and retrograde transport. The Golgi apparatus has a central role in processing and sorting cargos in the bi-directional trafficking. The Retention Using Selective Hooks (RUSH) system allows to synchronize the transport of cargos from the ER to downstream compartments and to systematically analyze the secretory routes. Owing to the interaction of streptavidin (Str) and streptavidin-binding peptide (SBP), the reporter protein can be retained in the ER and then released by the addition of biotin. However, biotin has a high affinity to streptavidin, impairing reversibility of the RUSH assay. With the Artificial Ligands of Streptavidin (ALiS), Golgi-to-ER retrograde transport can be monitored using RUSH upon their washout.The reversible RUSH assay was firstly set up to study two mechanisms of Golgi-to-ER retrograde transport, including the KDEL-mediated retrieval pathway and the glycosylation enzyme recycling pathway. Core streptavidin fused with the ER-retention signal (Str-KDEL), or with the invariant chain (Ii-Str) were used as hooks. Golgi-resident enzymes, ManII* (Mannosidase II)-SBP-EGFP or ST* (Sialyltransferase)-SBP-GFP served as Golgi-targeted RUSH reporters. Our kinetic analysis showed that the Golgi-to-ER transport of ManII* and ST* are both slower through the glycosylation enzyme recycling pathway than the KDEL-meditated retrieval pathway. To characterize the role of putative regulatory factors in KDEL-mediated retrograde transport, we performed RNAi experiments targeting to COG3 and Rab6. Our data showed that knockdown of COG3 resulted in delayed retrograde transport of ManII* and ST*, and the depletion of Rab6 led to impaired trafficking of ST*, consistent with the previous studies. Our data indicated that there are two distinct pathways regulating the retrograde transport of Golgi cargos, including KDEL-mediated ER retrieval and ER recycling of Golgi glycosylation enzymes.Lastly, we have generated endogenous tagged Golgi glycosylation enzymes using CRISPR-Cas9 or CRISPaint knock-in approaches and applied the reversible RUSH assays in the selected clones. The subcellular distribution of endogenous ManIIEN-SBP-mNeonGreen, GalNAc-T1EN-SBP-mNeonGreen and B4GalT1EN-SBP-EGFP were detected in Golgi, and we were able to synchronize the bidirectional transport through the expression of Str-KDEL
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Miranda, Kevin Charles. "Post-Golgi trafficking in the mammalian secretory pathway /." [St. Lucia, Qld], 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18194.pdf.
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Pecot, Matthew Y. "A declaration of independence the Golgi apparatus is here to stay /." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3225895.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed October 8, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 107-115).
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Poe, Tyler M., and Francine Marciano-Cabral. "Illumination of the Golgi apparatus of Pathogenic and Nonpathogenic Naegleria species." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6002.
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In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and Vero CCL-81, Chlorocebus sp. kidney epithelial cells, which were utilized as a positive control for Golgi expression. N. fowleri and N. lovaniensis whole-cell lysates had indications of a 110 kDa reduced protein, associated with the predicted molecular weights of the beta-COPI subunit of the COPI cis-Golgi vesicular transport complex with further Western immunoblot indication of a weak band around 25 kDa corresponding to rabbit polyclonal antibodies specific for ARF1. Serial Dilutions of Wheat Germ Agglutinin Alexa Fluor 488TM were performed on Vero cells, Naegleria fowleri 30894, and N. gruberi 30540 with 1:100 dilution of recommended stock dilution of WGA 488 determined for utilization in sequential immunofluorescence. Sequential immunofluorescence with Wheat Germ Agglutinin Alexa Fluor 488TM and then blocked with 3% BSA:PBS [wt/vol] dilution with subsequent incubation in rabbit anti-beta-COPI primary 1:250, and 1:1000 of Alexa Fluor 594 goat anti-rabbit secondary antibody exposure showed strong indications of organized cis- and trans-punctate Golgi body markers in close association in individual and dividing cells of Naegleria fowleri and conserved Golgi expression in the positive control Vero cells, but further experiments are necessary to verify this finding with N. fowleri.
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Nawazi, Fazlullah Salar Khan. "Golgi specificity and development of autoreactive B cells." View the abstract Download the full-text PDF version, 2008. http://etd.utmem.edu/ABSTRACTS/2008-019-Nawazi-index.html.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008.Title from title page screen (viewed on September 9, 2008). Research advisor: Marko Z. Radic, Ph.D. Document formatted into pages (xi,111 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 91-111).
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Zamzow, Daniel R. "Signaling capabilities of a novel H-Ras mutant from the Golgi apparatus." [Ames, Iowa : Iowa State University], 2006.
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Wiggins, Christine Anne Ruth. "Identification and characterisation of proteins from the Golgi apparatus of S. cerevisiae." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624979.
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Marotta, D. "Defining the role of the Golgi apparatus in juvenile NCL (Batten disease)." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1460387/.
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The neuronal ceroid lipofuscinoses (NCLs) are a group of severe neurodegenerative lysosomal storage disorders characterised by accumulation of autofluorescent ceroid lipopigments in most cells. NCLs are caused by mutations in at least fourteen recessively inherited human genes. The NCL genes encode both soluble and transmembrane proteins localised to the endoplasmic reticulum, Golgi apparatus or endosomal/lysosomal organelles. Mutations in the CLN3 gene result in juvenile neuronal ceroid lipofuscinoses (JNCL, Batten disease). JNCL represents the worldwide most common form of NCL. Currently more than 40 mutations have been characterised in the CLN3 gene. However, the most common mutation causes a 1-kb deletion. CLN3 encode a multi-pass type III transmembrane protein, which is conserved in single-celled eukaryotes such as the fission yeast Schizosaccharomyces pombe, suggesting a fundamental role for this protein in eukaryotic cells. CLN3 has been functionally linked to many different cellular processes, including lysosomal homeostasis, autophagy, lipid synthesis or modification, cytoskeleton organisation and trafficking. Despite these endeavours, the function of CLN3 remains unclear. The main goal of this project was to investigate the role of the Golgi apparatus in the pathogenesis of juvenile CLN3 disease. The role of CLN3 at the Golgi apparatus was studied in mammalian cells and in fission yeast model. The morphology of the Golgi complex was studied in fibroblast cell lines from patients and in HeLa cells depleted for CLN3 using RNAi. The observed changes in morphology were accompanied by manganese dyshomeostasis within the Golgi complex, ER stress and apoptosis. The morphology of the Golgi complex was studied in S. pombe using electron microscopy in order to confirm the changes observed in mammalian cells. Finally, drugs shown to ameliorate aspects of the yeast model of CLN3 disease were tested for their efficacy in mammalian cells as an early step in therapeutic development. In this study I have shown that both morphology and size of the Golgi apparatus result to be affected by the loss/depletion of CLN3. Moreover, the changes in Golgi complex morphology and size are accompanied by manganese dyshomeostasis within the Golgi complex with activation of ER stress and activation of the proapoptotic protein caspase 2. Together, these data suggest that the loss/depletion of CLN3 activates secretory stress pathways and cell death. A dysfunctional Golgi apparatus may be the key to uncover the role of CLN3 and find new targets for therapeutic development.
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Howe, Jonathon David. "Antiviral mechanisms of small molecules targeting the endoplasmic reticulum and Golgi apparatus." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:04368b4b-2fd3-4fc7-8f89-ec39cd87e37d.
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N-linked glycosylation is the most common form of post-translational modification in nature and is essential to almost all enveloped viruses, including members of the Flaviviridae family. The host cell N-linked glycoprotein processing pathway is utilised by these viruses and as such has long been identified as a potential target for the development of antiviral drugs. Here, the antiviral mechanisms of three classes of small molecules targeting the secretory pathway and altering viral envelope glycosylation are investigated, using the HCV surrogate model, BVDV. The antiviral activity of imino sugars, principally through α-glucosidase inhibition, is well-characterised and here, a group of novel adamantyl coupled imino sugars are investigated and demonstrated to inhibit ER α glucosidases, which correlates with their antiviral activity against BVDV. Additionally, BVDV is used to study the antiviral mechanism of action of nitazoxanide. Nitazoxanide, the parent compound of the thiazolide class of structures, is a broadly antimicrobial compound with antiviral activity against HBV, HCV, influenza, JEV and others. Here, nitazoxanide is shown to be antiviral against BVDV by inducing Ca2+ release from ATP-sensitive intracellular calcium stores, disrupting ER-Golgi trafficking and inhibiting complex glycan formation. Finally, the potential of Golgi endo-α-mannosidase as an antiviral target is explored, using the endomannosidase inhibitor glucose-isofagomine in conjunction with the imino sugar α-glucosidase inhibitor NAP-DNJ. Endomannosidase is shown to be a valid antiviral target for BVDV, both alone and in combination with α-glucosidase inhibition, and is utilised by viral glycoproteins to acquire complex glycan structure, even in the absence of α-glucosidase inhibition. Altogether, this work furthers our understanding of the varied antiviral mechanisms of small molecules targeting the secretory pathway, enhancing the search for novel antiviral drugs directed against host cell machinery.
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Kong, Anne Mandy 1973. "Cloning and characterisation of a novel 72 kDa inositol polyphosphate 5-phosphatase." Monash University, Dept. of Biochemistry and Molecular Biology, 2001. http://arrow.monash.edu.au/hdl/1959.1/9036.
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Guet, David. "Architecture of the Golgi apparatus and membrane trafficking probed by intracellular optical micromanipulation." Paris 6, 2012. http://www.theses.fr/2012PA066203.
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Le trafic membranaire est basé sur la formation d'intermédiaires de transports qui transitent d'un compartiment à un autre. Des études in vitro ont montré que des paramètres physiques tels que la courbure, la tensione et la composition des membranes influence le bourgeonnement et la fission de ces intermédiaires. Plus récemment le rôle du cytosquelette d'actine dans la fission de ces vésicules a été mis en avant. Au cours de mon doctorat je me suis intéresé à l'effet d'un stress mécanique sur la formation de vésicules in cellulo. Dans cette thèse je montre que l'utilisation de la microscopie confocale, couplée à une pince optique, permet de visualiser en temps réel des déformations induites par la sonde sur des membranes de l'appareil de Golgi exprimant des protéines GFP et de mesurer les forces nécessaire à cette déformation. Mes résultats montrent que la contrainte mécanique induit une déformation à longue portée, suggérant l'existence d'une matrice englobant l'appareil de Golgi. La dépolymérisation de l'actine, candidate pour cette matrice, entraîne une diminution des forces nécessaires aux déformations. Enfin, l'application de la contrainte mécanique entrapine un défaut de la formation de vésicules positives pour Rab6 et la formation de longues structures tubulairesMembrane transport is based on the formation of tubulo-vesicular intermediates traveling from one compartment of the cell to another along cytoskeletal tracks. In vitro studies have shown that physical parameters, such as membrane curvature, tension and composition, influence the budding and fission of transport intermediates. Recent studies in cells have highlighted the central role of the actin cytoskeleton in the fission of transport intermediates from the Golgi apparatus. Here I investigate the role of a mechanical stress on intracellular transport in cellulo. I focus on the mechanics of Golgi membranes and the formation of transport intermediates from the Golgi apparatus. Using confocal microscopy, I visualize the deformation of Rab6-positive and COPI-positive Golgi membranes applied by an internalized microsphere trapped in an optical tweezers, and simultaneously measure the corresponding forces. My results show that the force necessary to deform Golgi membranes drops when the actin cytoskeleton is depolymerized, suggesting that actin strongly contributes to the local rigidity of the Golgi apparatus. We also show that the applied stress has a long-range effect on Golgi membranes and induces a sharp decrease in the formation of vesicles and the formation of tubular structures from the Golgi apparatus
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Starr, Tregei Nicole. "Quantitative Studies of Intracellular Trafficking of Two Classes of Resident Golgi Apparatus Proteins." Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/27217.
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The research presented in this dissertation consists of two primary parts. The initial focus centered on understanding the distribution of Golgi resident glycosyltransferases between the ER and Golgi at steady-state. Retrograde trafficking of these Golgi proteins has been demonstrated experimentally mandating the existence of a dynamic equilibrium between the Golgi apparatus and ER. Our published studies also included the development of a quantitative method for analysis of data collected using fluorescent microscopy. The second part of this dissertation presents results pertaining to the quantification of a unique Golgi resident protein that cycles in the late endosome bypass pathway. Using the published method of analysis and techniques developed during the initial project, the anterograde and retrograde transport kinetics of this Golgi protein were determined and used to develop a compartmental model for pH sensitive trafficking in the bypass pathway. The spatial Golgi distribution of the protein during retrograde transport to the Golgi following endosomal exit was also investigated. This research lies at the interface of experimental cell biology and quantitative computational analysis. These experiments combined more traditional experimental biological approaches with more recent computational approaches to understanding cellular mechanisms. Additionally, development of a quantitative method of analysis validated the use of fluorescent microscopy as a quantitative tool for studying intracellular proteins.Ph. D.
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Fuchs, Evelyn. "Regulation of Membrane Traffic at the Golgi Apparatus by Rab GTPases and their GAPs." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-82832.
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Ali, Zahabia Shameem. "Investigating the role of AMPK in carbohydrate sensing and localisation at the golgi apparatus." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501761.
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Fuchs, Evelyn. "Regulation of membrane traffic at the golgi apparatus by rab GTPases and their GAPs." kostenfrei, 2007. http://edoc.ub.uni-muenchen.de/8283/.
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Deng, Yuping. "Studies of intraorganelle dynamics : the lysosome, the pre-lysosomal compartment, and the golgi apparatus /." Diss., This resource online, 1991. http://scholar.lib.vt.edu/theses/available/etd-07282008-134815/.
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Dahan, Sophie. "Intracellular proteinmembrane trafficking : evaluation of the Golgi and endosomal apparatus by cryoimmune electron microscopy." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29387.
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Protein trafficking events in the secretory and endosomal apparatus were evaluated in rat liver hepatocytes by EM immunogold cytochemistry. The cellular distribution of apolipoprotein E and other abundant hepatic secretory and endosomal proteins was quantitatively examined in ultrathin cryosections of liver hepatocytes. Under steady-state conditions, apoE was concentrated within the Golgi apparatus, along sinusoidal plasma membrane microvilli and within all components of the endosomal apparatus, as evaluated immunocytochemically and confirmed by quantitative immunoblotting of organelles isolated from liver homogenates.Within the secretory pathway, the hepatic Golgi apparatus was a site of protein concentration as evaluated by the gold labeling density of another major secretory protein of liver hepatocytes, albumin, which was concentrated $ sim$10-fold in the Golgi apparatus relative to the ER. Sorting of this secretory protein within pre-Golgi compartments was not observed. Within the Golgi apparatus, apoE was concentrated within Golgi saccular distensions while being predominantly absent from flattened saccular components; apoB was similarly segregated within peripheral distensions. In contrast albumin, as well as two other monomeric proteins, transferrin (Tf) and the polymeric immunoglobulin receptor (pIg-R) were distributed homogeneously throughout Golgi stacks. In an attempt to assess a key prediction of the vesicular transport hypothesis, small 60-90 nm vesicles in the immediate vicinity of Golgi apparatus, postulated to mediate intersaccular transport were examined for their content of cargo secretory or plasma membrane proteins. Lack of immunoreactive apoE, apoB, albumin, Tf, or pIgR, within small vasicular profiles suggests limits to current models of vesicle-mediated intra-Golgi transport.
Along the endocytic pathway, at the cell surface, apoE and pIgR were dispersely distributed along the sinusoidal microvilli. Quantitative analysis of the immunolabeling distribution of these proteins did not reveal concentration within plasma membrane pits. These findings which were confirmed by observations of cell surface labeling of two other ligands, Tf and apoB, are consistent with receptors and ligands gaining access to the endocytic machinery likely without receptor/ligand preclustering or prolonged clustering events within plasma membrane pits. Intracellularly, apoE was concentrated within endocytic structures which were double-labeled for apoE and internalized HRP. Large endocytic vesicles closely juxtaposed to Golgi stacks also revealed a high content of apoE. Together the endosomal labeling distribution of apoE as well as morphological features of endosomal components are consistent with the maturation model for endosomes.
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Capitanio, Paola. "Effects of familial Alzheimer's disease-linked presenilin 2 mutants on Ca2+ homeostasis of Golgi Apparatus sub-compartments." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423438.
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Alzheimer's Disease (AD) is a progressive neurodegenerative disorder and the most common form of senile dementia. The characteristic histopathological hallmarks of AD are the intracellular neurofibrillary tangles and the amyloid plaques, made of aggregated amyloid peptides (Aß), that deposit in the extracellular matrix of the brain. Aß peptides are the result of two sequential cleavages of the amyloid precursor protein (APP); Aß is eventually released by the α-secretase enzyme. The most abundant Aß peptide species, both physiologically produced throughout life, are Aß40 and Aß42, which is more insoluble and aggregation-prone.
Although most AD cases are sporadic, a small percentage of patients is affected by the hereditary form of AD (Familial Alzheimer's Disease, FAD), caused by dominant mutations in one of three genes. These genes code for the APP, presenilin-1 (PS1) and presenilin-2 (PS2); PSs are the catalytic subunits of the α-secretase enzyme complex but they also function in a α-secretase indipendent manner.
FAD-linked mutations in PSs lead to an increased Aß42/Aß40 ratio, that promotes Aß plaques deposition. Beside this effect on Aß production, many mutations in PS1 and PS2 have been extensively demonstrated to cause alterations in the intracellular Ca2+ homeostasis, thus making neurons more sensitive to excitotoxic stimuli and apoptosis.
The Golgi apparatus (GA) represents, together with the endoplasmic reticulum (ER), the major IP3-sensitive, rapidly mobilizable, intracellular Ca2+ store and its functionality is thus important for shaping cytosolic Ca2+ responses. Increasing evidence suggests that the GA is an heterogeneous Ca2+ handling organelle, equipped with a diverse molecular Ca2+ toolkit compared to the one expressed in the ER. For example, as Ca2+ uptake mechanisms, the GA expresses the classical sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) but also an additional Ca2+ pump, the secretory pathway Ca2+ ATPase1, SPCA1.
The use of a specific Cameleon Ca2+ sensor targeted to the trans-Golgi, allowed us to directly demonstrate the functional GA heterogeneity by showing the distinct behavior of this sub-compartment: it takes up Ca2+ almost exclusively via SPCA1 (and not by SERCA); it does not release Ca2+ in response to IP3 generation, but rather accumulates the cation as a consequence of the cytoplasmic Ca2+ rise.
As regard to the other GA compartments, we generated a new FRET-based Ca2+ indicator fused to the cis/medial-Golgi targeting sequence of the enzyme 1,6 N-acetylglucosaminyltransferase (C2gnT). The new probe very nicely co-localizes with the cis/medial-Golgi marker Giantin and thus was used to study Ca2+ dynamics in this compartment at single cell level.
The data collected suggest that the GA is unique in terms of Ca2+ homeostasis, with compartments that are separated by a few microns, and in very rapid equilibrium with each other, that still maintain quite substantial differences in terms of ion concentration and response to external stimuli.
The differences between the two GA sub-compartments, the medial and the trans-one, are confirmed by the specific effect on Ca2+ homeostasis of the expression of the FAD-linked PS2 T122R mutation. Cells expressing the mutated form of the protein show a decreased Ca2+ content in the cis/medial-Golgi but no effects on trans-Golgi Ca2+ homeostasis. PS2-T122R seems to inhibiting Ca2+ uptake in the cis/medial-Golgi by inhibiting SERCA pump activity while does not affect Ca2+ uptake, mediated by SPCA1, in the trans-Golgi.
As a major Ca2+ store, the GA could play an important role in AD and understanding the contribution of GA Ca2+ dysfunction in AD will significantly impact our ability to develop more effective therapies for the disease.La malattia di Alzheimer's (AD) è un disordine neurodegenerativo e la forma più comune di demenza senile. La caratteristica istopatologica di AD è la presenza di depositi neurofibrillari intracellulari e di placche amiloidi, costituite da aggregati di peptide amiloide (Aß), che si depositano nella matrice extracellulare del cervello. I peptidi Aß sono il risultato di due tagli sequenziali della Proteina Precursore dell'Amiloide (APP); Aß viene poi rilasciato dall'enzima α-secretasi. Le più abbondanti specie peptidiche di Aß, prodotte anche fisiologicamente per tutta la vita, sono Aß40 e Aß42, quest'ultimo più insolubile e più incline all'aggregazione. Sebbene la maggior parte dei casi di AD siano sporadici, una piccola percentuale di pazienti è affetta dalla forma ereditaria di Alzheimer (malattia familiare di Alzheimer, FAD), causata da mutazioni dominanti in uno dei geni codificanti per APP, presenilina-1 (PS1) e presenilina-2 (PS2); le PSs sono le subunità catalitiche del complesso enzimatico della α-secretasi ma funzionano anche in maniera indipendente da tale attività enzimatica. Le mutazioni in PSs legate a FAD portano ad un aumento nel rapporto Aß42/Aß40, che promuove la deposizione di placche amiloidi. Oltre a questo effetto, è stato ampiamente dimostrato che molte mutazioni in PS1 e PS2 provocano alterazioni della omeostasi del Ca2+ intracellulare, rendendo così i neuroni più sensibili agli stimoli eccitotossici e apoptotici. L'apparato di Golgi (GA) rappresenta, insieme al reticolo endoplasmatico (ER), il principale deposito intracellulare di Ca2+, IP3 sensibile, e la sua funzionalità è fondamentale per il controllo delle risposte citosoliche di Ca2+. Sempre maggiori evidenze suggeriscono che il GA sia un organello eterogeneo in termini di Ca2+ handling, essendo dotato di un diverso toolkit molecolare per il Ca2+ rispetto a quello espresso nell' ER. Ad esempio, come meccanismi di uptake per il Ca2+, il GA esprime la classica pompa SERCA (Sarco-Endoplasmic Reticulum Ca2+ ATPase) ma anche un ulteriore pompa, detta SPCA1 (Secretory Pathway Ca2+ ATPase1). L'utilizzo di uno specifico sensore per il Ca2+ specificatamente indirizzato al trans-Golgi, ci ha precedentemente permesso di dimostrare direttamente la eterogeneità funzionale del GA, mostrando il comportamento distinto di questo sub-compartimento: i meccanismi di uptake di Ca2+ sono mediati esclusivamente dalla SPCA1 (e non dalla SERCA); non rilascia Ca2+ in risposta alla generazione IP3, ma piuttosto si accumula il catione come conseguenza dell'aumento di Ca2+ citoplasmatico. Per quanto riguarda gli altri sub-compartimenti del GA, abbiamo generato un nuovo indicatore per il Ca2+ fuso alla sequenza di indirizzamento dell'enzima 1,6 N-acetylglucosaminyltransferasi (C2gnT) residente del cis/medial-Golgi. La nuova sonda co-localizza con il marcatore di cis/medial-Golgi Giantina e quindi è stata utilizzata per studiare le dinamiche di Ca2+ in questo sub-compartimento a livello di singola cellula. Complessivamente i dati ottenuti suggeriscono che il GA sia unico in termini di omeostasi del Ca2+, con tali sub-compartimenti separati da pochi micron, e in equilibrio molto rapido tra loro, ma comunque in grado di mantenere differenze consistenti in termini di concentrazione dello ione e risposta a stimoli esterni . Le differenze tra i due sub-compartimenti del GA sono confermate dall'effetto specifico sulla omeostasi del Ca2+ dell'espressione della forma mutata di PS2T122R legata alla malattia familiare di Alzheimer. Le cellule che esprimono tale proteina mostrano una diminuzione del contenuto di Ca2+ nel cis/medial-Golgi ma nessun effetto sull'omeostasi del Ca2+ nel trans-Golgi. PS2T122R sembra inibire l'assorbimento di Ca2+ nel cis/medial-Golgi, inibendo l'attività della pompa SERCA, mentre non influenza l'assorbimento di Ca2+, mediato dalla SPCA1, nel trans-Golgi. Il GA sembra quindi giocare un ruolo importante nella patogenesi di AD e comprendere il contributo di tale organello nella patogenesi di AD e la sua base fisiopatologica potrà avere un forte impatto sulla possibilità di sviluppare terapie più efficaci per AD.
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Fossati, M. "MECHANISMS OF PROTEIN TRANSPORT AT THE ER-GOLGI INTERFACE." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/214981.
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The Endoplasmic Reticulum represents the first station of the secretory path-way, where proteins destined to the cell surface or to some intracellular organelles are recruited in specific ER subdomains, the ER Exit Sites (ERES), and start to travel into transport carriers to reach the proper final destination. Even though cargoes are usual-ly recruited to ERES by a sequence-dependent mechanism, it is known that other fac-tors contribute to protein export from the ER. Using model fluorescent tail-anchored proteins our group previously demonstrated that the length/hydrophobicity of the transmembrane domain is an important factor determining recruitment to or exclusion from ERES: a protein with a short TMD (FP-17) is excluded from ERES and retained in the ER, while a longer TMD (FP-22) determines enrichment in ERES. In order to clarify the molecular mechanism underlying this TMD-dependent transport, we first compared the transport of an export signal-bearing (VSV-G DxE) membrane protein with our model protein FP22, which lacks an export signal. FP22 and VSV-G accu-mulate together at ERES, but VSVG reaches the plasma membrane more rapidly than FP22. To investigate the basis of this difference, we combined cDNA microinjection to temperature blocks and live-cell imaging approaches that allowed us to analyze the transport at early steps of the secretory pathway at the ER-Golgi interface. At 20°C, a temperature at which only the transport between the ER and the Golgi is allowed, all of the VSVG accumulates in the Golgi, while FP-22 remains distributed between the ER and the Golgi. After bleaching the Golgi fraction of FP22 we observed a rapid, energy-dependent, fluorescence recovery, indicating an efficient ER to Golgi transport even in the absence of the export signal and suggesting that FP22 may be re-cycled between the two compartments. In agreement, a rapid emptying of the Golgi was observed after ER bleaching (accompanied by a fluorescence recovery of the ER fraction). To investigate whether this phenomenon is restricted to our model protein only or it is more general event, we then tested the behavior of a signal-deleted form of VSV-G (VSV-G AxA). Similarly to FP22, VSVG AxA is distributed between the Golgi and the ER at 20°C and Golgi fluorescence rapidly decreases after ER bleach-ing, suggesting a new role of the ER export signal, which is important not only in re-cruiting cargoes at the ERES, but also in preventing their recruitment into futile cy-cles between the Golgi and the ER, which delay their arrival to the cell surface.
To further characterize the mechanism of TMD-dependent sorting, we then in-vestigated the role of membrane curvature; our group previously demonstrated that FP-22 is segregated from FP-17 in specific ER subdomains, which are characterized by membrane curvature (ERES and ER tubules). In collaboration with Bruno Goud and Jean-Baptiste Manneville (Institute Curie, Paris), we created highly curved do-mains using membranes composed of a uniform lipid composition (POPC, palmitoyl-oleyl-phosphatidylcholine) or ER lipids extracted from rat liver microsomes, and we analyzed the distribution of our two model proteins in flat and curved domains. Our results indicate that the two proteins are uniformly distributed in curved membranes and strongly suggest that the membrane curvature alone cannot drive the TMD-dependent partitioning of membrane proteins in ERES and ER tubules.
Taken together, our data contribute to clarify the role of two fundamental fac-tors influencing the transport of membrane proteins along the secretory pathway that were never investigated before.
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Foote, Christopher. "The role of the AP-1 adaptor complex in trafficking between the trans-Golgi Network and endosomal system." Diss., Columbia, Mo. : University of Missouri-Columbia, 2005. http://hdl.handle.net/10355/4172.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2005.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (November 7, 2006) Vita. Includes bibliographical references.
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Compton, Shannon Leigh. "Functional analysis of PRAF1 and its effect on corticotrophic ACTH secretion." Auburn, Ala., 2007. http://repo.lib.auburn.edu/07M%20Dissertations/COMPTON_SHANNON_44.pdf.
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Weigert, Roberto. "Biochemical analysis of the factors controlling the process of membrane tubule formation from the Golgi complex." Thesis, Open University, 2000. http://oro.open.ac.uk/58143/.
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Membranous tubules are very abundant structures in living cells and form or are part of most intracellular organelles. The Golgi apparatus is mainly formed by tubules, which adopt different geometries and conformations. However, their physiological role has not yet been established and this is mainly due to the almost absolute lack of knowledge about the biochemical mechanisms regulating their formation, maintenance and disruption. The aim of this thesis was to investigate in a systematic way these mechanisms. The first step has been to set up an in vitro morphological assay suitable for the visualisation of Golgi-associated tubules in isolated Golgi stacks. This assay was based on electron microscopy and specifically on negative staining of whole-mount preparations. It allowed both qualitative and quantitative analysis of the morphological changes of Golgiassociated tubules after in vitro incubations. This assay was then used for screening several molecules or experimental conditions for their effect on tubular homeostasis. Among them, the most significant was BARS (BFA-dependent ADP-Ribosylation Substrate), a protein previously implicated in the maintenance of Golgi architecture. BARS has been found to cause the selective breakdown of the tubular part of the Golgi complex promoting fission events which convert the tubular structures into clusters of vesicles. This effect correlated with the enzymatic activity of BARS, which acts as an acyl-CoA dependent lysophosphatidic acid acyl transferase (LPAAT), increasing phosphatidic acid (PA) levels in Golgi membranes. This suggests that local modifications of the composition of the lipid bilayer is a possible mechanism for the fission of membranous tubules.
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Young, Robin Elizabeth. "Secretion of plant cell wall polysaccharides by the Golgi apparatus in Arabidopsis thaliana seed coat cells." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/11573.
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The plant cell wall determines cell shape and is essential for plant growth during development. Pectin is an important component of cell walls and, like many wall polysaccharides, is synthesized in the Golgi apparatus and secreted by vesicles. In Arabidopsis thaliana seed coats, pectin-rich mucilage is secreted in a polarized fashion during a specific stage of development. How the Golgi apparatus in seed coat cells accommodates the large increase in pectin-rich mucilage provides a unique window into the cellular machinery that supports cell wall polysaccharide biosynthesis and secretion.
Examination of seed coat cells, using cryo-fixation and transmission electron microscopy and electron tomography, showed that Golgi stacks undergo dramatic changes in structure during mucilage production. Initiation of mucilage biosynthesis also correlated with increased numbers of Golgi stacks per cell. To understand if these cellular changes were dependent on pectin biosynthesis, the cell structure of a reduced mucilage mutant, mum4, was studied by similar methods and revealed that, while the morphology of Golgi stacks was dependant on mucilage, the increased stack number was not.
To determine what proportion of the scattered Golgi stacks were producing mucilage, immunogold labeling with the novel mucilage-specific antibody CCRC-M36 was used to detect the pectin cargo. The large percentage of labeled Golgi stacks found suggests that many stacks produce pectin synchronously, rather than a subset of specialist Golgi. To test if a pectin modifying enzyme, MUM2, is co-secreted with pectin, a tagged MUM2 was engineered and introduced into mum2 mutants, where it rescued the mutant phenotype. However, the tag was not detectable using antibodies in immunofluorescence.
Although mucilage was secreted to the top of the cell, antibody label demonstrated that pectin-producing stacks were randomly distributed throughout the cytoplasm, indicating that the destination of cargo has little effect on location of the Golgi stack producing it. The mechanism of targeting of vesicles with the domain of the plasma membrane exclusively at the mucilage pocket is unknown, although the correlation of a population of densely staining vesicles and abundant cortical microtubules in the cell cortex at the site of secretion was documented.
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Millarte, Valentina [Verfasser]. "Signaling at the Golgi Apparatus During Cell Migration and Implication for Cancer Cell Metastasis / Valentina Millarte." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1080908919/34.
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Hiding, Johan. "Functional characterization of the secretory pathway and the role of COPI vesicles /." Göteborg : Göteborg University, Institute of Biomedicine, Department of Medical Genetics, Sahlgrenska Academy, Göteborg University, 2007. http://hdl.handle.net/2077/8502.
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Szul, Tomasz J. "The role of GBF1 in Golgi biogenesis and secretory traffic." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2009p/szul.pdf.
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Grabski, Robert. "Using RNA interference to study the function of the tethering protein p115 in ER-Golgi traffic." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008p/grabski.pdf.
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Bellouze, Sarah. "Mécanismes moléculaires de la fragmentation de l' appareil de Golgi dans les maladies du neurone moteur." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4080.
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La fragmentation de l'appareil de Golgi représente un des changements les plus précoces et les plus répandus dans les maladies neurodégénératives. Afin de comprendre les mécanismes moléculaires de ces changements, j'ai étudié deux modèles expérimentaux de maladie du neurone moteur. 1. Les souris pmn (progressive motor neuronopathy) : Celles-ci sont atteintes d'une forme très grave de dégénérescence des neurones moteurs et des défauts moléculaires sont liés à une mutation faux-sens d'une protéine localisée au niveau du Golgi, la chaperonne des tubulines TBCE, identifiée par (Martin, Jaubert et al. 2002; Schaefer, Schmalbruch et al. 2007). Au cours de ma thèse, nous avons identifié des anomalies importantes du Golgi dans les neurones moteurs lombaires de souris pmn et déterminé leur relevance fonctionnelle ainsi que les mécanismes moléculaires. D'après les immunomarquages et la modélisation 3D des membranes, la fragmentation et l'atrophie du Golgi dans les neurones lombaires moteurs pmn ressemblent à celles rapportées dans la SLA et se produit dans des cinétiques similaires. Les analyses en microcopie électronique montrent que l'empilement des citernes golgiennes est progressivement remplacé par des petites vésicules. Les analyses biochimiques révèlent : 1/ une redistribution cytosolique des protéines d'arrimage tel que GM130, 2/ une diminution des protéines β-COP et 3/ une augmentation considérable des protéines golgiennes d'amarrage v-SNARE GS15 et GS28 contrôlant la fusion des vésiculesFragmentation of the Golgi apparatus represents one of the earliest and most constant pathological changes in neurodegenerative diseases. To understand the molecular mechanisms of these changes I investigated two experimental models of motor neuron diseases. 1. pmn mice with progressive motor neuronopathy. The pmn mice were chosen since they suffer from a very aggressive form of motor neuron degeneration and since their molecular defects represents a missense mutation in a Golgi-localized tubulin chaperone TBCE, as shown by previous (Martin et al 2002, Schäfer et al 2007). In the last years, we identified severe Golgi abnormalities in motor neurons of pmn mice and dissected out their functional relevance and molecular mechanisms. According to immunolabelings and 3D membrane modelings, Golgi fragmentation and atrophy in lumbar pmn motor neurons resembled those reported in human ALS and proceeded with similar kinetics. Electron microscopy illustrated that Golgi cisternae were progressively transformed into small vesicles. Biochemical analyses revealed : 1/ a cytosolic redistribution of tethering factor such as GM130, 2/ a decrease in β-COP protein level and 3/ a massive increase in the Golgi v-SNARE proteins GS15 and GS28 controlling vesicle fusion. These pathological changes were due to loss of TBCE expression since they could be rescued by transgenic expression of wildtype TBCE but not mimicked by sciatic nerve axotomy. They involved defective dynamics of Golgi-derived microtubules rather than accumulation of misfolded tubulins as shown by the differential effects of TBCE-depletion, Nocodazole and a folding-incompetent tubulin mutant
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Heymann, Julia. "Chlamydia infection impairs host cell motility via CPAF-mediated Golgi fragmentation." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16564.
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Chlamydien sind obligat intrazelluläre Bakterien, die sich in einem membranumschlossenen Kompartiment namens Inklusion vermehren. Nach Infektion fragmentiert der Golgi-Apparat der Wirtszelle in kleine Membranstapel. Dies verbessert die Aufnahme von Sphingolipiden und ist deshalb für die chlamydiale Vermehrung essentiell. Die infektionsinduzierte Golgi-Fragmentierung geschieht nach Spaltung des Golgi-Matrix-Proteins Golgin-84. In dieser Arbeit konnte, durch den Vergleich mit bekannten Substraten und Inhibitorstudien, die chlamydiale Protease CPAF (Chlamydia protease-like activity factor) als das Enzym identifiziert werden, das diese Spaltung induziert, abhängig von der Anwesenheit zweier Rab-Proteine, Rab6 und Rab11, die den zellulären Vesikeltransport kontrollieren und zur Inklusion rekrutiert werden. Die Fragmentierung des Golgi-Apparates verhinderte dessen Relokalisierung während der Zellpolarisierung nach Einbringen eines migratorischen Stimulus. Sowohl infizierte als auch Golgin-84-depletierte Zellen migrierten langsamer und randomisiert in einem Motilitätsassay. Die Relokalisierung des Golgi-Apparates konnte durch seine Stabilisierung mittels WEHD oder Rab-Depletion wieder gewonnen werden, was die Zellmotilität teilweise wieder herstellte. Darüber hinaus konnte gezeigt werden, dass die Infektion außer der Golgi-Reorientierung die Signaltransduktion durch GTPasen beeinflusst. Die Aktivität von Cdc42 in infizierten Zellen war erhöht und die Interaktionen mit vielen ihrer Effektoren laut quantitativer Massenspektrometrie stark verändert. Die Ergebnisse dieser Arbeit zeigen, dass CPAF die für Chlamydien lebenswichtige Golgin-84 Prozessierung und Fragmentierung des Golgi-Apparates auslöst. Dies verringert die Mobilität der Wirtszelle, vor allem da der Golgi-Apparat während der Polarisierung nicht mehr ausgerichtet werden kann, des Weiteren durch Modulierung der Protein-Protein-Interaktionen von Cdc42.Chlamydia are obligate intracellular human pathogens that proliferate inside a membrane-bound compartment called the inclusion. In infected cells, the Golgi apparatus is fragmented into small ministacks that are aligned around the inclusion. This facilitates uptake of host cell sphingolipids and is essential for chlamydial development. Infection-induced Golgi fragmentation happens after processing of the Golgi matrix protein golgin-84. This work could, via comparison with well-known substrates and inhibitor studies, identify the chlamydial protease CPAF (Chlamydia protease-like activity factor) as the enzyme accountable for this cleavage. Golgi Fragmentation depended on two Rab proteins, Rab6 and Rab11, which control vesicle transport and are recruited to the Chlamydia inclusion. As a consequence of Golgi fragmentation, cells lost the capacity to reorient the Golgi apparatus during polarization after a migratory stimulus. Both infected and golgin-84 depleted cells with a permanently fragmented Golgi apparatus displayed decelerated and furthermore randomized migration in a motility assay. Relocalization of the Golgi apparatus could be restored via stabilizing WEHD treatment or Rab depletion which partly rescued cell motility. Moreover, it could be shown that migration signaling via small GTPases was influenced by Chlamydia infection. Infected cells exhibited activation of the small polarity GTPase Cdc42. Numerous interactions with downstream effectors were strongly altered in infected cells according to quantitative mass spectrometry. Particularly, the binding of Cdc42 to migration-associated effectors was decreased. The results of this work show that CPAF, by processing of golgin-84, induces Golgi fragmentation which is vitally important for Chlamydia. This disturbs host cell motility because the Golgi apparatus cannot be reoriented during polarization and, additionally, via the modulation of protein-protein-interactions of Cdc42.
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Bailly, Anne-Laure. "Rôle de GRASP-55 dans la spermatogenèse et la différenciation hématopoïétique." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4112.
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Les molécules d’adhésion jonctionnelles JAM-B et JAM-C forment une paire récepteur/ligand impliquée dans la régulation de nombreux mécanismes biologiques dont l’inflammation, l’hématopoïèse et la spermatogénèse. Dans la moelle osseuse, l’interaction entre JAM-C et JAM-B, respectivement exprimée par les cellules souches hématopoïétiques (CSH) et les cellules stromales, joue un rôle dans la rétention et la quiescence des CSH. Dans le testicule, JAM-C participe à la polarisation des spermatides en différenciation en interagissant avec JAM-B exprimée par les cellules de Sertoli. GRASP55 (Golgi ReAssembly and Stacking Protein of 55 kDa), identifiée au laboratoire comme un interacteur intracellulaire des protéines JAMs, est une protéine de l’appareil de Golgi participant à l’architecture et la dynamique de celui-ci ainsi qu’au transport protéique non-conventionnel.Le but de mon travail de thèse a été d’étudier le rôle de GRASP55 in vivo par des approches génétiques et pharmacologiques. Nous avons ainsi pu mettre en évidence que l’expression de GRASP55 par la spermatide ronde permet la localisation polarisée de JAM-C et le déroulement correct de la spermatogénèse. A contrario, GRASP55 n’est pas essentiel à l’hématopoïèse en conditions basales ou de stress. Toutefois, la délétion de GRASP-55 dans les cellules leucémiques diminue le progression de la pathologie in vivo. Ces résultats montrent un rôle non redondant de GRASP55 dans la spermatogenèse et la prolifération de cellules leucémiques et ouvrent des pistes possibles pour un ciblage thérapeutique de GRASP55 en hématologieThe junctional adhesion molecules JAM-B and JAM-C form a receptor / ligand pair involved in regulation of many biological mechanisms including inflammation, hematopoiesis and spermatogenesis. In the bone marrow, the interaction between JAM-C and JAM-B, expressed by hematopoietic stem cells (HSC) and stromal cells respectively, is involved in HSC retention and quiescence. Similarly, in the testis, JAM-C participates in the polarization of differentiated spermatids by interacting with JAM-B expressed by Sertoli cells. GRASP55 (Golgi ReAssembly and Stacking Protein of 55 kDa), identified in our laboratory as a new intracellular interactor of JAM, is a Golgi apparatus protein involved in Golgi architecture and dynamics as well as unconventional secretion.The aim of my thesis was to study the role of GRASP55 in vivo by genetic and pharmacological approaches. We demonstrate that GRASP55 expression by round spermatid allows polarized localization of JAM-C and the correct course of the spermatogenesis. In contrast, GRASP55 is not essential for hematopoiesis in basal or stress conditions. However, deletion of GRASP-55 in leukemic cells decreases the progression of the pathology in vivo. These results show a non-redundant role of GRASP55 in the spermatogenesis and proliferation of leukemic cells and allow us to consider GRASP55 as a potential target in hematology
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Panić, Bojana. "The small GTPases Arl1p/Arl1 and Arl3p/ARFRP1 act in a pathway for targeting proteins to the Golgi apparatus." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616124.
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Green, Harold G. "Ultrastructural and cytochemical characteristics of the Golgi apparatus in epithelial principal cells of the initial segment of the rat epididymis." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59246.
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Epididymides post-fixed in reduced osmium were used to study the morphological characteristics of the Golgi apparatus of principal cells of the initial segment. Others, fixed in glutaraldehyde, were treated to detect nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyro-phosphatase (TPPase), and cytidine mono-phosphatase (CMPase) activities within Golgi compartments and glucose-6-phosphatase activity localized in cisternae of ER.The Golgi apparatus of these cells is composed of numerous saccular and tubular elements. On the cis face of the Golgi there is an osmiophilic tubular network. Of the numerous subjacent saccules, the first is dilated and unreactive for phosphatases, the next 3 show NADPase activity and the last 4 are TPPase positive. On the trans aspect of the stack there are 3 or 4 CMPase positive trans Golgi networks, one showing a peeling off configuration while the other 2 or 3 are completely separated from the stack. The TGN's often contain nodular distensions suggestive of secretion granule formation and occasionally present a shrivelled appearance.
Cisternae of ER showing buds are present on the cis face of the stacks.
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Bruinsma, Paul. "The role of the yeast COG3, VPS35, and YDR141C proteins in membrane trafficking /." free to MU campus, to others for purchase, 2002. http://wwwlib.umi.com/cr/mo/fullcit?p3074381.
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Massarweh, Ahmad. "Dolichol linked Oligosaccharide Diphosphatase : a potential regulator of dolichol linked oligosaccharides." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066447.
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CONTEXTE: Les " Type I Congenital disorders of glycosylation " (CDG-I) comportent des déficits de biosynthèse de l'oligosaccharide lié au dolichol (DLO) qui est nécessaire pour la N-glycosylation des protéines. Ces déficits induisent : 1) une hypoglycosylation des protéines qui serait à l'origine de la pathologie ; et 2) une accumulation de DLO tronqués à partir desquels, par un mécanisme encore inconnu, des structures oligosaccharidiques libres phosphorylées (OSP) sont générées dans le cytosol. Afin de comprendre le rôle de ce processus dans le CDG, il était donc nécessaire de caractériser l'activité qui est à l'origine des OSP.RESULTATS: J'ai caractérisé biochimiquement une DLO diphosphatase (DLODP) qui génère des OSP et du dolichol phosphate à partir de DLO. L'activité DLODP co-fractionne avec un marqueur de l'appareil de Golgi (AG) mais pas avec les enzymes réticulaires qui utilisent le dolichol phosphate. Cette localisation inattendue de DLODP m'a conduit à étudier la génération des OSP dans les cellules en utilisant la bréfeldine A (BFA) qui fusionne l'AG avec le RE. La BFA ne modifie pas les taux de DLO tronqués ni ceux des OSP cytoplasmiques dans un modèle cellulaire de CDG-I. Cependant, dans ces cellules et dans les cellules témoins, la BFA induit une forte augmentation des OSP dans le système endomembranaire à partir de DLO non-tronqués.CONCLUSION: L'identification de différents pools d'OSP, topologiquement distincts et pouvant être modulés de façon indépendante, révèle la multiplicité des mécanismes pour la génération d'OSP et suggère que la DLODP Golgienne n'est pas forcément l'enzyme responsable de la génération des OSP dans le contexte de CDG-IBACKGROUND: Type I congenital disorders of glycosylation (CDG-I) are caused by genetic defects in the biosynthetic pathway for the dolichol-linked oligosaccharide (DLO) that is required for protein N-glycosylation. These mutations result in the accumulation of truncated DLO and protein hypoglycosylation. Although protein hypoglycosylation is thought to be the main pathogenic factor in CDG-I, the role of truncated DLO intermediates in cellular homeostasis is not clear. Truncated DLO intermediates are known to give rise to cytoplasmic oligosaccharyl phosphates (OSP) by an uncharacterized mechanism. To understand this DLO editing process biochemical and molecular characterization of the activity that generate OSP is needed.RESULTS: I biochemically characterized a DLO diphosphatase (DLODP) that generates OSP and dolichol phosphate from DLO. Subcellular fractionation of mouse liver homogenates demonstrated a microsomal activity that co-distributes with a Golgi apparatus (GA) marker but not with endoplasmic reticulum (ER)-situated dolichol phosphate utilizing enzymes. This unexpected localization of DLODP prompted me to study OSP generation in cells using brefeldin A (BFA), which fuses the GA with the ER. BFA did not affect the levels of truncated DLO or cytoplasmic OSP, present in a cellular model of CDG-I. However, in these, and control cells, BFA caused striking increases of OSP within the endomembrane system. CONCLUSION: the identification of topologically distinct, independently modulated, OSP pools indicates multiple mechanisms for OSP generation and suggest that the GA-situated DLODP may not be the enzyme responsible for OSP generation in CDG-I
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Serra, Peinado Carla. "Papel del citoesqueleto de actina en la regulación de la H+-ATPasa vacuolar de complejo de Golgi." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/663846.
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La vía secretora se caracteriza por la acidificación progresiva de sus orgánulos, este gradiente es crucial para funciones tales como la modificación postraduccional de proteínas o el trafico de membranas. El principal responsable de generar y mantener este gradiente es la H+-ATPasa vacuolar (V-ATPase), que transporta protones desde el citosol hacía el interior del Golgi. Esta bomba está compuesta de dos dominios, el dominio V1 y el V0, a su vez ambos están formados por varias subunidades. Se ha descrito que las subunidades B y C del dominio V1 contienen dominios de unión a actina.
Existen significantes similitudes entre los efectos subcelulares producidos por la despolimerización de actina y la inhibición farmacológica de la V-ATPasa: Alteración de transporte vesicular Golgi-Retículo endolplasmatico y Golgi-membrana plasmática, alcalinización del complejo de Golgi, dilatación de las cisternas de Golgi. Teniendo en cuenta que dos subunidades de la V-ATPasa tienen la capacidad de unirse a los microfilamentos de actina, nosotros hipotetizamos que estos podrían participar en la homeostasis del pH de Golgi a través de la regulación de la V-ATPasa, particularmente la actina podría estar manteniendo la asociación de los dominios V1 y V0.
Generamos un constructo de la subunidad B conjugado con GFP (B2-GFP) que se incorporaba en el dominio V1. Observamos que este constructo se localizaba en los compartimentos distales del complejo de Golgi y que translocaba al citosol al despolimerizar la actina. Diferentes ensayos bioquímicos nos sirvieron para confirmar que la despolimerización de actina inducía la disociación de los dominios V1 y V0 de la V-ATPasa. Además, detectamos interacción entre la actina y las subunidades B y C. Finalmente, está descrito que la V-ATPasa se localiza en los dominios ricos en colesterol de la membrana plasmática y que el citoesqueleto de actina juega un papel importante en la organización de estos dominios, lo que observamos fue que la desorganización de los dominios ricos en colesterol inducía una subida del pH de Golgi.
Con todo, concluimos que la actina regula el pH de Golgi a través del mantenimiento de la asociación de los dos dominios de la ATPasa gracias a su unión a las subunidades B y C además de su papel en el mantenimiento de los dominios ricos en colesterol.We previously reported that agents that depolymerize actin filaments promote the alkalization of the Golgi stack and the trans-Golgi network. Vacuolar-type H-translocating ATPase (V-ATPase) is responsible of proton translocation and acidification of Golgi lumen. V-ATPase is a multisubunit complex composed of two domains (V1 and V0). Moreover, two subunits of V1 domain contain actin binding sides, subunit B and C. In this work we hypothesize that actin filaments could have a role in the maintaining of V1 and V0 domain association. We have generated a GFPtagged subunit B2 construct that is incorporated into the V1 domain, this construct localizes at distal Golgi compartments and translocate to cytosol upon actin depolymerization. Several biochemical assays confirmed that microfilaments distruption induces dissociation of V1-V0 domains. Moreover, we detected interaction between subunits B-C and actin filaments. Finally, V-ATPase is localized in lipid raft domains of plasma membrane and actin filaments participate in organization of these domains. We observed that lipid raft disorganization promotes an increase of intra-Golgi pH. Overall, we conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0 domains of V-ATPase through the binding of microfilaments to subunits B and C and preserving the integrity of lipid raft.
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Ross, Kyla Turpin. "Quantitative Analysis of Feedback During Locomotion." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/14110.
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It is known that muscles possess both intrinsic and reflexive responses to stretch, both of which have been studied extensively. While much is known about heterogenic and autogenic reflexes during XER, these have not been well characterized during locomotion. In this study, we mapped the distribution of autogenic and heterogenic feedback in hindlimb extensor muscles using muscle stretch in the spontaneously locomoting premammillary decerebrate cat. We used natural stimulation and compared stretch-evoked force responses obtained during locomotion with those obtained during XER. The goal was to ascertain whether feedback was modulated between the two states. We found that heterogenic feedback pathways, particularly those emanating from MG, remained inhibitory during locomotion while autogenic feedback specifically in MG increases in gain. Furthermore, increases in MG gain were due to force-dependent mechanisms. This suggests that rather than an abrupt transition from inhibition to excitation with changes in motor tasks, these pathways coexist and contribute to maintaining interjoint coordination. Increases in autogenic gain provide a localized loading reflex to contribute to the completion of the movement. The results of these experiments are clinically significant, particularly for the rehabilitation of spinal cord injured patients. To effectively administer treatment and therapy for patients with compromised spinal reflexes, a complete understanding of the circuitry is required.
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Marchini, Claudia Maria Meirelles. "O papel funcional da enzima fosfolipase D2 (PLD2) nas células da linhagem de mastócitos RBL-2H3." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-04122008-073156/.
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Os mastócitos participam do sistema imunológico liberando mediadores farmacologicamente ativos. A principal via de ativação dos mastócitos é através do receptor de alta afinidade para a imunoglobulina E (FcRI). A ativação dos mastócitos via FcRI culmina com a liberação de mediadores. A enzima PLD atua sobre fosfolipídios hidrolisando a fosfatidilcolina em ácido fosfatídico e colina. A PLD é ativada após o estímulo via FcRI e possui um papel importante na transdução do sinal em mastócitos. Existem duas isoformas da enzima PLD, a PLD1 e a PLD2 que são expressas, diferentemente, de acordo com o tipo celular. Ambas as isoformas podem estar expressas numa mesma célula, apenas uma ou nenhuma. Neste estudo foram utilizadas células RBL-2H3 transfectadas para a super expressão PLD2 nas formas catalítica ativa (CA) e inativa (CI). O papel da PLD2 foi examinado nestas células com o objetivo de elucidar sua atuação no processo de secreção incluindo o aparelho de Golgi e os grânulos secretores. As células CA e CI possuem maior atividavidade de -hexosaminidase total, porém quando estimuladas mostram uma deficiência na liberação desta enzima, quando comparadas com as células selvagens. A PLD2 nas células CA, CI, VET e RBL-2H3 está localizada no citosol, sendo abundante na região justanuclear, principalmente nas células CI, sugerindo uma associação com o aparelho de Golgi. A dupla marcação com o mAb AA4, que imunomarca gangliosídeos derivados do GD1b da membrana plasmática e com anti-PLD2, mostrou que esta enzima não se localiza na membrana plasmática. A dupla marcação com anti-PLD2 e anti-GM130 mostrou que as áreas de maior concentração da PLD2 se co-localizam com o aparelho de Golgi, especialmente nas células CI. A marcação com anti-GM130 e os experimentos com microscopia eletrônica de transmissão mostraram que o aparelho de Golgi está organizado nas células CA e desorganizado nas células CI, onde se encontra disperso no citoplasma. Ainda, as células CI expressam menos GM130 em comparação com as demais linhagens celulares. Quando a produção de PA pela PLD está inibida pelo 1-Butanol, as células CA apresentam as mesmas características fenotípicas das células CI. A incubação das CI com PA resulta na reestruturação do aparelho de Golgi. A manutenção estrutural do aparelho de Golgi, também está relacionada com os microtúbulos. Nas células CI o centro organizador de microtúbulos é dificilmente identificado. Os microtúbulos nas células CI são desordenados em comparação com as demais linhagens celulares. Estes resultados mostram que a produção de PA pela PLD2 é importante na organização de microtúbulos e na manutenção da estrutura do aparelho de Golgi. As alterações celulares relacionadas com os microtúbulos e o aparelho de Golgi afetam o processo secretor nestas células e, provavelmente, em outros tipos de células secretoras. Estes achados poderão levar a novas estratégias terapêuticas para controlar a liberação de mediadores durante processos alérgicos e inflamatórios.Mast cells are components of the immune system that liberate a wide variety of pharmacologically active mediators. The principle method of activating mast cells is through the high affinity receptor for IgE (FcRI). This activation then culminates with the release of mediators. Phospholipase D (PLD) acts on phospholipids, hydrolyzing phosphatidylcholine to phosphatidic acid (PA) and choline. PLD is activated following stimulation via FcRI and plays an important role in signal transduction in mast cells. PLD has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type where none, one or both may be expressed. RBL-2H3 cells, a mast cell line, transfected to super express catalytically active (CA) and inactive (CI) forms of PLD2 were used in the present study. The role of PLD2 was examined in these cells in order to clarify the action of PLD2 in the secretory process. Although the CA and CI cells posses a greater total -hexosaminidase activity, when stimulated these cells release less -hexosaminidase than cells transfected with empty vector or wild type RBL-2H3 cells. In all cell lines, PLD2 was dispersed throughout the cytoplasm with a concentration in the juxtanuclear region suggesting an association of PLD2 with the Golgi apparatus. Double labeling with anti-PLD2 and mAb AA4, which recognizes gangliosides derived from GD1b on the plasma membrane, showed that PLD2 was not associated with the plasma membrane. When the cells were double labeled with anti-PLD2 and anti-GM130, which labels the cis-Golgi saccules, PLD2 does colocalize with the Golgi apparatus, especially in CI cells. Labeling with anti-GM130 alone as well as experiments employing transmission electron microscopy revealed that the Golgi apparatus is well organized in the CA cells, but is disorganized and dispersed in the cytoplasm in the CI cells. By Western Blotting, the CI cells also expressed less GM130 than the other cell lines. When the production of PA by PLD2 was inhibited by 1-Butanol, the Golgi apparatus of the CA cells presented the same phenotypic characteristics as that of the CI cells. Conversely, incubation of the CI cells with PA resulted in the reorganization of the Golgi apparatus. The structural maintenance of the Golgi apparatus is also related to microtubules. In the CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized in the cytoplasm as compared to the other cell lines. These results show that the production of PA by PLD2 is important in the arrangement of the microtubules and in maintaining the structure of the Golgi apparatus. Alterations in the distribution of the microtubules and the structure of the Golgi apparatus in the CI cells affect the secretory process in these cells, and such alterations may affect the secretory process in other cell types as well. The findings presented here may lead to new therapeutic strategies to control the production and release of mediators during allergic and inflammatory processes.
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Lebon, Sophie. "Implication de la DYMECLINE et de GRASP65 dans les golgipathies neurodéveloppementales." Electronic Thesis or Diss., Université Paris Cité, 2024. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=5768&f=73915.
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Mon doctorat porte sur l'étude de deux maladies génétiques récessives qui touchent le développement cérébral postnatal et résultent de variants dans des gènes impliquant l'appareil de Golgi. La première maladie étudiée, le syndrome de Dyggve-Melchior-Clausen (DMC) a conduit à impliquer en 2003 la première protéine golgienne, la DYMECLINE, dans une microcéphalie postnatale, et a ensuite mené au concept de Golgipathies neurodéveloppementales. Les atteintes neurodéveloppementales du DMC sont associées à une déficience intellectuelle et à des anomalies squelettiques spécifiques également postnatales (dysplasie spondylo-epi-métaphysaire) et résultent de la déficience du gène DYM codant pour la DYMECLINE dont l'absence induit un défaut de transport antérograde entre le réticulum endoplasmique et l'appareil de Golgi notamment dans les neurones. Un variant clinique du DMC sans microcéphalie ni déficience intellectuelle, mais dont les caractéristiques osseuses sont identiques, la dysplasie de Smith McCort (SMC), peut résulter soit de variants moins délétères du gène DYM (SMC1) soit de variants du gène codant la RAB-GTPase RAB33B (SMC2) suggérant une relation entre les deux protéines. Dans ce travail, j'ai montré que ces deux protéines co-localisent au cis-Golgi, interagissent physiquement, que la DYMECLINE est recrutée au Golgi par RAB33B et intervient dans le contrôle de l'autophagie dans des cellules non neuronales. Dans les neurones déficients pour Dym, les deux protéines sont en revanche faiblement co-localisées et l'autophagie n'est pas perturbée, mais des défauts de transport rétrograde de la membrane plasmique vers le Golgi ont été identifiés, associés à des anomalies de croissance dendritique et des défauts de maturation synaptique. La seconde partie de ma thèse porte sur l'identification d'un variant biallélique du gène GORASP1 chez un patient présentant un nouveau syndrome neurodéveloppemental caractérisé par des atteintes de la substance blanche, neurosensorielles, neuromusculaires et squelettiques. GORASP1 code pour la protéine golgienne GRASP65, connue pour son rôle dans la structure du Golgi, dans la glycosylation des protéines et dans le contrôle de l'entrée en mitose. Malgré ces fonctions apparemment essentielles et ubiquitaires, le gène n'a été impliqué dans aucune pathologie humaine. A partir à la fois des fibroblastes du patient et de cellules RPE où j'ai introduit par CRISPR/Cas9 une mutation imitant le variant du patient, j'ai montré que la protéine GRASP65 n'est plus présente dans les cellules mutées et j'ai identifié dans ces cellules des défauts de glycosylation et de mitose. Ces défauts n'empêchent cependant pas les cellules RPE de proliférer normalement. En étudiant un autre mutant généré par hasard dans les cellules RPE et qui s'est avéré produire une protéine tronquée en C-terminal mais stable, j'ai observé un phénotype de croissance cellulaire plus sévère que lorsque la protéine est totalement absente, suggérant des effets dominants négatifs de la protéine tronquée. En revanche ce mutant stable n'a pas montré de défauts de glycosylation. Cette étude a permis d'impliquer GRASP65 dans une maladie neurodéveloppementale et suggère qu'une absence totale de protéine est parfois moins délétère qu'une protéine tronquée stableMy PhD focuses on the study of two recessive genetic diseases that affect postnatal brain development and result from variants in genes involving the Golgi apparatus. The first disease studied, Dyggve-Melchior-Clausen syndrome (DMC), led in 2003 to the involvement of the first Golgi protein, DYMECLIN, in postnatal microcephaly, and subsequently to the concept of neurodevelopmental Golgipathies. In DMC, neurodevelopmental impairments are associated with intellectual disability and specific skeletal defects, of postnatal onset as well (spondylo-epi-metaphyseal dysplasia) and result from a deficiency in the DYM gene encoding DYMECLIN, this absence of which induces a defect in anterograde transport between the endoplasmic reticulum and the Golgi apparatus, notably in neurons. Smith McCort dysplasia (SMC), a clinical variant of DMC without microcephaly or intellectual deficiency, but with identical skeletal features, can result either from less deleterious variants of the DYM gene (SMC1) or from variants of the gene encoding the RAB-GTPase RAB33B (SMC2), suggesting a relationship between the two proteins. In this work, I have shown that these two proteins co-localize at the cis-Golgi, interact physically, that DYMECLIN is recruited to the Golgi by RAB33B and is involved in the control of autophagy in non-neuronal cells. However, in Dym-deficient neurons, the two proteins are weakly co-localized and autophagy is not disrupted, but defects in retrograde transport from the plasma membrane to the Golgi have been identified, associated with abnormalities in dendritic growth and defects in synaptic maturation. The second part of my thesis concerns the identification of a biallelic variant in the GORASP1 gene in a patient with a new neurodevelopmental syndrome characterized by white matter, neurosensory, neuromuscular and skeletal abnormalities. GORASP1 encodes the Golgi protein GRASP65, known for its role in Golgi structure, protein glycosylation and control of mitosis entry. Despite these apparently essential and ubiquitous functions, the gene has not been implicated in any human pathology so far. Using both patient's fibroblasts and RPE cells in which I introduced by CRISPR/Cas9 a mutation mimicking the patient's variant, I showed that the GRASP65 protein is no longer present in mutated cells, and identified glycosylation and mitosis defects in these cells. However, these defects do not prevent RPE cells from proliferating normally. Studying another mutant generated incidentally in RPE cells, which turned out to produce a C-terminally truncated but stable protein, I observed a more severe cell growth phenotype than when the protein is totally absent, suggesting dominant negative effects of the truncated protein. In contrast, this stable mutant showed no glycosylation defects. This study implicates GRASP65 in a neurodevelopmental disease and suggests that a total absence of the protein is sometimes less deleterious than a stable truncated protein
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Marais, Claire-Line. "Rôle de la SNARE Memb11 comme « récepteur » de la GTPase Arf1 à l’appareil de Golgi chez Arabidopsis thaliana." Thesis, Bordeaux 2, 2013. http://www.theses.fr/2013BOR22105/document.
Full textAbstract:
Les protéines SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) sont essentielles pour la fusion membranaire. J'ai étudié chez Arabidopsis thaliana la SNARE Memb11 de l’appareil de Golgi qui intervient au début de la voie sécrétoire à l'interface Réticulum endoplasmique (RE)-appareil de Golgi. Dans les cellules de mammifères, l'orthologue de Memb11 (Membrine) est un « récepteur » potentiel de la GTPase Arf1 à la membrane golgienne. Cette dernière est impliquée dans le recrutement de la machinerie COPI nécessaire au transport rétrograde de l'appareil de Golgi vers le RE. Le but de ce travail était de déterminer si Memb11 pouvait interagir avec Arf1 dans les cellules végétales. Des anticorps dirigés contre la partie cytosolique de Memb11 ont été obtenus et ont été utilisés sur tissus végétaux pour réaliser des immunomarquages en microscopie électronique à transmission et des immunoprécipitations sur extraits de plantes. Il a été démontré que Memb11 est située au niveau de la membrane cis-golgienne et qu'elle co-immunoprécipite avec Arf1, suggérant ainsi que Arf1 peut interagir avec Memb11. J'ai confirmé l'interaction de Memb11 et Arf1 au niveau de l'appareil de Golgi par des expériences de BiFC (Bimolecular Fluorescence Complementation) in vivo. Cette interaction est spécifique puisque ni Memb12 (90% d'identité avec Memb11) ni Sec22 interagissent avec Arf1. Grâce à une approche de bioinformatique structurale, j'ai déterminé les régions de Memb11 (différentes de Memb12) qui pourraient être critiques pour l'interaction et j’ai commencé à tester in vivo les mutants correspondants par BiFC. En outre, des expériences d’immunoprécipitations avec des protéines recombinantes produites in vitro suggèrent que la forme d’Arf1 liée au GTP interagit avec Memb11The SNARE proteins (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) are critical for membrane fusion in the secretory pathway. I have studied the Golgi SNARE Memb11 in Arabidopsis thaliana cells. Memb11 is involved at the ER-Golgi interface. In mammalian cells, the ortholog of Memb11 (Membrin) is the potential “receptor” of the GTPase Arf1 in the Golgi membrane. This protein is involved for the recruitment of the COPI machinery, required for retrograde transport from the Golgi to the ER. The aim of this work was to determine whether Memb11 can interact with Arf1 in plant cells. Antibodies against the cytosolic part of Memb11 were obtained and were applied on plant tissues to perform immunolabeling by transmission electron microscopy and immunoprecipitation (IP) studies. It has been shown that Memb11 is located at the cis-Golgi and that it co-immunoprecipated with Arf1, suggesting that Arf1 may interact with Memb11. I confirmed the interaction of Memb11 and Arf1 at the Golgi by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was specific since neither Memb12 (90% identity with Memb11) nor Sec22 interacted with ARF1. Thanks to a structural bioinformatic approach, I determined the regions in Memb11 (different from Memb12) that could be critical for the interaction and started to test corresponding mutants in vivo by BiFC. In addition, IP experiments with recombinant proteins produced in vitro suggest that the GTP-bound form of ARF1 interacts with Memb11