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WASP restricts active Rac to maintain cells' front-rear polarizationAmato, C., Thomason, P.A., Davidson, A.J., Swaminathan, Karthic, Ismail, S., Machesky, L.M., Insall, R.H. 28 February 2020 (has links)
Yes / Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif. / Cancer Research UK grants A15672, A24450, and multidisciplinary grant A20017.
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GFAP Polarity and Primary Cilia in Astrocytes of Mouse BrainElliott, Jonathan David 05 1900 (has links)
Often in front-back, left-right, and top-bottom, cell polarity is a basic property of tissues and organs and essential for the development of multicellular organisms. In the central nervous system, neurons are a paragon of polarity, receiving action potentials in their apically located dendrites and propagating them down a single axon extending from the basal pole of neuronal somas, ultimately ending in basally situated axon termini. In contrast, astrocytes are often considered relatively unpolarized, in keeping with the meaning of their name, "star cells." However, astrocytes do exhibit polarity in the distribution of glial fibrillary acidic protein (GFAP) and the location of the primary cilium. These features may be polarized beginning with the birth of astrocytes, when newly born pairs of daughter cells are mirror images of each other with the most distant somatic poles having both the primary cilium and the highest concentration of GFAP. The present study is a systematic analysis which addresses these aspects of astrocyte polarity: heterogeneity across brain regions and ages; influence of cilium deficiency; and orientation with respect to brain architecture and migration.
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Analysis of the role of the atypical cadherin Fat2 during tissue elongation in the developing ovary of Drosophila melanogaster / Analyse der Rolle des atypischen Cadherins Fat2 bei der Gewebestreckung während der Ovarentwicklung von Drosophila melanogasterAurich, Franziska 29 May 2017 (has links) (PDF)
Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown.
In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation.
Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system. / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert.
Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch.
Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.
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Analysis of the role of the atypical cadherin Fat2 during tissue elongation in the developing ovary of Drosophila melanogasterAurich, Franziska 10 April 2017 (has links)
Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown.
In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation.
Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system.:1 ABSTRACT I
2 ZUSAMMENFASSUNG II
3 TABLE OF CONTENTS III
4 LISTS 7
4.1 List of Abbreviations 7
4.2 List of figures 9
5 INTRODUCTION 11
5.1 Tissue morphogenesis during development 11
5.1.1 Tissue organization by differential cell affinity 11
5.1.2 Cell adhesion is mediated by cadherins12
5.1.3 The cytoskeleton drives cell shape changes 13
5.1.4 Planar polarity is required for tissue-level directionality 17
5.2 Models of tissue elongation 19
5.2.1 Germ-band extension in Drosophila melanogaster 19
5.2.2 Primitive streak formation in the chick embryo 21
5.2.3 Neural tube formation in Xenopus 22
5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24
5.3.1 Oogenesis in Drosophila 24
5.3.2 Egg chamber as a model for tissue elongation 27
5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29
5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32
5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34
5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35
6 AIMS OF THE THESIS 38
7 MATERIALS AND METHODS 39
7.1 Fly husbandry 39
7.2 Used fly stocks 39
7.3 Phenotypic markers 40
7.4 Ovary dissection for fixation 40
7.5 Antibody stainings 41
7.6 Used antibodies 42
7.7 Drug treatment 42
7.8 Microscopy of fixed samples 43
7.9 Live imaging 43
7.9.1 Imaging of the basal F-actin oscillations 43
7.9.2 Imaging of egg chamber rotation 44
7.10 Generation of the transgenic fosmid constructs 44
7.10.1 General materials required for molecular genetics in E.coli 46
7.10.2 Step 1: Amplification of the tagging cassette 47
7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49
7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50
7.10.5 Step 4: Removal of the KanR gene 50
7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50
7.10.7 Step 6: Integration of the transgene into the fly genome 52
7.11 Image analysis and quantifications 53
7.11.1 Statistics 53
7.11.2 Aspect ratio measurements 54
7.11.3 Quantification of GFP localization55
7.11.4 Quantification of tissue-wide Collagen IV alignment 55
7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57
7.11.6 Analysis of periodicity of F-actin oscillations 58
7.11.7 Quantification of the rotation velocity of egg chambers 60
8 RESULTS 61
8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61
8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61
8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63
8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65
8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67
8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70
8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73
8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73
8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76
8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78
8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81
8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85
8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88
8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90
8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92
8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293
8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96
8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99
9 DISCUSSION 103
9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104
9.2 Egg chamber elongation correlates with a functional molecular corset 107
9.3 Fat2 promotes egg chamber elongation by its extracellular region 109
9.4 Alternative mechanisms potentially drive egg chamber elongation 111
9.5 New model of egg chamber elongation 114
9.6 Future perspectives 116
9.7 Impact on tissue morphogenesis in general 119
10 ACKNOWLEDGEMENTS 120
11 REFERENCES 121
12 APPENDIX 134
12.1 Script for “FFTAlignment.m" 134
12.2 Script for “Test1” 143
12.3 Script for “AverageCellAlignment.m" 143 / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert.
Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch.
Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.:1 ABSTRACT I
2 ZUSAMMENFASSUNG II
3 TABLE OF CONTENTS III
4 LISTS 7
4.1 List of Abbreviations 7
4.2 List of figures 9
5 INTRODUCTION 11
5.1 Tissue morphogenesis during development 11
5.1.1 Tissue organization by differential cell affinity 11
5.1.2 Cell adhesion is mediated by cadherins12
5.1.3 The cytoskeleton drives cell shape changes 13
5.1.4 Planar polarity is required for tissue-level directionality 17
5.2 Models of tissue elongation 19
5.2.1 Germ-band extension in Drosophila melanogaster 19
5.2.2 Primitive streak formation in the chick embryo 21
5.2.3 Neural tube formation in Xenopus 22
5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24
5.3.1 Oogenesis in Drosophila 24
5.3.2 Egg chamber as a model for tissue elongation 27
5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29
5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32
5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34
5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35
6 AIMS OF THE THESIS 38
7 MATERIALS AND METHODS 39
7.1 Fly husbandry 39
7.2 Used fly stocks 39
7.3 Phenotypic markers 40
7.4 Ovary dissection for fixation 40
7.5 Antibody stainings 41
7.6 Used antibodies 42
7.7 Drug treatment 42
7.8 Microscopy of fixed samples 43
7.9 Live imaging 43
7.9.1 Imaging of the basal F-actin oscillations 43
7.9.2 Imaging of egg chamber rotation 44
7.10 Generation of the transgenic fosmid constructs 44
7.10.1 General materials required for molecular genetics in E.coli 46
7.10.2 Step 1: Amplification of the tagging cassette 47
7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49
7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50
7.10.5 Step 4: Removal of the KanR gene 50
7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50
7.10.7 Step 6: Integration of the transgene into the fly genome 52
7.11 Image analysis and quantifications 53
7.11.1 Statistics 53
7.11.2 Aspect ratio measurements 54
7.11.3 Quantification of GFP localization55
7.11.4 Quantification of tissue-wide Collagen IV alignment 55
7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57
7.11.6 Analysis of periodicity of F-actin oscillations 58
7.11.7 Quantification of the rotation velocity of egg chambers 60
8 RESULTS 61
8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61
8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61
8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63
8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65
8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67
8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70
8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73
8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73
8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76
8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78
8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81
8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85
8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88
8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90
8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92
8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293
8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96
8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99
9 DISCUSSION 103
9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104
9.2 Egg chamber elongation correlates with a functional molecular corset 107
9.3 Fat2 promotes egg chamber elongation by its extracellular region 109
9.4 Alternative mechanisms potentially drive egg chamber elongation 111
9.5 New model of egg chamber elongation 114
9.6 Future perspectives 116
9.7 Impact on tissue morphogenesis in general 119
10 ACKNOWLEDGEMENTS 120
11 REFERENCES 121
12 APPENDIX 134
12.1 Script for “FFTAlignment.m" 134
12.2 Script for “Test1” 143
12.3 Script for “AverageCellAlignment.m" 143
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Caractérisation par microscopie électronique des étapes précoces de l'entrée du virus de l'hépatite C dans les hépatocytes / Unraveling the details of the entry of hepatitis C virus into hepatocytic cells by electron microscopy imagingPerrault, Marie 22 November 2010 (has links)
L'infection par le virus de l'hépatite C (HCV) reste aujourd'hui une cause majeure d'hépatite chronique, de cirrhose du foie et de carcinome hépatocellulaire. L'attachement cellulaire et l'entrée de HCV sont médiés par les protéines d'enveloppe E1 et E2. De nouveaux récepteurs ont été récemment identifiés mais l'entrée du virus dans les hépatocytes reste énigmatique et n'a jamais été visualisée. Nous avons tout d'abord caractérisé le modèle des pseudo-particules HCV (HCVpp) encryomicroscopie électronique en transmission (cryo-MET). Ce sont des particules sphériques de 100 nm de diamètre portant à leur surface E1 et E2. Nous avons ensuite visualisé l'entrée des HCVpp dans les hépatocytes en MET conventionnelle en utilisant des lignées d'hépatome et des hépatocytes primaires humains(PHH). Ces derniers maintiennent leur polarité en culture comme en témoigne la persistance de canalicules biliaires, tels que dans les hépatocytes natifs. Après synchronisation à 4°C avec les cellules, les HCVpp sont retrouvées liées aux prolongements cellulaires via des 'piliers', et sont ensuite internalisées à 37°C par endocytose dépendante de la clathrine. Ces 'piliers', actuellement en cours d'identification par immunomarquages, sont internalisés avec les HCVpp dans les hépatocytes au sein de vésicules de clathrine ; ce suivi est effectué par des approches de congélation haute pression et de tomographie électronique. Enfin, les évènements d'endocytose des HCVpp dans les PHH se sont avérés rares, avec une cinétique ralentie comparée aux lignées cellulaires. Ces études en MET soulignent l'importance d'utiliser un modèle cellulaire physiologique polarisé pour l'étude du mécanisme d'entrée de HCV. / Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Receptor recognition, cell binding and membrane fusion rely on HCV envelope proteins E1 and E2. New receptors were recently discovered; however HCV entry into hepatocytes remains largely unknown and has not yet been visualized. At first, we characterized HCV pseudoparticles (HCVpp) by cryo-transmission electron microscopy (cryo-TEM). They appeared as regular spherical structures of ca. 100-nm, with E1 and E2 at their surface. By conventional TEM, we then visualized HCVpp entry into hepatocytes, using hepatoma cells and primary human hepatocytes (PHH) as a more physiological cell model.PHH maintain their polarity in culture as attested by TEM observation of persistent bile canaliculi. At 4°C, viral particles were primarily found attached to microvilli at the cell surface via molecular bridges and, after warming to 37°C, they were internalized by endocytosis in clathrin-coated pits and vesicles. Using freeze substitution and electron tomographyapproaches, these bridges were found intimately surrounding HCVpp inside the clathrincoated vesicles, suggesting a concomitant internalization. The nature of these bridges is currently under investigation by immunogoldlabeling approaches. Finally, we reproducibly observed less HCVpp internalization events in PHH compared to hepatoma cells, and the kinetics of these events seemed delayed, probably due to PHH polarity. To conclude, ourTEM approach proved powerful to visualize HCV entry, and highlights the importance of studying a physiological cell model to understand HCV entry mechanism.
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Molecular mechanisms regulating B lymphocyte polarization / Mécanismes moléculaires régulant la polarisation des lymphocytes BObino, Dorian 16 June 2016 (has links)
Dans les organes lymphoïdes secondaires, les lymphocytes B acquièrent des antigènes immobilisés à la surface de cellules voisines. L’engagement du BCR (récepteur des cellules B) avec de tels antigènes induit la formation d’une synapse immunologique et la polarisation des lymphocytes B. Cette polarisation inclut le repositionnement du centrosome à la synapse immunologique ainsi que le recrutement et la sécrétion locale des lysosomes qui sont nécessaires à l’extraction, l’apprêtement et la présentation des antigènes sur les molécules du complexe majeur d’histocomptabilité de classe II (CMH-II) aux lymphocytes T CD4+ pré-activés. Des travaux précurseurs menés dans le laboratoire ont permis de mettre en évidence les premiers acteurs moléculaires impliqués dans ce processus. Cependant, le mécanisme précis gouvernant la polarisation du centrosome demeure encore aujourd’hui inconnu. Le travail réalisé pendant cette thèse avait pour objectif d’identifier de nouveaux régulateurs contrôlant la polarisation du centrosome dans les lymphocytes B après engagement du BCR avec des antigènes immobilisés. De plus, au regard du rôle grandissant joué par le microenvironnement tissulaire dans l’activation des lymphocytes B ainsi que dans la modulation de leurs fonctions, nous avons étudié l’effet de la protéine extracellulaire Galectine-8 sur la régulation de la capacité des lymphocytes B à se polariser et à extraire et présenter des antigènes immobilisés. Le travail présenté dans ce manuscrit montre que la présence du complexe Arp2/3 au centrosome des lymphocytes B non activés permet la nucléation locale de filaments d’actine qui permettent, grâce à leur interaction avec le complexe LINC, de lier le centrosome au noyau. L’activation des lymphocytes B induit la déplétion partielle du complexe Arp2/3 du centrosome qui est recruté à la synapse immunologique par la protéine HS1. Ceci induit une diminution de la nucléation d’actine au centrosome entraînant la séparation entre le centrosome et le noyau et permettant la polarisation du centrosome vers la synapse. De plus, nous montrons que la présence de la protéine Galectine-8 dans le milieu extracellulaire favorise le recrutement et la sécrétion des lysosomes à la synapse immunologique, conférant aux lymphocytes B une meilleure capacité à extraire et présenter des antigènes immobilisés. Nos résultats mettent en évidence des mécanismes inattendus régulant la polarisation des lymphocytes B en réponse à une stimulation antigénique et soulèvent des questions intéressantes concernant la régulation coordonnée de ces mécanismes qui confèrent aux lymphocytes B la capacité d’extraire, d’apprêter et de présenter des antigènes immobilisés efficacement. / In secondary lymphoid organs, B cells acquire antigens that are tethered at the surface of neighboring cells. Engagement of the B cell receptor (BCR) with such immobilized antigens leads to the formation of an immune synapse and the subsequent polarization of B cells. This includes the repositioning of the centrosome towards the immune synapse as well as the recruitment and local secretion of lysosomes required for efficient antigen extraction, processing and presentation onto class II major histocompatibility complex (MHC-II) molecules to primed CD4+ T cells. Pioneer work performed in the lab has highlighted the first molecular players involved in this process. However, the precise mechanism governing centrosome polarization remains to be fully elucidated. The work performed during this thesis aimed at identifying new regulators supporting centrosome polarization in B lymphocytes upon BCR engagement with immobilized antigens. In addition, in view of the emerging role played by the tissue microenvironment in shaping B cell activation and functions we investigated whether extracellular Galectin-8 modulates the ability of B cells to polarize, extract and present immobilized antigens. We show here that, in resting lymphocytes, centrosome-associated Arp2/3 (actin related protein-2/3) locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC (linker of nucleoskeleton and cytoskeleton) complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its HS1-dependent recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. In addition, we show that extracellular Galectin-8 favors lysosome recruitment and secretion at the immune synapse, hence providing B cells with an enhanced capacity to extract and present immobilized antigens. Our findings highlight unexpected mechanisms that tune B cell polarity in response to antigenic stimulation and raise exciting questions concerning the coordinated regulation of these mechanisms to provide B cells with the capacity to efficiently extract, process and present surface-tethered antigens.
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Curcumin Protects against Renal Ischemia by Activating the Unfolded Protein Response and Inducing HSP70Lee, Sarah Angeline 03 November 2009 (has links)
The purpose of this study was to establish whether curcumin protects renal proximal tubule cells against ischemic injury, determine whether this postulated cytoprotective effect is mediated through the upregulation of HSP70, and investigate whether the mechanism by which curcumin induces HSP70 expression and confers its protective effect is through activation of the Unfolded Protein Response. LLC-PK1 cells were cultured on collagen-coated filters to mimic conditions of in vivo renal proximal tubule cells and induce cell polarization. Injury with and without curcumin treatment was studied by using chemically-induced ATP-depletion which mimics renal ischemic injury. Cell injury was assessed using a TUNEL assay in order to evaluate DNA cleavage associated with ischemia-induced apoptosis and actin staining used to assess cytoskeletal disruption. Renal ischemic damage was further investigated by determining detachment of the Na-K ATPase from the basolateral membrane, which represents loss of cell polarity. Cells were incubated with curcumin in a dose- and time-response fashion and subsequent levels of HSP70 expression were assessed. Cells were then incubated with AEBSF, an inhibitor of the Unfolded Protein Response (UPR) and HSP70 and BiP/GRP78 (an ER resident chaperone that is upregulated by the UPR) expression levels were evaluated. Results demonstrated that treatment with curcumin during two hours of injury results in significantly less injury-related apoptosis and cytoskeletal disruption compared to control injured cells. It was demonstrated that curcumin induces HSP70 in both a dose- and time-response fashion. Moreover, curcumin treatment resulted in profound stabilization of Na-K ATPase on the basolateral membranes as there was significantly less Na-K ATPase detachment in cells treated with curcumin during two hours of injury compared to control injured cells. Finally, treatment with AEBSF inhibited HSP70 upregulation in curcumin-treated cells as well as inhibiting the GRP78 over-expression otherwise demonstrated in curcumin-treated cells. Protection of proximal tubule cells against renal ischemic injury by curcumin was therefore indicated to be mediated by the activation of the UPR through which HSP70 is upregulated. Curcumins activation of the UPR and induction of HSP70 explains the stabilization of Na-K ATPase on the cytoskeleton and also provides a potential mechanism explaining many of curcumins therapeutic and protective qualities.
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Functional analysis of the Bazooka protein in the establishment of cell polarity in Drosophila melanogaster / Funtionelle Analyse des Bazooka-Proteins während der Etablierung der Zellpolarität in Drosophila melanogasterKrahn, Michael 18 June 2009 (has links)
No description available.
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Regulation Of Long-Range Planar Cell Polarity By Fat- Dachsous SignalingSharma, Praveer Pankaj 14 January 2014 (has links)
Planar cell polarity (PCP) is the organization of cellular characteristics within the plane of a tissue. PCP manifests both structurally, as in the directionality of insect bristles or mammalian skin hair, or dynamically, as in vertebrate neurulation, gastrulation, and oriented cell division in the kidney. Two well-conserved pathways are known to regulate PCP in invertebrates and in vertebrates: the Frizzled/PCP pathway and the Fat-Dachsous (Ft-Ds) pathway. The latter consists of the cadherins Ft and Ds, along with the Golgi kinase Four-jointed (Fj) and the transcriptional co-repressor Atrophin (Atro). Ft and Ds can bind each other, suggesting a mechanism for signal transduction. Fj phosphorylates Ft and Ds, modulating their binding affinities for each other. Atro is proposed to link Ft-Ds signaling with downstream events in the nucleus during eye development. The details of Ft-Ds binding, and the consequences of their interactions with other members of the pathway are poorly understood.
In this work, I quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. My findings suggest that the Ft-Ds pathway regulates PCP independently of asymmetric cellular accumulation of Ft or Ds. I found that Atro has a position-specific role in regulating polarity in the eye, that Fj dampens clonal polarity signals, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. My observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.
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Regulation Of Long-Range Planar Cell Polarity By Fat- Dachsous SignalingSharma, Praveer Pankaj 14 January 2014 (has links)
Planar cell polarity (PCP) is the organization of cellular characteristics within the plane of a tissue. PCP manifests both structurally, as in the directionality of insect bristles or mammalian skin hair, or dynamically, as in vertebrate neurulation, gastrulation, and oriented cell division in the kidney. Two well-conserved pathways are known to regulate PCP in invertebrates and in vertebrates: the Frizzled/PCP pathway and the Fat-Dachsous (Ft-Ds) pathway. The latter consists of the cadherins Ft and Ds, along with the Golgi kinase Four-jointed (Fj) and the transcriptional co-repressor Atrophin (Atro). Ft and Ds can bind each other, suggesting a mechanism for signal transduction. Fj phosphorylates Ft and Ds, modulating their binding affinities for each other. Atro is proposed to link Ft-Ds signaling with downstream events in the nucleus during eye development. The details of Ft-Ds binding, and the consequences of their interactions with other members of the pathway are poorly understood.
In this work, I quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. My findings suggest that the Ft-Ds pathway regulates PCP independently of asymmetric cellular accumulation of Ft or Ds. I found that Atro has a position-specific role in regulating polarity in the eye, that Fj dampens clonal polarity signals, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. My observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.
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