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Haglund, Kaisa. "Ubiquitination and Receptor Endocytosis." Doctoral thesis, Uppsala University, Ludwig Institute for Cancer Research, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4259.
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<p>Protein ubiquitination is an evolutionary conserved mechanism that controls a wide variety of cellular functions. Polyubiquitinated proteins are generally degraded in the proteasome, whereas monoubiquitination controls various other cellular processes, including endocytosis and endosomal sorting.</p><p>Termination of signaling by activated receptor tyrosine kinases (RTKs) largely occurs via their endocytosis and subsequent lysosomal degradation, processes accompanied by receptor ubiquitination. Cbl family proteins are major ubiquitin ligases that promote RTK ubiquitination and downregulation. We showed that epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors are monoubiquitinated at multiple sites following their ligand-induced activation and that a single ubiquitin is sufficient for both receptor internalization and degradation. Cbl also controls EGF receptor (EGFR) downregulation by binding to CIN85, which recruits endophilins to EGFR/Cbl complexes. In the complex with activated EGFRs, Cbl directs monoubiquitination of CIN85, and the entire complex is targeted for degradation in the lysosome. We propose that multiple monoubiquitination of activated receptors and associated protein complexes ensures proper receptor sorting towards the lysosome. Importantly, the functions of Cbl are also negatively controlled in order to maintain cellular homestasis. Sprouty2 blocks EGFR downregulation by sequestering Cbl from activated EGFRs. We showed that Sprouty2 also associates with CIN85 and that this binding is required for efficient inhibition of EGFR ubiquitination and endocytosis. </p><p>Cbl is also implicated in other aspects of RTK signaling, including organization of the actin cytoskeleton. We found that growth factor receptor signals promote lamellipodia formation in neuronal cells via a complex containing Cbl, the adaptor protein ArgBP2 and Pyk2. The lamellipodia formation required intact lipid rafts and the recruitment of Crk and PI(3)K to tyrosine phosphorylated Cbl.</p><p>In conclusion, our findings contribute to a better understanding of monoubiquitin signals in downregulation of RTKs and point at a role of Cbl in the regulation of cytoskeleton dynamics.</p>
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Webster, Paul. "Endocytosis by African trypanosomes." Thesis, Brunel University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280722.
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Kalkman, Edward R. I. C. "Endocytosis in filamentous fungi." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/1970.
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Endocytosis is little understood in filamentous fungi. For some time it has been controversial as to whether endocytosis occurs in filamentous fungi. A comparative genomics analysis between Saccharomyces cerevisiae and 10 genomes of filamentous fungal species showed that filamentous fungi possess complex endocytic machineries. The use of the endocytic marker dye FM4-64, and various vesicle trafficking inhibitors revealed many similarities between endocytosis in the filamentous fungus Neurospora crassa, and endocytosis in budding yeast and mammalian cells. Actin polymerization was found to be crucial for endocytosis in N. crassa, and the microtubule cytoskeleton seemed to be necessary for long distance movement of putative early endosomes. Brefeldin A (BFA) blocked vesicular transport to the Spitzenkörper. Three putative endocytic proteins (WASP, clathrin light chain and Rab5) were labelled with fluorescent proteins in N. crassa. WASP-GFP was found to localise to motile, punctate structures in the plasma membrane just behind the hyphal apex in growing hyphae. This localisation changed to the hyphal apex when growth was temporarily arrested, indicating a possible role in endocytosis and polarized growth. Clathrin light chain-GFP was found to be concentrated in a region just behind the Spitzenkörper, which is consistent with there being a high concentration of clathrinmediated endocytosis in this region. Clathrin light chain-GFP also labelled putative Golgi and this labelling was found to be BFA sensitive, whereas BFA did not have a detectable effect on FM4-64 internalisation and organelle staining. GFP-Rab5 labelled putative early endosomes and decorated microtubules. Knock-outs of putative endocytic proteins in N. crassa, generated as part of the Neurospora genome consortium gene knock-out project, were analysed for defects in endocytosis. 14 out of 17 gene knock-outs were found to be ascospore lethal. The Rab5 knock-out was viable, but did not show a detectable effect on the endocytic internalisation of FM4-64 or its pattern of staining. However, it did exhibit a defect in sexual crossing.
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Gagliardi, M. "Endocytosis and wingless signalling." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1353109/.
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Wingless (Drosophila Wnt-1) is secreted glycoprotein that triggers an evolutionary conserved signal transduction pathway. The role of endocytosis in Wnt/Wingless signaling is not clearly understood and highly debated. In my thesis I explore the role that endocytosis/endocytic trafficking has on Wingless signalling activation and termination. In the canonical pathway Wingless binds to a member of the Frizzled family of seven-pass transmembrane receptor (Frizzled1 or Frizzled2) and to Arrow. Formation of this trimeric complex leads to the inactivation of the Armadillo degradation complex and translocation of Armadillo into the nucleus where it contributes to the activation of target genes. I show that internalization of the ligand-receptor complex is not required for signalling activation. I also show that Wingless has different effects on the trafficking route of its receptors: it induces the degradation of Frizzled2 and the recycling of Arrow. To identify post translational modifications that regulate Arrow trafficking I conducted an RNAi screen in Drosophila S2R+ cells for de-ubiquitylating enzymes (DUBs) and ubiquitin conjugating enzymes (E2) that modulate signalling. To carry out this screen, improvements on the current TOPFlash Wnt/Wingless signalling reporter were made. I also directly assessed the role of endocytic trafficking on signalling using a chemical inhibitor of endocytosis, Dynasore. I find that Dynasore inhibits signalling by causing a strong decrease in Armadillo levels. Future experiments will determine whether it is the stability or the rate of production of Armadillo that is affected.
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Rehman, Michael. "Analysis of endocytosis at eisosomes." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-135951.
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Jha, Ankita. "Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0393/document.
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Au cours de la gastrulation de l’embryon de Drosophile, l’activation apicale du cytosquelette d’acto-myosine orchestre la constriction apicale dans le mésoderme en invagination ainsi que l’intercalation cellulaire dans l’ectoderme en extension. Un contrôle quantitatif de l’activité des GPCRs et, par conséquent, de l’activation de Rho1 est à l’origine des différences de déformation des cellules du mésoderme et de l’ectoderme mais ces mécanismes demeurent incompris. L’activité du GPCR Smog se concentre respectivement en deux compartiments distincts à la surface de la membrane plasmique (PM) et dans ses invaginations (PMI). Au moyen de la FCS, nous avons étudié la surface de la PM et pu montrer que Smog oligomérise en homo-clusters en réponse à son activation par le ligand Fog. L’endocytose de Smog est facilitée par la kinase Gprk2 et sa protéine adaptatrice la β-Arrestine-2 qui retire Smog actif de la PM. Lorsque que la concentration de Fog est élevée ou que l’endocytose est réduite, Smog s’organise en homo-clusters et s’accumule au niveau des PMI qui agissent comme des centres d’activation de Rho1. Une concentration plus importante d’homo-clusters de Smog et un nombre plus important PMI dans le mésoderme par comparaison avec l’ectoderme. Répartition dynamique de Smog actif à la surface de la PM ou dans ses invaginations impacte directement sur la signalisation Rho1. Les PMI accumulent de hauts niveaux de Rho1-GTP suggérant qu’elles forment des centres de signalisation. La concentration de Fog et l’endocytose de Smog sont des processus régulateurs couplés qui contrôlent la différence quantitative d’activation de Rho1 dans le mésoderme et l’ectoderme de la Drosophile<br>During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Here, we show that cell-surface G-protein coupled receptor, Smog activates G-proteins, Rho1 and Rho-kinase that is required for apical constriction and cell-cell intercalation. Quantitative control over GPCR activity and thereby Rho1 activation underlies differences in deformation of the mesoderm and ectoderm cells but the mechanisms remain elusive. We show that GPCR-Smog activity is concentrated on two different apical plasma membrane compartments i.e. the surface and the plasma membrane invaginations. Using FCS, we probe the surface of the plasma membrane (PM) and show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is facilitated by the kinase Gprk2 and the adaptor protein β-Arrestin-2 that clears active Smog from the surface of PM. When Fog concentration is high or endocytosis is low, Smog arranges in homo-clusters and accumulates in plasma membrane invaginations (PMI), that are hubs for Rho1 activation. Lastly, we find high Smog homo-cluster concentrations and numerous apical PMIs in the mesoderm compared to the ectoderm. We identify that dynamic partitioning of active Smog on the surface of the PM or PMI directly impact on Rho1 signaling. PMIs accumulate high Rho1-GTP suggesting they form signaling centers. Fog concentration and Smog endocytosis form coupled regulatory processes that regulate quantitative differential Rho1/MyoII activation in the Drosophila mesoderm and ectoderm
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Shurety, Wenda. "Apical endocytosis in Caco-2 cells." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242912.
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Karagiannis, Sophia. "The process of endocytosis of CD23." Thesis, King's College London (University of London), 1995. https://kclpure.kcl.ac.uk/portal/en/theses/the-process-of-endocytosis-of-cd23(553202e7-a9c8-444e-b0a5-f7561d1e6297).html.
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Nichols, II James Tucker. "DSL-ligand endocytosis and notch signaling." Diss., Restricted to subscribing institutions, 2008. http://proquest.umi.com/pqdweb?did=1692099791&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Wilbur, Jeremy D. "Conformational switches regulate clathrin mediated endocytosis." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324583.
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Ohlwein, Nina. "Endocytosis controlled by monolayer area asymmetry." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16403.
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Endozytose erfordert hohe Membrankrümmung und führt zu Flächenänderungen der Membranhälften. Dies kann durch eine Oberflächendifferenz zwischen den Schichten initiiert werden, die durch geänderte Lipidzusammensetzungen hervorgerufen werden kann. Daher wurde die Hypothese aufgestellt, dass Lipid-Transporter zu Beginn der Endozytose für veränderte Flächenverhältnisse verantwortlich sind. Um den Einfluss veränderter Flächen auf Endozytose zu untersuchen, wurden die Oberflächenverhältnisse der Membran durch Zugabe von Phospholipiden verändert und anschließend Endozytose gemessen. Abhängig von der Sorte wurden die Lipide nur in die äußere Schicht eingebaut oder auch auf die innere Seite transportiert, wodurch die entsprechende Seite vergrößert wurde. Die Zugabe verschiedener Aminophospholipide, die auf die innere Membranseite transportiert werden, führte zu gesteigerter „bulk flow“ Endocytose in K562-Zellen. Darüber hinaus deuten die Ergebnisse darauf hin, dass Clathrin-vermittelte Endozytose von Hep2-Zellen ebenfalls stimuliert wurde. Umgekehrt hatte die Zugabe von Lipiden, die auf der äußeren Hälfte bleiben, reduzierte „bulk flow“- oder Clathrin-vermittelte Endozytose in verschiedenen Zelllinien zur Folge. Bemerkenswert ist, dass auch Clathrin-vermittelte Endozytose durch die Lipidzugabe beeinflusst wurde, obwohl gerade in diesem Weg viele Proteine involviert sind, die Krümmung induzieren können. Dies passt zu einem neuen Modell wie Lipidtransporter in Endozytose involviert sind. Durch den Transport von Lipiden und die zusätzliche Interaktion mit Endozytoseproteinen, könnten diese Transporter zwei Mechanismen zur Erzeugung von Krümmung miteinander verbinden: Membrankrümmung induziert durch eine Flächenasymmetrie zwischen den Membranhälften und durch Wechselwirkung mit Proteinen. Die Ergebnisse dieser Arbeit deuten darauf hin, dass die für Endozytose notwendige Krümmung durch die durch Lipidtransport induzierte Flächenasymmetrie der Membranschichten unterstützt wird.<br>Endocytic engulfment requires high local membrane curvature and causes significant area changes of the membrane leaflets. This can be initiated by differences between the surface areas of the two monolayers related to leaflet specific modulation of lipid composition. Thus, it was proposed that lipid translocators, pumping phospholipids from the outer to the inner leaflet, account for monolayer area asymmetry as an early step in endocytosis. To elucidate the influence of this asymmetry on endocytosis, surface area relation was altered by adding exogenous phospholipids to living cells and changes in endocytic activity were quantified. Depending on the lipid species, exogenous lipids were only incorporated into the outer layer or subsequently translocated across the plasma membrane thereby increasing either the outer or inner surface area. Addition of different analogues of aminophospholipids, which are translocated to the inner leaflet, led to an enhancement of bulk flow endocytosis in K562 cells. Moreover, our data indicate that clathrin-mediated endocytosis of Hep2 cells was stimulated as well. Inversely, addition of phospholipids, which remain on the outer layer, reduced bulk flow or clathrin-mediated endocytosis in various cell lines. Notably, also clathrin-mediated endocytosis was influenced by the addition of lipids, although many proteins noted for their ability to induce membrane curvature are known to be implicated in this pathway. This corroborates a recent model how aminophospholipid translocases are implicated in endocytosis. Upon translocating lipids and additionally interacting with endocytic accessory proteins, lipid translocators could integrate two processes to generate curvature: membrane bending based on monolayer area asymmetry and protein-related mechanisms. Collectively, findings in the present study suggest that curvature generation in endocytosis is supported by the induction of monolayer area asymmetry mediated by the translocation of lipids.
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Ivashenka, Alena. "GL-Lect Endocytosis in In-Vivo Model Systems." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS420.
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Une multitude de voies endocytiques existent à la surface de la membrane plasmique, ce qui conduit à l'endocytose de la majeure partie des membranes ainsi que de leurs protéines associées, des molécules de signalisation, des facteurs de croissance et autres cargos (Smith et al. 2017). Pendant des décennies, la voie majeure dite clathrine dépendante a été la plus étudiée, Cette voie d’endocytose se caractérise par la polymérisation de molécules de clathrine et de protéines adaptatrices au niveau de récepteurs liés à leurs ligands, entrainant la courbure de la membrane, avant sa scission et son endocytose (Smith et al. 2017). Récemment, plusieurs mécanismes alternatifs ont été découverts facilitant l'absorption endocytique des protéines cargo et des récepteurs membranaires, même en l'absence de la voie clathrine dépendante (Mayor et al.2014). Un modèle d'endocytose qui ne requiert pas de clathrine mais à la place des galectines et des glycolipides a été proposé par le groupe de Ludger Johannes (Lakshminarayan et al. 2014). Les galectines sont des lectines qui se lient aux bêta-galactosides et qui, à ce jour, regroupent 15 membres (galectine-1 à galectine-15) chez les mammifères et sont retrouvées dans de nombreux types de cellules et tissus (Leffler et al., 2004). Les galectines sont probablement sécrétées extra-cellulairement par une voie inconnue et non-classique (Hughes, 1999). Les glycosphingolipides (GSL) sont des constituants membranaires ubiquitaires, divisés en fractions neutres ou acides. Le terme GSL s'applique aux composés contenant au moins un monosaccharide et une céramide. Il est à noter que l’enzyme UDP-glucose céramide glucosyltransférase (Ugcg) catalyse l’étape initiale de la biosynthèse de GSLs à base de glycosylcéramides.Notre modèle de travail actuel, impliquant les glycolipides et les lectines, a été qualifié d'hypothèse GL-Lect (Johannes et al. 2016), et préliminairement soutenu par des données expérimentales comme décrit par Lakshminarayan et al. en 2014. Il peut être décrit comme suit:i) le monomère Gal3 se lie aux glycoprotéinesii) Gal3 commence alors à oligomériseriii) Gal3 oligomérisé a la capacité de se lier aux glycosphingolipides, ce qui peut induire la formation de clusters de GSLsiv). Ces clusters Gal3-GSL induisent une invagination de la membrane plasmique, une endocytose des protéines cargo liées à Gal3 et la formation subséquente de CLIC (clathrin-independent carriers ), endosomes pré-précoces.Selon ce modèle, l’oligomère Gal3 est capable de se lier aux GSLs et d’induire une déformation de la membrane de la même manière qu’une autre lectine, la sous-unité pathogène shiga toxine-B (STxB). Par conséquent, les deux processus pourraient être résumés sous la même hypothèse GL-Lect, où GL représente les glycosphingolipides (Gb3 pour STxB et des GSLs non identifiés pour Gal3) et Lect corresponds aux lectines (STxB, Gal3 et éventuellement d'autres). Comprendre si les voies d'internalisation indépendantes de la clathrine, mais dépendantes de la galectine 3, sont conservées, non seulement pour les modèles in vitro mais également in vivo, est un défi majeur dans le domaine du trafic cellulaire.Nous avons caractérisé, pour la première fois, un nouveau mécanisme dans l’intestin facilitant le transport endocytique d’un cargo. Ce mécanisme est conduit par la Galectine 3 et agit dans les entérocytes intestinaux dans le processus analogue de transcytose et dépend des glycosphingolipides. En effet, nous avons découvert que la lactotransferrine (LTF), un cargo de Gal3 que nous avons identifié par Mass spec, dépendait fortement de la Galectine3 pour son endocytose efficace, et des GSLs pour son mode de distribution analogue à la transcytose. Sur la base de ces découvertes dans l'épithélium intestinal de souris, nous avons établi un système modèle in vivo fonctionnel dans lequel le mécanisme endocytique récemment proposé, appelé dans notre laboratoire GL-Lect, a été étudié physiologiquement<br>A host of endocytic pathways exist at the surface of eukaryotic cells, which lead to the internalization of the bulk of membranes along with membrane proteins, signaling receptors, growth factors, and other cargoes (Smith et al. 2017). For decades, the clathrin-mediated pathway has been the major well characterized endocytic process where clathrin polymerizes along with the associated adaptor proteins to include ligand-bound receptors, leading to membrane bending, membrane scission, and endocytosis (Smith et al. 2017). Recently, multiple alternative mechanisms have been uncovered which facilitate the endocytic uptake of cargo molecules and membrane receptors even in the absence of clathrin machinery (Mayor et al.2014). A model of endocytosis that doesn’t require clathrin but rather sugar-binding galectins and glycolipids has been proposed by my host laboratory (Lakshminarayan et al. 2014). Galectins constitute a family of beta-galactoside–binding lectins, which to date consists of 15 members in mammals. Galectins are broadly distributed in a variety of cells and tissues (Leffler et al. 2004). They are translocated from the cytosol to the extracellular space by a process of non-classical secretion (Hughes 1999). Glycosphingolipids (GSLs) are ubiquitous membrane constituents that are subdivided in neutral or acidic fractions. The term GSLs applies to compounds that contain at least one monosaccharide and a ceramide. Of note, the enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step for the biosynthesis of glycosylceramide-based GSLs.Our current working model, which involves glycolipids and lectins, was termed the GL-Lect hypothesis (Johannes et al. 2016). It is backed up by experimental data as described in Ref. (Lakshminarayan et al. 2014) and can be described as follows:i) Monomeric Gal3 binds to glycoproteinsii) Gal3 then starts to oligomerizeiii) Oligomerized Gal3 has the capacity to bind to glycosphingolipids and this may induce clustering of GSLsiv) Gal3-GSL cluster are inducing the invagination of the plasma membrane to generate tubular endocytic pits from which clathrin-independent carriers (CLICs, which are pre-early endosomes) are generated.Oligomeric Gal3 is indeed able to bind to GSLs and to induce membrane deformation (Lakshminarayan et al. 2014) in a similar way the pathogenic lectin Shiga toxin-B subunit (STxB) does. Therefore, both processes could be summarized under the same hypothesis, the GL-Lect hypothesis, where GL stands for the glycosphingolipids (Gb3 for STxB and gangliosides for Gal3) and Lect summarizes the lectins (STxB, Gal3 and possibly others as well).Understanding if this clathrin-independent but Gal3-dependent internalization mechanism is conserved not only in vitro model systems but in vivo is a main challenge in the field of trafficking.We characterized for the first time that in the gut a new mechanism facilitates endocytic uptake of cargo. This mechanism is driven by Galectin3 and operates in intestinal enterocytes for transcytosis like process and is glycosphingolipid dependent. Indeed, we have found that the lactotransferrin (LTF), a Gal3 cargo that we have identified by Mass spec, strongly required Gal3 and GSLs for its efficient endocytosis and its transcytosis like distribution pattern, respectively. Based on these findings in mouse intestinal epithelium, we established a functional in vivo model system where the newly proposed endocytic mechanism termed in our lab as GL-Lect, was physiologically investigated
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Kowanetz, Katarzyna. "Adaptor Proteins in Regulation of Receptor Endocytosis." Doctoral thesis, Uppsala University, Ludwig Institute for Cancer Research, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4477.
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<p>Ligand-induced endocytosis of receptor tyrosine kinases (RTKs) is a dynamic process governed by numerous protein-protein and protein-lipid interactions. This is a major mechanism of signal termination and is also frequently impaired in cancer. The Cbl family of ubiquitin ligases has been shown to play a key role in downregulation of RTKs, by directing their ligand-induced ubiquitination and subsequent lysosomal degradation. My thesis work has led to the identification of novel, ubiquitin-ligase independent, functions of Cbl in receptor endocytosis. We demonstrated that the adaptor protein CIN85 links Cbl with epidermal growth factor receptor (EGFR) internalization. The three SH3 domains of CIN85 interact with Cbl/Cbl-b in a phosphotyrosine dependent manner, whereas its proline-rich region constitutively binds endophilins, known regulators of plasma membrane invagination. The SH3 domains of CIN85 recognize an atypical proline-arginine (PxxxPR) motif present in Cbl and Cbl-b. Moreover, we showed that numerous endocytic regulatory proteins, among them ASAP1 and Dab2, interact with CIN85 via their PxxxPR motifs. The SH3 domains of CIN85 are able to cluster and exchange its effectors at subsequent stages of EGFR endocytosis, thus participating in the control of receptor internalization, recycling and degradation in the lysosome. We proposed that CIN85 functions as a scaffold molecule implicated in control of multiple steps in downregulation of RTKs. </p><p>Furthermore, we identified two novel Cbl- and ubiquitin-interacting adaptor proteins named Sts-1 and Sts-2 (Suppressors of T-cell receptor signaling). Ligand-induced and Cbl-mediated recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization and subsequent block of receptor degradation followed by prolonged mitogenic signaling pathways. Our results indicate that Sts-1 and Sts-2 represent a new class of negative regulators of Cbl functions in receptor endocytosis. </p><p>In conclusion, this thesis describes novel mechanisms by which Cbl, coupled to its effectors, orchestrates trafficking of RTKs. Detailed understanding of how these processes are controlled under physiological as well as under pathological conditions may be important for future therapeutic approaches.</p>
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Liu, Guangbo, Alyssa N. Coyne, Fen Pei, Spencer Vaughan, Matthew Chaung, Daniela C. Zarnescu, and J. Ross Buchan. "Endocytosis regulates TDP-43 toxicity and turnover." NATURE PUBLISHING GROUP, 2017. http://hdl.handle.net/10150/626460.
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Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. ALS-affected motor neurons exhibit aberrant localization of a nuclear RNA binding protein, TDP-43, into cytoplasmic aggregates, which contributes to pathology via unclear mechanisms. Here, we demonstrate that TDP-43 turnover and toxicity depend in part upon the endocytosis pathway. TDP-43 inhibits endocytosis, and co-localizes strongly with endocytic proteins, including in ALS patient tissue. Impairing endocytosis increases TDP-43 toxicity, aggregation, and protein levels, whereas enhancing endocytosis reverses these phenotypes. Locomotor dysfunction in a TDP-43 ALS fly model is also exacerbated and suppressed by impairment and enhancement of endocytic function, respectively. Thus, endocytosis dysfunction may be an underlying cause of ALS pathology.
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Falconer, Helen Louise. "Ca²⁺-dependent interactions in synaptic vesicle endocytosis." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/29095.
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Endocytosis proteins contain a number of different specialised domains which control protein-protein interactions at specific steps. A variety of these domains were used in a proteomic screen using GST-pull down assays from nerve terminal lysates to investigate whether Ca<sup>2+</sup> controlled any specific interactions. Many interactions appeared to occur only in the presence of Ca<sup>2+</sup>, however this was attributed to non-specific binding due to the presence of ZnSO<sub>4</sub>. Two new interactions were identified that bind SH3 domains only in the absence of Ca<sup>2+</sup>, the adaptor protein caskin I and a protein known as “similar to KIAA0856”. Since SH3 domain containing proteins had Ca<sup>2+</sup>-dependent interactions, these were examined in more detail. Most proteins bound to SH3 domains only in the absence of Ca<sup>2+</sup>. These proteins included caskin I, synaptojanin, amphiphysin I, dynamin I and synapsin I. However an unidentified 90 kDa band bound only in the presence of Ca<sup>2+</sup> suggesting Ca<sup>2+</sup> positively and negatively regulates interactions. These interactions were characterised further by examining the amount of Ca<sup>2+</sup> required to stimulate or inhibit the above associations. Ca<sup>2+</sup> binding of endophilin II has been proposed to control its protein-protein interactions. A number of complementary techniques were used to examine the Ca<sup>2+</sup> affinity of SH3 domains including endophilin. These techniques included <sup>45</sup>Ca<sup>2+</sup> overlay assays, tyrosine and tryptophan fluorescence and equilibrium dialysis. From all of the fusion proteins investigated the SH3 domains appeared to bind <sup>45</sup>Ca<sup>2+</sup> however the full length proteins did not. Tyrosine and tryptophan fluorescence showed a Ca<sup>2+</sup> dependent fluorescence shift suggesting a conformational change on the addition of Ca<sup>2+</sup>, meaning that Ca<sup>2+</sup> may bind to the SH3 domains. Finally equilibrium dialysis showed that no endophilin GST fusion protein bound to Ca<sup>2+</sup>. From these results it is still open to question whether endophilin binds Ca<sup>2+</sup>.
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Lundmark, Richard. "Sorting nexin 9 in clathrin-mediated endocytosis." Doctoral thesis, Umeå : Department of Medical Biochemistry and Biophysics, Umeå University, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-197.
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Ball, S. L. R. "Endocytosis and recycling of angiotensin II receptors." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596314.
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The main finding of this work is evidence that the AT<sub>1</sub>R is constitutively active and can undergo endocytosis and subsequent recycling in the absence of agonist. The application of a putative inverse agonist was able to increase the relative surface density in the presence of an inhibitor of novel protein synthesis. The internal pool partially colocalised with markers for early endosomes and the Golgi Apparatus, and was estimated to be 20% of the surface receptor density. This work has determined the presence of extracellular proteases that cleave the ang II into smaller peptide fragments within experimental assay timeframes. The putative cleavage product, ang III, was determined to induced AT<sub>1</sub>R endocytosis if present in sufficient concentrations. Simple mathematical models have been used to describe the changes in the surface receptor number in the presence of drugs, one of which is the Two-compartment model’. This model assumes that there are only two compartments in which the receptor can exist, the plasma membrane and inside the cell. It ignores trafficking time via endosomes to intracellular organelles such as sorting endosomes and lysosomes. A modified version of the two-compartment model was derived to include agonist depletion by internalisation or degradation and the effect of a basal level of constitutive internalisation. This work has determined that the internalisation profile of the AT<sub>1</sub>R can be fitted to the two-compartment model, although it is inadequate to describe the AT<sub>1</sub>R recycling profile and suggests that there is more than one internal receptor recycling pool. This work has also attempted to perform a detailed characterisation of an AT<sub>1</sub>R antagonist ZD 7155 in terms of receptor responses, and it effects upon ligand internalisation and receptor trafficking. It was concluded that insurmountable antagonism is a result of slow dissociation kinetics, but also may be due to residual antagonist during experimental procedures.
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Holt, M. G. "Exocytosis and endocytosis in a retinal neurone." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604197.
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The synaptic vesicle cycle in the nerve terminal consists of exocytosis and neurotransmitter release, endocytosis and vesicle regeneration. However, the exact mechanisms which underlie this cycle are still largely unknown. This dissertation describes the use of fluorescence microscopy to study aspects of vesicle cycling in the synaptic terminal of retinal depolarizing bipolar cells isolated from goldfish retina. Endocytosis at the synapse may proceed via one of two pathways: through the direct reformation of small vesicles, or through the formation of large cisternae. However, the mechanisms responsible for forming these larger compartments are unclear. In chapter 3, it is shown that following exocytosis membrane is retrieved via small vesicles and large vacuoles in bipolar cells. Vacuoles were heterogeneous in size and their formation was dependent on P1 3-kinase and F-actin, whereas formation of small synaptic vesicles was not. Vacuoles were also transported away from the plasma membrane by an actin-dependent mechanism, stimulated by calcium influx. Bulk membrane retrieval in the bipolar cell therefore exhibits the properties of macropinocytosis observed in non-neuronal cells. The bipolar cell can maintain high levels of neurotransmitter release over periods of many minutes, in response to sustained stimulation. The bipolar cell also contains high amounts of PKCα. A role for this enzyme in continuous exocytosis was investigated and the results presented in chapter 4. To maintain continuous exocytosis required an elevated free Ca<SUP>2+</SUP> level in the synaptic terminal of approximately 1 μM. Inhibition of PKC led to a reduced Ca<SUP>2+</SUP> level during stimulation, blocking exocytosis. In addition, PKCα may also have other roles in the exocytic process, such as modulating the mobility of synaptic vesicles or regulating the sensitivity of the exocytic machinery to Ca<SUP>2+</SUP>. In conclusion to the chapter, experiments are described which could help distinguish between these possible functions of PKCα in the bipolar cell. The mobility of synaptic vesicles is likely to play an important role in the regulation of synaptic transmission. This is especially true in the bipolar cell, because of its ability to support continuous exocytosis.
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Moore, Rachel. "Regulation of JAK/STAT signalling by endocytosis." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/22459/.
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The JAK/STAT pathway is a highly evolutionarily conserved signal transduction pathway, whose activation can lead to a broad range of cellular outcomes. The pathway is used repeatedly during multiple developmental stages and in adult tissue, and therefore tight regulation is required to enable accurate responses in a context specific manner. Internalisation and endocytic trafficking of signalling components provides a mechanism whereby spatial compartmentalisation can enable distinct signalling outputs. Within this study I have investigated the role of endocytosis in the regulation of the Drosophila melanogaster JAK/STAT pathway, and demonstrated that internalisation and endocytic trafficking differentially regulates target genes. Although the JAK/STAT pathway is transcriptionally competent and can regulate the expression of particular targets when the activated receptor is at the cell surface, receptor endocytosis and localisation to distinct endosomes is required for the expression of other targets. This appears to be context-dependent, as high levels of ligand stimulation overcomes endocytic regulation. STAT92E, the Drosophila JAK/STAT transcription factor, is a target of endocytic regulation. Although it is efficiently activated and undergoes nuclear translocation when endocytosis is perturbed, it is not capable of regulating a subset of target genes and therefore further STAT92E interacting partners and/or post translational modification must be required to fine-tune its transcriptional competency during endocytic trafficking. Utilising mass spectrometry I identified a novel STAT92E phosphorylation site, at threonine 702. Mutation of this threonine to prevent its phosphorylation, resulted in inhibition of STAT92E signalling and nuclear translocation, and also prevented phosphorylation of a highly conserved tyrosine residue at position 704, which is crucial for ligand activated JAK/STAT signalling outputs. Therefore, this study has enhanced our understanding of mechanisms that can modulate JAK/STAT activity. I have revealed an important role for endocytosis in fine- tuning Drosophila JAK/STAT signalling outputs and also identified a novel phosphorylation site which is crucial in the activity of STAT92E.
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Thorley, Jennifer. "Investigating mechanisms of Hepatitis C virus endocytosis." Thesis, University of Birmingham, 2014. http://etheses.bham.ac.uk//id/eprint/5042/.
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Many viruses exploit and, in some cases, promote host cell endocytic pathways for infection. These pathways include caveolar and clathrin-mediated endocytosis, as well as macropinocytosis. The entry mechanisms of many viruses are not clear cut, with more than one pathway implicated in some cases. Hepatitis C virus (HCV) is a hepatotropic virus associated with liver disease, fibrosis, cirrhosis and hepatocellular carcinoma. There are four co-receptors or “entry factors” for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI) and the tight junction proteins Claudin 1 (CLDN1) and Occludin (OCLN). Clathrin-dependent endocytosis of HCV has been demonstrated in hepatoma cell lines and has also been shown to be the route of entry for co-receptor CD81; however, other endocytic pathways have not been considered. This thesis investigates a role for caveolae in HCV entry. In addition, it has recently become apparent that the epidermal growth factor receptor (EGFR) is required for viral entry into hepatoma cells and that stimulation with EGF results in increased entry and infection. This thesis investigates the role of EGFR in the endocytosis and trafficking of HCV receptors/entry factors, with a particular focus on CD81, using live-cell and super-resolution imaging techniques.
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Madziva, Michael Taurai. "Mechanisms of M4 muscarinic acetylcholine receptor endocytosis." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619733.
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Olesen, Lene Elsnab. "AP2 binding proteins in clathrin-mediated endocytosis." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613672.
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Marks, Bruno. "Mechanisms and regulation of synaptic vesicle endocytosis." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621839.
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Hodson, Nicola Anne. "Identifying novel components of clathrin-mediated endocytosis." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648718.
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Lu, Mingfeng, and Mingfeng Lu. "Papillomavirus L2-Dependent Endocytosis and Subcellular Trafficking." Thesis, The University of Arizona, 2016. http://hdl.handle.net/10150/621363.
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Human papillomaviruses (HPV) are among the most common sexually transmitted infections and are responsible for 5% of all human cancers. HPV type 16 is the most prevalent of the high-risk HPVs (a subgroup of HPVs with potential to cause cancer), accounting for ~55% of HPV-associated cancers. HPV16 is a nonenveloped virus, composed of the major capsid protein L1, the minor capsid protein L2, and a circular double-stranded DNA genome (vDNA) condensed with human histones. HPV initially infects undifferentiated basal keratinocytes and viral replication is dependent on epithelial differentiation. Like many other DNA viruses, HPV must deliver its vDNA to the host cell nucleus to successfully replicate. Initial binding of HPV16 to host cells is through L1 interactions with cell surface heparan sulfate receptors. Shortly after virus binding, L2 is believed to undergo furin cleavage-dependent conformational changes, resulting in spanning of the protein across the local membrane and exposure of the central and C-terminal regions of L2 (which was lumenal and and inaccessible before furin cleavage) to the host cell cytosol. L2 is critical for transport of the L2/vDNA from endosomes to the trans-Golgi network (TGN). We hypothesize that furin-dependent early L2 spanning, through the direct binding and recruitment of cytosolic sorting factors, may contribute to viral endocytosis and subcellular retrograde trafficking (trafficking from endosomes to Golgi) of vDNA. We have developed a Tac receptor (CD25 or IL2 receptor, a transmembrane cell surface protein) chimera system to study L2-dependent endocytosis and trafficking. In this system the Tac ecto- and transmembrane domains are fused to the ~400 amino acid portion of L2 that is likely cytosolic upon L2 spanning. Through transient expression of Tac-L2 chimera we use anti-Tac ectodomain antibodies to label and track cell surface populations by immunofluorescence and confocal microscopy. We have also adopted this system to study endocytosis through a cell surface biotinylation approach. Both approaches suggest that L2 may enhance endocytosis and preliminary evidence suggests that the Tac-L2 chimera may recruit the cytosolic retromer complex (the host cytosolic factors help protein retrograde trafficking) to preferentially traffic to the TGN. Retromer-dependent trafficking of cargo from early endosomes to the TGN is known to involve certain members of the sorting nexin family, specifically the SNX-BAR proteins. We performed a small siRNA screen and identify SNX6 and SNX32 (aka SNX6b) as SNX-BAR proteins that may be specifically involved in retrograde trafficking of HPV16 L2/vDNA during infection. Future work will focus on the mechanisms through which L2 and SNX6 influence HPV16 entry and trafficking.
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Kokotos, Alexandros Christoforos. "Molecular identity of activity-dependent bulk endocytosis." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/25509.
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At the neuronal synapse, neurotransmitter-filled synaptic vesicles (SVs) fuse with the presynaptic plasma membrane during activity. Following exocytosis, SVs must be retrieved for neurotransmission to be maintained. Several modes of SV recycling have been identified. During mild neuronal activity, clathrin-mediated endocytosis has been regarded as the dominant SV retrieval mode, however the recently identified ultrafast endocytosis mode may also be important in this condition. During elevated activity, activity-dependent bulk endocytosis (ADBE) is the dominant SV retrieval pathway. In ADBE, large invaginations are formed from the plasma membrane, which then undergo scission to create bulk endosomes. In a second distinct step, SVs bud from these endosomes and specifically repopulate the reserve SV pool. However, since its first identification, only few molecules have been shown to participate in ADBE. The aim of this PhD was to identify novel molecules and elucidate the molecular mechanism of ADBE. To achieve this, two independent biochemical approaches were designed to purify and enrich bulk endosomes from primary neuronal cultures. In the first approach, bulk endosomes and SVs were labelled with a dye, FM1-43, using a strong stimulus. Cells were broken mechanically and the post nuclear supernatant, that contains all intracellular organelles, was collected. The supernatant was then subjected to subcellular fractionation using discontinuous Nycodenz gradients. This stimulated sample was always processed in parallel with a basal sample, where no neuronal stimulus was applied, in order to visualise activity dependent FM loading. After different fractionation protocols were applied, bulk endosomes were efficiently separated from SVs, as revealed by tracking fluorescence in different fractions. The fractionation results were further validated by electron microscopy, where bulk endosomes and SVs were labelled with horseradish peroxidase and purified using the established protocol. Immunoblotting against selected SV cargo proteins from stimulated bulk endosome and SV samples, indicated the specific and preferential localisation of VAMP4 on bulk endosomes, in contrast to other SV cargo. The molecular identity of bulk endosomes was also approached by submitting the bulk endosome fractions to semi-quantitative mass spectrometry. This analysis revealed many different proteins that were identified in bulk endosome samples and quantification approaches further indicated proteins that can be localised on bulk endosomes and have a potential role in ADBE. A second magnetic isolation approach was designed, to purify bulk endosomes using a completely different methodology. In this case, bulk endosomes were specifically labelled with iron nanoparticles, which are preferentially taken up by bulk endosomes since they are larger than SVs. The cells were broken as before and post nuclear supernatant was acquired. In this case, the supernatant was submitted to magnetic isolation that separated iron beads labelled structures from all other intracellular organelles. An extensive immunoblotting analysis of magnetic bulk endosomes validated that VAMP4 and syndapin I, two essential ADBE proteins, were enriched in these purified samples. These magnetic bulk endosomes were also analysed using semi-quantitative MS and revealed many proteins with a potential role in ADBE. Significant overlap between the two independent methods was observed, further validating these approaches. Combining these two methods with bioinformatics tools allowed the identification of the molecular signature of ADBE as well as novel key candidates for this process. Specific molecules were investigated for their role in ADBE and SV recycling using a variety of different real-time fluorescent imaging assays. A major focus was on rab small GTPases. High molecular weight dextran uptake was used to specifically study the role of these proteins in ADBE, as it preferentially reports uptake via larger bulk endosomes. A pH sensitive chimeric protein, synaptophysin-pHluorin, was used to investigate the role of these proteins in CME. Additional imaging assays were used to answer emerging questions regarding the function and localisation of these targets in the presynapse. Using these approaches, rab11A and rab35 were found to promote ADBE and accelerate clathrin-mediated endocytosis. This effect was specific to high intensity stimulation, while SV exocytosis was not affected. Further research on the role of both novel and established ADBE molecules will provide key future insights into the mechanism of both bulk endosome generation/scission and subsequent SV reformation. A very promising group is rab proteins and now evidence for their implication in SV recycling is presented here. Identification and characterisation of new targets will allow to investigate the role of ADBE in neurotransmission in both physiology and pathophysiology.
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Le, devedec Dahiana. "Role of Confinement on Clathrin-mediated Endocytosis." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS234.
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L’endocytose dépendante de la clathrine (EDC) est la principale voie d’internalisation des récepteurs de surface et de leurs ligands. L’internalisation se fait suite à l’invagination de la membrane plasmique vers l’intérieur de la cellule suite à la formation, dans un premier temps, de puits recouverts de clathrine (PRCs) qui bourgeonnent ensuite en vésicules recouvertes de clathrine dans le cytosol. L’EDC est un processus très dynamique qui a lieu en l’espace de 30 sec-1mn. Elle est impliquée dans de multiples fonctions et permet ainsi à la cellule de réguler l’expression de ses protéines en surface, de répondre aux signaux de prolifération ou migration envoyés par l’environnement immédiat via l’activation de voies de signalisation spécifiques ou encore de réguler le renouvellement des composants de la membrane plasmique. De par son importance, des dérégulations de l’endocytose dépendante de la clathrine ont déjà été observées dans les cancers. Ces modifications peuvent impliquer directement l’EDC en modifiant ses composants ou indirectement lors d’altérations de récepteurs régulés par celle-ci. La progression tumorale est elle-même régulée par de multiples facteurs, notamment l’accumulation de mutations qui ont des conséquences sur les cellules cancéreuses elle-même ou bien sur l’environnement immédiat, formant ainsi la « niche tumorale ». Ces changements agissent réciproquement sur la progression tumorale afin de l’amplifier. Lors de la croissance tumorale, les cellules cancéreuses recrutent des fibroblastes qui vont participer au remodelage et à l’augmentation de la rigidité autour de la tumeur. La rigidité de la matrice extracellulaire est détectée par les cellules ce qui envoie des signaux déclencheurs de prolifération et de migration en conséquence. Cette détection passe essentiellement par les intégrines à la surface membranaire qui vont s’agréger et induire des cascades de signalisation impliquées dans ces réponses. Ces intégrines peuvent se regrouper dans deux types de structures, les adhésions focales et les structures recouvertes de clathrine. En ce qui concerne ces dernières, il a été démontré précédemment que la rigidité du substrat augmente sa force d’interaction avec les intégrines, et empêche ainsi l’internalisation des vésicules recouvertes de clathrine, on parle alors d’ « endocytose frustrée ». Cette rétention des structures recouvertes de clathrine à la surface provoque une signalisation soutenue à la surface au lieu de l’arrêter par dégradation ultérieure des récepteurs dans les lysosomes. Le laboratoire a démontré que les structures de clathrine frustrées capturent ainsi différent récepteurs conduisant à une signalisation accrue dans la voie de la MAP Kinase Erk. Mon projet de thèse repose sur ces observations en s’intéressant plus particulièrement au rôle d’une autre modification induite par la croissance tumorale, le confinement. En effet, en se multipliant de manière incontrôlée dans un environnement spatialement restreint, les cellules tumorales se retrouvent soumises à des forces de compression. Les résultats mis en évidence au cours de ma thèse ont montré que le confinement provoque, comme la rigidité, une frustration des structures de clathrine qui ne sont donc plus capables de soutenir l’endocytose des récepteurs. De plus, la compression cellulaire induit le clivage d’un pro-ligand de l’EGFR, le HB-EGF, ce qui conduit à l’activation paracrine de l’EGFR et à l’activation de la voie Erk. En effet, l’absence de facteurs de croissance dans le milieu ainsi que l’inhibition de ce clivage démontrent la nécessité de la mise en place de ce mécanisme. En résumé, le confinement induit le clivage du pro-ligand HB-EGF, qui à son tour va activer le récepteur à l’EGF. En parallèle, l’endocytose est ralentie et provoque une signalisation accrue à la membrane. Ces deux évènements coopèrent pour mener à une très forte activation de la voie Erk<br>Clathrin-mediated endocytosis (CME) is the major route of endocytosis for many cargos in eukaryotic cells. Endocytosis takes place at clathrin-coated pits (CCPs), small assemblies of clathrin and clathrin adaptors randomly distributed at the plasma membrane. Clathrin polymerization induces the progressive bending of the plasma membrane resulting in the formation of a vesicle budding off into the cytosol. CME is a highly dynamic process with an average lifetime of CCPs in the order of 30 seconds. In this manner, CME fulfills a range of different functions and enables cells to regulate the surface expression of proteins, to sample the cell’s environment for growth and guidance cues, to control the activation of signaling pathways and to turn over membrane components by sending these components for degradation in the endo-lysosomal pathway. A deregulation of the endocytic pathways was previously shown to be involve in cancer. These modifications can affect CME directly by modifying its actors, or indirectly with mutations on receptors or cargoes undertaken by CME. Tumor progression is dependent of several factors, the first one involving the accumulation of mutations which results in modifications in the cells themselves or on their surrounding environment by changing its biochemical and physical properties, leading to the formation of the tumor niche. These changes reciprocally foster cancer progression. During tumor growth, fibroblasts will be recruited around tumor cells, leading to the remodeling of the microenvironment and to an increase of rigidity nearby the tumor. This stiffness is sensed by the cells and send signals for proliferation and migration as a result. Stiffness sensing engages mainly integrins at the cell surface which will aggregate and initiate signaling cascades accountable for these responses. Integrins are capable of clustering into two types of structures: focal adhesions and clathrin-coated structures (CCSs). Regarding CCSs, it was shown previously that high stiffness strengthen the interaction between integrins and the substrate, hence preventing the budding off of the vesicle, and this is referred to as “frustrated endocytosis”. This holding of CCSs at the cell surface promotes a sustained signaling at the plasma membrane instead of a signal termination after internalization and further degradation in lysosomes. My PhD project relied on these previous findings, with a particular focus on another mechanical alteration observed in tumors, the confinement. Indeed, during the uncontrolled proliferation of cancer cells in a spatially restricted area, cells become subjected to compressive forces. The results I obtained indicate that confinement leads to frustrated endocytosis and hence to sustained signaling from the plasma membrane. In addition, compression also leads to HB-EGF shedding at the cell surface, and the resulting EGF product activate the EGFR in a paracrine manner, thus leading to the activation of the MAP kinase Erk signaling pathway. Indeed, both the absence of EGFR ligands in the medium and the inhibition of the shedding demonstrate the necessity of this mechanism in EGFR activation. To sum up, confinement induces the shedding of the EGFR pro-ligand HB-EGF necessary to EGFR activation in these conditions. Simultaneously, endocytosis is delayed and frustrated endocytosis leads to sustained signaling at the cell surface. Together, these events cooperate to strongly activate the Erk pathway. These findings highlight the interplay between the physical feature of the tumor environment and signaling pathways known to govern tumor growth
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Gad, Helge. "Synaptic vesicle endocytosis studied in a living synapse /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4287-0/.
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Martinović, Vladan. "Protein complex formation and membrane remodelling in clathrin-mediated endocytosis." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709269.
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Hammich, Hussein. "Etude fonctionnelle et structurale de la GTPase Rab35 et de ses effecteurs : vers le mécanisme de contrôle de la dynamique de l'actine dans l'endocytose et la cytocinèse." Electronic Thesis or Diss., Paris Sciences et Lettres (ComUE), 2017. https://theses.hal.science/tel-02412888.
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Rab35 est une petite GTPase de la superfamille des Ras protéines. Chez l’Homme, plus de 60 Rabs jouent un rôle clef « d’interrupteur moléculaire » dans la régulation du trafic membranaire. Des progrès significatifs ont été réalisé sur le rôle de certaines Rabs, cependant il reste encore un travail important de compréhension fonctionnelle et du mécanisme d’action de celles-ci au sein de la cellule. L’actuel projet a pour but d’étudier Rab35, un régulateur essentiel d’une voie de recyclage vésiculaire vers la membrane plasmique, aussi impliqué dans la régulation de l’actine lors de la dernière étape de la division cellulaire. Rab35 agit via le recrutement et la régulation de protéines spécifiques appelées effecteurs. Chez l’humain, des mutations chez les partenaires de Rab35 entrainent de rares maladies connues sous le nom du syndrome de Birt-Hogg-Dube et syndrome de Lowe. Ce projet en étroite collaboration avec le laboratoire d’Arnaud Echard (Institut Pasteur) consiste à mieux comprendre le rôle cellulaire et fonctionnel de deux nouveaux effecteurs de Rab35 : MICAL1 et MiniBAR. Lors de la division cellulaire, MICAL1 pourrait réduire le niveau d’actine avant l’abscission car il possède un domaine catalytique monooxygénase démontré comme étant un facteur de désassemblage de l’actine. MICAL1 agit directement sur les filaments d’actine et oxyde des résidus spécifiques permettant la dépolymérisation de ces derniers. Mais il est actuellement inconnue si MICAL1 joue un rôle dans la division cellulaire. Par ailleurs, nos collaborateurs ont découvert un nouvel effecteur de Rab35 appelé MiniBAR. Ils ont récemment décrit MiniBAR comme étant un partenaire spécifique de Rab35 et contenant un domaine BAR (connue pour lier et courber les membranes). Ce domaine, adjacent au domaine d’interaction de Rab35, lie spécifiquement Rac1 une autre GTPase bien connue pour son rôle dans la régulation de l’actine. De ce fait MiniBAR pourrait être un lien entre les deux GTPases coordonnant le processus de remodelage de l’actine dans la cellule.Le laboratoire de Motilité Structurale dirigé par Anne Houdusse est spécialisé dans l’étude structurale des moteurs moléculaire et GTPases qui contribuent aux différentes fonctions dans la cellule par l’interaction avec leur différents cargos et effecteurs. L’élaboration de ce projet consiste à caractériser d’un point de vue structural et fonctionnel les complexes entre Rab35 et ses effecteurs. Laboratoire spécialisé dans la détermination de structure à haute résolution par cristallographie aux rayons X, ce travail mènera à la résolution de complexes macromoléculaires afin de désigner des mutants spécifiques pour des études fonctionnelles dans la cellule en étroite collaboration avec l’équipe d’Arnaud Echard, leader dans l’étude cellulaire de Rab35<br>Rab35 is an essential regulator of a recycling pathway back to the plasma membrane, that is also required for the post-furrowing terminal steps during cytokinesis that are associated with F-actin depolymerisation [1]. Rab35 performs its role in the cell via recruitment and regulation of specific effector proteins. Recently the lab of our collaborator Arnaud Echard (Pasteur institute, Paris) has identified and is currently studying by cell biology approaches two novel Rab35 effectors - MICAL1 and MiniBAR proteins. MICAL1 may restrict actin levels before cytokinesis abscission, because it harbours a monooxygenase catalytical domain and has been shown to be an F-actin-disassembly factor [2]. But it is unknown whether MICAL1 has a function in cell division. Another effector of Rab35 currently uncharacterized was identified and named MiniBAR by our collaborators. They recently described that MiniBAR specifically binds to active Rab35, that it contains an unnoticed, putative BAR domain (known to sense membrane curvature) and that this domain, adjacent to the Rab35-binding domain, binds specifically to GTP-bound Rac1 - a well known actin remodelling regulator. So, MiniBAR may function as a linker between the two small GTPases coordinating actin-remodelling processes in the cell. The aim of the project is to perform extensive structural/functional characterization of complexes between the Rab35 GTPase and its interacting effector proteins.References:[1] - Dambournet and al, Nat Cell Biol, 2011. Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.[2] - Giridharan SS and Caplan S, Antioxid Redox Signal, 2014. MICAL-family proteins: Complex regulators of the actin cytoskeleton
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Claverie, Léa. "Dynamique d'échange de la dynamine mesurée dans les cellules vivantes pendant la formation de vésicules d'endocytose." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0053.
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L'endocytose dépendante de la clathrine (EDC), c’est-à-dire la formation de vésicules recouvertes de clathrine (VRC) à partir de la membrane plasmique, est un processus essentiel dans les cellules eucaryotes. Au cours de l’EDC, la GTPase dynamine est recrutée au cou de la VRC naissante où elle s'oligomérise en hélice. Les changements de conformation induits par l'hydrolyse du GTP catalysent la scission du cou vésiculaire. Ce processus a été étudié en détail par reconstitution in vitro sur des tubules membranaires, mais il doit être établi dans des cellules vivantes, où les interactions de la dynamine avec d'autres protéines comme l'amphiphysine sont critiques. L'imagerie TIRF (Total Internal Reflection Fluorescence) avec le protocole pH pulsé (ppH) sur cellules vivantes permet la détection de la formation de VRC avec une résolution spatiale (~100 nm) et temporelle (2 s) élevée. Ce protocole a révélé que la dynamine présente un recrutement biphasique aux puits recouverts de clathrine (PRC) en maturation avec un pic au moment de la scission mais les paramètres de son recrutement dans les cellules vivantes restent peu clairs. Pour déterminer ces paramètres, j’ai utilisé des techniques d’imagerie sur cellules vivantes pour étudier le recrutement de la dynamine à l’échelle globale et à l’échelle de la molécule unique lors de perturbations aiguës de sa fonction. Mes résultats de thèse ont montré que la dynamine est recrutée à la membrane plasmique, diffuse à l'extérieur des PRC et y est transitoirement piégée. De plus, j’ai déterminé avec des dynamines mutées (1) que le domaine PRD de la dynamine est crucial pour son recrutement aux PRC ; (2) que le domaine PH est important pour la scission vésiculaire mais par pour son recrutement aux PRC ou à la membrane plasmique. Enfin, j’ai observé que la dynamine s'échange en permanence avec un pool extra-PRC, ce qui permettrait son recrutement ultérieur par l'ajout de nouveaux sites de liaison et sa capacité à rétrécir le cou des vésicules suite à l’hydrolyse du GTP. En conclusion, ces données suggèrent qu’aux PRC, les molécules de dynamine (1) sont constamment échangées ; (2) diffusent à des taux similaires tout au long du processus de formation, maturation et scission des vésicules; et (3) l'activité GTPase de la dynamine contribue à la maturation et à la scission des VRC<br>Clathrin-mediated endocytosis (CME), the formation of clathrin-coated vesicles (CCV) from the plasma membrane, is an essential process in eukaryotic cells. During CME, the GTPase dynamin is recruited to the neck of nascent CCV where it oligomerizes into helical filaments. Conformational changes induced by the hydrolysis of GTP catalyze the scission of the vesicle neck. This process has been studied in detail with in vitro reconstitution on membrane tubules but it needs to be established in living cells, where interactions between dynamin and other proteins such as amphiphysin are critical. Live cell total internal reflection fluorescence (TIRF) imaging with the pulsed pH (ppH) assay allows the detection of CCV formation with high spatial (~100 nm) and temporal (2 s) resolutions. It has revealed that dynamin is recruited to maturing clathrin-coated pits (CCP) in two phases with a peak at the time of scission but the parameters of its recruitment in living cells remain unclear. To determine these parameters, we have performed live cell imaging of dynamin recruitment at collective and single molecule levels during acute perturbations of its function. My PhD results showed that dynamin is recruited to the plasma membrane, diffuses outside of CCP and is trapped at CCP. Furthermore, we determined with mutated dynamins that (1) the PRD domain of dynamin is crucial for its recruitment at CCP; (2) the PH domain is important for vesicular scission but not for recruitment to CCP or to the plasma membrane. Finally, I observed that dynamin exchanges with an extra-CCP pool at all times: this would allow for its further recruitment by addition of new binding sites and its ability to narrow the vesicle neck after GTP hydrolysis. Altogether, these data suggest that in CCP dynamin molecules (1) are constantly exchanged; (2) diffuse at similar rates throughout the entire process of vesicle formation, from maturation until scission; and (3) that dynamin’s GTPase activity contributes to CCP maturation and scission
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Bertot, Laëtitia. "Le rôle des protéines courbant les membranes dans l’endocytose indépendante de la clathrine suivie par le récepteur de l’interleukine 2." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS570/document.
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L’endocytose permet l’internalisation d’éléments présents dans le milieu extracellulaire tels que les nutriments. Ce processus prend place dans la membrane plasmique. Les courbures de la membrane jouent un rôle essentiel dans l’endocytose pour générer une invagination initiale, un puits, puis une vésicule qui se sépare ensuite de la membrane plasmique pour fusionner avec les compartiments intracellulaires. Il existe plusieurs voies d’endocytose qui peuvent être classées selon des critères tels que la taille des vésicules produites, la médiation par un récepteur ou la présence d’un manteau recouvrant les vésicules. La voie d’endocytose la mieux caractérisée est celle dépendante de la clathrine. Mon laboratoire d’accueil travaille sur l’entrée du récepteur de l’interleukine 2 (IL-2R). Ce récepteur peut entrer de façon constitutive ou induite en présence de son ligand l’IL-2. Les deux voies sont indépendantes de la clathrine. Lors de mon arrivée dans le laboratoire, ces voies étaient encore peu caractérisées, notamment les facteurs induisant les courbures membranaires restaient à identifier. Ces facteurs doivent être particulièrement impliqués car les vésicules contenant l’IL-2R sont dépourvues de manteaux. Un crible par interférence à ARN, réalisé avant mon arrivée avait permis de proposer des protéines candidates pouvant courber les membranes. La première partie de ma thèse a consisté à confirmer l’importance de certaines protéines issues de ce crible puis à étudier leurs rôles dans la voie constitutive de l’IL-2R. Parmi ces protéines confirmées, deux familles de facteurs étaient particulièrement intéressantes pour leur capacité à courber les membranes, les phospholipases D et les endophilines. Ces dernières ont permis d’identifier une nouvelle voie d’entrée nommée « Fast Endophilin Mediated Endocytosis » FEME dans laquelle l’endophiline joue un rôle essentiel et qui est empruntée par de nombreux récepteurs transmettant le signal. La voie FEME partage plusieurs facteurs communs avec la voie d’endocytose induite de l’IL-2R. Pour finir, mes travaux de thèse ont porté sur l’orchestration de l’endophiline et de la dynamine dans la voie d’endocytose constitutive de l’IL-2R. Ces deux facteurs sont impliqués en fin d’endocytose, pour scinder les vésicules de la membrane plasmique. Cependant, ces deux protéines n’ont pas la même orchestration. Nos travaux montrent une action distincte de l’endophiline et de la dynamine dans les voies d’endocytose dépendante et indépendante de la clathrine<br>Endocytosis allows the uptake of elements from the extracellular fluid such as nutriments. This process takes place at the plasma membrane. The membrane curvatures play an important role in endocytosis for the production of initial invagination to form a pit that will be then separate from the plasma membrane and will go to the intracellular compartments. Several routes of endocytosis exist and can be classified depending on vesicles size formed, receptor mediated endocytosis or coat on vesicles. The well-known characterized endocytosis pathway is the clathrin mediated one. My lab is working on interleukin 2 receptor (IL-2R) entry. This receptor can enter either constitutively or upon induction by the ligand IL-2. Both uptake pathways are independent of clathrin. When I arrived in the lab, those pathways were still under characterization, in particular the factors inducing the membrane curvature. Their role should be important since IL-2R containing vesicles are coated free. A small interfering RNA screen performed before my phD, allowed to identify new candidates. The first part of my thesis was to verify the involvement of some of them in the IL-2R constitutive pathway and then to study their role in this pathway. Among them, 2 families of proteins were particularly interesting as they can curve membranes, phospholipases D and endophilins. The endophilin allowed the discovery of a new route called “Fast Endophilin Mediated Endocytosis” (FEME) in which it plays an essential role and which is used by numerous receptors that transmit signal. The FEME pathway shares several factors that are common with the IL-2 induced endocytosis pathway. Then, I conducted a work on the orchestration of endophilin and dynamin during the constitutive IL-2R endocytosis. Both factors are recruited at the end of the mechanism, to separate the vesicles from the plasma membrane. However, both proteins do not have the same orchestration. Our works show a distinct action of endophilin and dynamin in clathrin dependent and independent endocytosis
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Hunjan, Anoop Singh, and Anoop Singh Hunjan. "Chronic Arsenite Exposure in Lung Epithelium Modulates Endocytosis." Thesis, The University of Arizona, 2017. http://hdl.handle.net/10150/626765.
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Arsenic exposure in humans has been implicated in the development of a myriad of non-cancerous and cancerous diseases. A reductionist approach to understanding this unusual phenomenon would suggest that arsenic-induced perturbation of a small number of fundamental biological processes could manifest as this diverse array of disease endpoints. Endocytosis is a fundamental cellular process involved in the internalization and transport of various extracellular molecules and membranous components. BEAS-2B, a human bronchial epithelial cell line, was used to characterize the effects of chronic arsenite exposure on endocytosis. Fluorophore-labeled bovine albumin, human transferrin, and human low-density lipoprotein (LDL) were the substrates utilized to measure endocytosis in BEAS-2B cells. The uptake of albumin in unexposed BEAS-2B cells is both dose-dependent and temperature sensitive. Chronic arsenite exposure in BEAS-2B cells increased the uptake of albumin by 3.4-fold after 8 hours of uptake relative to unexposed BEAS-2B cells. Pharmacological studies utilizing endocytosis inhibitors suggested that the uptake of albumin in both unexposed and arsenite-exposed BEAS-2B cells occurs through a combination of receptor-mediated endocytosis and macropinocytosis. Chronic arsenite exposure in BEAS-2B cells also increased the endocytic uptake of transferrin by 2.9-fold at 30 minutes and LDL by 1.3-fold at 2 hours relative to unexposed BEAS-2B cells. Together, the data suggests that chronic arsenite exposure can increase the rate of endocytosis. This novel finding could add mechanistic insight to the conundrum of arsenic-associated human diseases.
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Evergren, Emma. "Coordination of endocytosis at the synaptic periactive zone /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-683-2/.
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Taylor, David Richard. "Mechanisms of endocytosis of the cellular prion protein." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434253.
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Hinze, Claudia. "Changes in endocytosis and trafficking during proliferative quiescence." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10060713/.
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A significant proportion of cells in the adult human body, including naïve lymphocytes, hepatocytes, stem cells and cancer stem cells, reside in an actively maintained state of proliferative quiescence (G0). Quiescent cells exit the cell cycle under certain conditions and resume proliferation upon appropriate stimuli, which distinguishes them from senescent or terminally differentiated cells. Little is known about how molecular processes, such as endocytosis, are regulated upon cell cycle exit. The aim of this study was to measure endocytic pathways during quiescence and study how their regulation contributes to quiescence maintenance. A quiescence induction protocol was optimised for hTERT-immortalised RPE1 (retina pigmented epithelial) cells to compare endocytosis between quiescent and proliferating RPE1 cells. A SILAC mass spectrometry screen comparing proteome and phosphoproteome between G0 and G1 RPE1 cells revealed changes in total protein levels and phosphorylation of endocytic proteins during G0. Confocal microscopy, Western blotting, flow cytometry and high throughput imaging techniques were used to measure clathrin-mediated (CME) and -independent endocytosis in quiescent and continuously proliferating RPE1 cells. Total levels of core proteins of the clathrin machinery were increased during G0, but uptake of the classical CME cargoes transferrin, EGF and LDL were decreased. CME activity during quiescence is cargo-specific, as could be shown for elevated Lamp1 endocytosis. Endocytosis of clathrin-independent cargoes such as oxidised LDL and Cholera toxin was highly active in quiescent cells, as was uptake of the acropinocytosis cargoes dextran and BSA. Elevated BSA uptake, however, did not promote mTORC1-mediated survival in a nutrient-(amino acid-) deprived environment. Moreover, BSA endocytosis was mediated by AP2. Finally, quiescence survival signalling via integrins was found to be dependent on endocytosis and recycling. Together, this study identified differentially regulated endocytic pathways and suggestd a role for integrin trafficking to maintain proliferative quiescence.
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Glebov, Oleg. "Study of clathrin-independent endocytosis in higher eukaryotes." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613725.
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Narayanan, Radhakrishnan. "Genetic analysis of endocytosis at the Drosophila synapse." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/279946.
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Endocytosis plays an essential role in maintaining a pool of synaptic vesicles for sustained neurotransmitter release. Synaptic vesicles are internalized and fuse with endosomes, and are subsequently reassembled to be ready for another round of exocytosis. Here I describe in two distinct studies the function of endosomes at synapses and regulation of dynamin, a protein essential for endocytosis, using the Drosophila synapse as a model. To study the function of endosomes at synapses I analyzed the localization and function of two Drosophila endosomal proteins, Hook and Deep orange (Dor), at the larval neuromuscular junction. I present here the first genetic evidence of a role for endocytic trafficking in plasticity of the synapse. I also found that mutations in hook and dor affect the number of varicosities at the nerve terminal without affecting synaptic vesicle recycling, indicating that Hook and Dor proteins play a role in later stages of endocytosis at the synapse. Dynamin is a GTPase that is essential for internalization of synaptic vesicles from the plasma membrane. Flies carrying shi ts mutations have a conditional defect in dynamin function. Molecules that regulate GTP loading (guanine-nucleotide exchange factors-GEFs) and GTPase activity (GTPase activating proteins-GAPs) of dynamin are unknown. Here I describe the identification of such molecules/domains by analyses of enhancer and suppressor mutations identified in previously conducted genetic screens. I show here that the enzymatic activity of Nucleoside diphosphate kinase (NDP Kinase), a source of GTP encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, is essential for dynamin function at synapses. Dynamin is also regulated by an intramolecular GTPase effector domain (GED) and I have identified separate mutations in shi, which map to the GED, that suppress endocytic defects in shits2. Overall, these data indicate a model in which the stability of dynamin: GTP is opposingly regulated by an unusual GEF activity of NDP kinase and a GAP activity in dynamin; in addition these findings indicate the possibility of an intriguing therapy for nm23 tumor progression.
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St, Pierre Christine A. "Endocytosis, Phagocytosis, and Innate Immune Responses: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/488.
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In this dissertation, the roles of endocytosis and phagocytosis pathways in a variety of clinically relevant scenarios were examined. These scenarios include antibody-mediated internalization of cell surface proteins, titanium wear-particle uptake in failed joint replacements, and polymeric microparticle uptake and immune responses for drug delivery or adjuvant use.
The use of antibodies specific for cell surface proteins has become a popular method to deliver therapeutics to target cells. As such, it is imperative to fully understand the ability of antibodies to mediate internalization and endosomal trafficking of the surface protein that it recognizes, so that drug delivery can be optimized. By comparing the internalization and endosomal localization of two different antibody-bound proteins, the transferrin receptor (TfR) and rabies G, we have found that there is a specific antibody-mediated internalization pathway that occurs when an antibody binds to a cell surface protein. Interestingly, the internalization pathway induced by antibody binding is different than that seen with recycling receptor internalization after ligand binding. This may have broad implications for the future development of antibody-based therapeutics.
Joint replacement failure is a major clinical problem. Studies have indicated that a large amount of metal and polyethylene wear debris is found in the synovial membrane and tissue surrounding failed replacements. Through examination of the immune response following uptake of titanium particles, our results suggest that titanium wear-particle induced inflammation and subsequent joint replacement failure may be due to activation of the NLRP3 inflammasome, leading to increased IL-1ß secretion and IL-1 associated signaling. These findings introduce IL-1 as a target for potential therapeutics for patients exhibiting significant inflammation.
Polymeric microparticles have been widely used in a variety of therapeutic applications, including drug delivery and vaccine adjuvants. It is essential to understand the ability of such particles to either activate or inhibit an immune response following uptake. Through comparison of particles with varying surface morphology, we have determined that particles with regions of high surface curvature (budding) are more immunogenic than particles with low surface curvature (spherical). Budding particles were more rapidly phagocytosed and induced higher levels of the inflammasome-associated cytokine, IL-1ß, when exposed to mouse macrophages. Additionally, budding particles induced a more rapid neutrophil response in vivo, when compared to spherical particles. These findings have broad implications for the development of future targeting vehicles for delivery of vaccines, drugs, proteins, and siRNA therapeutics.
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Yoshida, Hiroyuki. "Compensated endocytosis of LDL by hamster cells co-expressing the two distinct mutant LDL receptors defective in endocytosis and ligand binding." Kyoto University, 1999. http://hdl.handle.net/2433/181746.
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Eschenbrenner, Etienne. "Rôles des tétraspanines Tspan5 et Tspan15 dans le contrôle de l'endocytose et du niveau d'expression de la métalloprotéase ADAM10." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS500/document.
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Les tétraspanines sont des protéines à quatre domaines transmembranaires ayant la capacité d’interagir avec des partenaires et de les intégrer au sein d’un réseau dynamique d’interactions nommé tetraspanin web. Parmi elles, les TspanC8 représentent une sous-famille partageant un même partenaire, ADAM10.Cette protéase, essentielle au développement, est responsable du clivage de nombreux substrats dont le récepteur Notch, plusieurs cadhérines et facteurs de croissance. ADAM10 est également impliquée dans la régulation de plusieurs pathologies, telles que la maladie d’Alzheimer ou des cancers.Les précédentes recherches du laboratoire ont montré que les TspanC8 régulent la fonction d’ADAM10 à travers son expression et sa compartimentation membranaire. Les travaux exposés dans cette thèse montrent que plusieurs TspanC8 régulent également l’activité d’ADAM10 à travers une endocytose et une stabilité différentielle et en explorent les mécanismes sous-jascents. Ils démontrent également l’existence d’une compétition entre TspanC8 pour l’association avec ADAM10, et portent une réflexion sur l’utilisation de tags et sur les biais expérimentaux qui peuvent en découler<br>Tetraspanins are a family of proteins containing four-transmembrane domains that can interact with partners to include them in a dynamic network of interactions named tetraspanin web. Among them, the TspanC8 tetraspanin subfamily is known to share one partner, the ADAM10 metalloprotease.The ADAM10 protease is essential to development and is responsible for the shedding of a number of substrates, including the Notch receptor ectodomain, several cadherins and growth factors. ADAM10 is also implicated in the regulation of several pathologies including Alzheimer’s disease and carcinogenesis.Previous studies from our laboratory show that TspanC8 family members regulate the function of ADAM10 through its expression and its membrane compartmentalisation.Data presented in this thesis demonstrate that several TspanC8s also regulate ADAM10’s activity through differential stability and endocytosis, and explore the subjascent mechanisms. We also show that TspanC8 family members compete for association with ADAM10; thus, we bring elements of reflexion the use of tags and subsequent experimental bias
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Stunt, Richard John. "The role of the cytoplasmic domain in the localisation of CTLA-4 and CD28." Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274324.
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Roosen, Gert. "Design and development of a synthetic shuttle vector for DNA delivery." Thesis, University of Liverpool, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365910.
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Karakikes, Ioannis. "Association of the invariant chain (CD74) and human leucocyte antigen (HLA) DR : a single molecule approach." Thesis, University of Essex, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274374.
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Paolucci, Clara. "Role of nitric oxide in the maturation process of human dendric cells." Thesis, Open University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273309.
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Taylor, Marcus. "The molecular dynamics of clathrin mediated endocytosis in live cells." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609138.
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Kozik, Patrycja. "Sorting signals and machinery in the endocytic pathway." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609631.
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Vonderheit, Andreas. "Visualization of the endocytosis pathway of Semliki forest virus /." [S.l.] : [s.n.], 2004. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=15853.
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Kirkham, Matthew John. "The role of caveolin in endocytosis and caveolae biogenesis /." [St. Lucia, Qld.], 2006. http://adt.library.uq.edu.au/public/adt-QU20060713.115922/index.html.
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Samuels, Anne Lacey. "Endocytosis in elongating root tip cells of Lobelia erinus." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/29279.
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Endocytosis was measured along the axis of differentiation of epidermal and cortical root cells of Lobelia erinus from meristematic to fully expanded vacuolate cells. Both lanthanum and lead were tested as markers of endocytosis; lanthanum proved to be more effective. Lanthanum treatment produced electron dense deposits in the apoplast of the root, as well as coated pits, coated vesicles, smooth vesicles and multivesicular bodies within the cells. X-ray microanalysis was used to confirm the lanthanide nature of the deposits.
In both secretory (meristematic and elongating cells actively depositing new cell wall material) and non secretory (mature vacuolate) cells the amount of endocytosis occurring was measured by counting the' number of lanthanum labelled vesicles/um² /cell. The amount of endocytosis correlated very well with the cell wall secretory activity. The highest amount of endocytosis was found in the elongating cells, with meristematic having an intermediate value. Mature, vacuolate cells had the least endocytosis. The relationship between endocytosis and secretory activity suggests that endocytosis may be acting to remove excess membrane material added during exocytosis of secretory vesicles.
Cytochemical tests for polysaccharides were performed on both conventional transmission electron microscopy preparations and ultrarapidly frozen, freeze substituted preparations. The ultrastructural preservation was superior using the cryotechnique. The organelles involved in secretion of cell wall components were compared with the organelles associated with endocytosis in chapter 1. The Golgi showed distinct dictyosome polarity, small peripheral vesicles and an elaborate trans Golgi network. Vesicles in the cytoplasm displayed diverse staining properties. One population of larger, densely staining vesicles located on the trans Golgi and in the cortical cytoplasm was interpreted to be secretory vesicles.
Microtubules were disrupted with colchicine to test the importance of these cytoskeletal elements in endocytosis. Immunofluorescence was used to determine the concentration of colchicine which disrupted cortical microtubules in these cells. After colchicine treatment, there were fewer endocytotic vesicles and less lanthanum label in the multivesicular body, suggesting microtubules are involved in endocytosis during elongation. Cell wall pore size was tested using electron dense apoplast markers. Partial enzyme digestions were used to find which components of the cell wall determine porosity. Pectin was found to be the most important component, while cellulose and protein did not seem to effect porosity in these primary roots. The porosity of the wall was interpreted in terms of the intact wall ultrastructure.
Ultrarapid freezing, freeze substitution were used to reexamine endocytosis in elongating cells. Using lanthanum as a marker for endocytosis, the results of the earlier, conventional TEM study were supported and extended. In addition to coated and smooth vesicles, multivesicular bodies were labelled. The instantaneous preservation of the endomembrane system allowed the localization of label in the partially coated reticulum and in vesicles associated with the Golgi as well. The partially coated reticulum and multivesicular body are proposed to represent plant endosomes. When interpreted in conjunction with the secretion study, it can be suggested that the heterogenous populations of vesicles occur in the cytoplasm of the elongating Lobelia erinus root cells.<br>Science, Faculty of<br>Botany, Department of<br>Graduate