The ecology, species distribution, and population structure of Armillaria was investigated in the forests of Massachusetts. From 64 plots at 16 sites, 640 isolates of Armillaria were collected from six forest types (northern hardwoods, mixed oak, pitch pine, white pine, white pine/mixed oak, and eastern hemlock). Armillaria gallica proved to be the most abundant species, making up 316/640 (52%) of all isolations. This was followed by A. solidipes (219/640; 34%), A. mellea (46/640; 7%), A. calvescens (36/640; 6%), A. gemina (16/640; 3%), and A. sinapina (7/640; 1%). Armillaria gallica was routinely encountered causing significant decay of the lower bole on living hardwood hosts, especially oaks. The population structure of 153 isolates of A. gallica collected from mixed oak forests was investigated using amplified fragment length polymorphisms (AFLPs). From a total sampling area of 4.51 ha, 38 AFLP genotypes were discovered, yielding a figure of eight genets per hectare with the average A. gallica genet occupying 0.13 ha. When the effects of hydrolyzable tannins on in vitro growth were compared between A. calvescens and A. gallica, it was A. gallica that appeared better at oxidizing and metabolizing commercial tannins (tannic acid and gallic acid) along with black oak root bark extracts. This was determined through measurements of colony area and dry biomass, and suggests that A. gallica may be a better adapted pathogen of oak. In order to more accurately distinguish between isolates of A. calvescens and A. gallica, a three-gene phylogeny was reconstructed, using partial sequences of the elongation factor 1-alpha (tef1), RNA polymerase II (rpb2) and nuclear large subunit (nLSU) genes. After comparing 12 isolates each of A. calvescens and A. gallica that originated from across northeastern North America, only the tef1 gene could accurately distinguish these two species. Five single nucleotide polymorphisms were present between the two species and maximum likelihood and maximum parsimony methods grouped A. calvescens and A. gallica into monophyletic clades.

Actin is a highly abundant protein in most eukaryotic cells. The assembly of actin monomers to double helical filaments is crucial for many cellular functions, including cell movement and cell division. Actin filament assembly in cells occurs in a crowded intracellular environment consisting of various molecules, including cations and organic osmolytes. Recent studies show that cation binding stiffens actin filaments, and a small organic osmolyte trimethylamine-N-oxide (TMAO) modulates filament assembly. However, how cations and TMAO combined affect actin filament mechanics is not understood. We hypothesize that depending on the concentrations of cations and osmolytes, there will be different effects on the stiffness and assembly of actin filaments. In this study, using TIRF we evaluate actin filament mechanics and assembly. Our findings indicate that when TMAO is present alone, it can increase the elongation rate and stiffness of actin filaments, however the inclusion of potassium levels alongside TMAO reduces the persistence length of actin filaments, suggesting a decrease in filament stiffness compared to the influence of TMAO alone. Furthermore, the elongation rate of actin filaments decreases when both TMAO and potassium ions are present. This study will help us better understand how cations and osmolytes together can affect actin filament mechanics in the living cells.

No description available.

No description available.

No description available.

No description available.

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.

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

Abstract: Molecular and cellular insights into IKAP and Elongator functions As the first step in the complex process of gene expression, the transcription of genes from DNA to RNA by RNA polymerase II is subject to a multiplicity of controls and is thereby the endpoint of multiple cell regulatory pathways. We focused here on the molecular and cellular functions of IKAP and by extension of Elongator complex, initially found associated with the hyperphosphorylated RNA polymerase II during the elongation stage of transcription. IKAP is required for the assembly of Elongator subunits into a functional complex. Elongator has a histone acetyltransferase (HAT) activity associated with one of its subunits, named hELP3. In agreement with a potential role in transcript elongation, Elongator is associated with nascent RNA emanating from the elongating RNA polymerase II along the transcribed region of several yeast genes and chromatin immunoprecipitation experiments have also demonstrated an association of Elongator with genes in human cells. Different mutations in the human IKBKAP gene, encoding IKAP/hELP1, cause familial dysautonomia, a severe neurodevelopmental disease with complex clinical characteristics. Affected individuals are born with the disease and abnormally low numbers of neurons in peripheral nervous ganglions. To gain insight into the role played by IKAP and the Elongator complex in the transcription of genes and concomitantly learn about the molecular defects underlying the FD, an RNA interference approach was used to deplete the IKAP protein in human cells. In yeast, disruption of ELP1 (yeast homolog of human IKAP) is known to destabilize the ELP3 catalytic subunit, which leads to loss of Elongator integrity. Our experiments performed in human cells revealed that the levels of hELP3 protein is also affected by IKAP depletion after RNAi. We took advantage of this cellular loss-of-function model to identify genes whose transcription requires IKAP, by microarray experiments. Among the identified candidates, several were previously described to be involved in cell motility, or actin cytoskeleton remodelling. Because cell motility is of crucial importance for the developing nervous system, and therefore of obvious relevance to FD, the potential role of IKAP in cell motility was characterized at the cellular level. Several cell motility/migration assays demonstrated that the IKAP depletion has functional consequences so that IKAP-depleted cells showed defects in migration. Particularly, the reduced cell motility of neuronal-derived cell lines may be highly relevant to the neurodevelopmental disorder that affects FD patients. Whether or not the defects in cell migration resulted of impaired transcriptional elongation of the IKAP-dependent genes was investigated by chromatin immuno-precipitation technique. These experiments indicated that IKAP depletion leads to a decreased histone H3 acetylation in the transcribed region of its target genes in the context of Elongator complex. These acetylation defects are correlated with a decrease of the RNA polymerase II recruitment through the transcribed region of target genes, whereas the recruitment on the promoter is mostly unaffected. These results indicate that Elongator affects transcript elongation in vivo, but not the recruitment of the RNA polymerase II to the promoter. These very specific effects of IKAP/hELP1 depletion on histone acetylation and RNA polymerase II density across target genes are consistent with a direct effect of Elongator on transcriptional elongation in vivo and point to a function for Elongator in histone acetylation during transcript elongation. Résumé: Caractérisation des rôles biologiques de la protéine IKAP et du complexe Elongator La transcription des gènes de lADN en ARN est fondamentale pour lexpression des protéines et la capacité de nos cellules à sadapter à leur environnement. Ce processus finement régulé est catalysé par un enzyme, lARN polymérase II, vers lequel convergent une multitude de voies de signalisation. Dans le cadre de ce travail, nous nous sommes intéressés aux fonctions moléculaires et cellulaires de la protéine IKAP et du complexe Elongator. IKAP est la protéine qui assemble les sous unités dElongator en un complexe fonctionnel. Le complexe Elongator est associé à lARN polymérase II hyper-phosphorylée pendant létape délongation de la transcription et possède une activité histone acétyltransferase associée à une de ses sous unités, appelée ELP3. Chez la levure, Elongator est recruté an niveau des ARNs naissants, qui émanent directement de lARN polymérase II au niveau de la région transcrite des gènes étudiés. De plus, des expériences dimmunoprécipitation de la chromatine ont mis en évidence la présence du complexe Elongator au niveau de plusieurs gènes humains. Différentes mutations au niveau du gène IKBKAP, codant pour la protéine IKAP, sont responsables de la dysautonomie familiale, une maladie génétique qui affecte le développement du système nerveux périphérique. En effet, les individus affectés présentent une diminution de la densité de neurones au niveau des ganglions nerveux périphériques. Lobjectif de nos travaux est de comprendre davantage le rôle de la protéine IKAP et du complexe Elongator dans la transcription des gènes et ainsi, dinvestiguer les mécanismes moléculaires responsables dans la physiopathologie de la dysautonomie familiale. Un modèle de perte de fonction pour la protéine IKAP a dabord été généré par interférence dARN. Des travaux réalisés chez la levure indiquent que la protéine ELP1 (homologue de IKAP chez la levure) est essentielle pour la stabilité de la sous unité catalytique du complexe, la protéine ELP3. Les expériences réalisées sur notre modèle humain démontrent que le taux de la protéine ELP3 est également affecté par la déplétion dIKAP causée par linterférence dARN. Ce modèle de perte de fonction a été utilisé afin détablir la liste des gènes dont lexpression est contrôlée par la protéine IKAP, par des expériences de microarrays. Parmi les candidats identifiés, plusieurs ont été décrits comme impliqués dans la migration cellulaire et le remodelage du cytosquelette dactine. Le processus de migration des cellules est fondamental au cours du développement du système nerveux et par conséquent particulièrement relevant dans le contexte de la dysautonomie familiale. Limplication dIKAP dans la migration cellulaire a été investigué par différents tests de fonction qui montrent que la diminution dIKAP dans différentes lignées cellulaires entraîne une réduction significative de leur capacité migratoire. Ces résultats suggèrent que la diminution du nombre de neurones observée dans les ganglions périphériques des patients atteints de la dysautonomie familiale pourrait résulter dune altération de leur capacité à migrer au cours du développement. Enfin, des expériences dimmunoprécipitation de la chromatine ont été menées en utilisant notre modèle afin de déterminer dans quelle mesure le déficit de migration observé en labsence dIKAP serait la conséquence dun défaut de la fonction dElongator au niveau de lélongation de la transcription des gènes. Les résultats nous ont montré que la diminution dexpression dIKAP entraîne une réduction de lacétylation des histones H3 dans la région transcrite de ses gènes cibles. De plus, ce déficit dacétylation est directement corrélé avec un désengagement progressif de lARN polymérase II le long de la région transcrite de ces gènes. Par conséquent, ces résultats démontrent que le complexe Elongator affecte lélongation des transcrits in vivo, mais pas le recrutement de lARN polymérase II au niveau du promoteur. Ces effets très spécifiques de labsence dIKAP sur lacétylation des histones et lengagement de la polymérase II dans la transcription des gènes cibles montrent quElongator exerce un rôle direct au niveau de lélongation de la transcription de ces gènes. De plus, ces résultats suggèrent que la fonction dElongator serait dacétyler les histones au cours de lélongation transcriptionnelle in vivo.

La morphogenèse des tissus dans les organismes multicellulaires est très importante pour le développement et certaines pathologies. La morphogenèse tissulaire est dirigée par des forces bio-mécaniques générées par des moteurs moléculaires tels que la myosine et transmis via le cytosquelette et les structures d'adhésion à l'intérieur et entre les cellules. La contractilité de la myosine, souvent en mode oscillatoire, a été étudiée principalement au niveau du domaine apical des cellules épithéliales au cours du développement mais très peu au niveau de leur domaine basal. L'oscillation de la myosine basale est importante pour le contrôle de l'élongation du tissu durant l'oogenèse chez la Drosophile. Bien que la voie Rho1-ROCK-myosin-MBS soit connue pour contrôler l'activité de la myosine, le mécanisme précis de ce contrôle n'a pas été élucidé. Le but de mon projet de thèse est de répondre à deux questions : Quels sont les facteurs en amont de cette voie ? Comment cette voie de signalisation crée et maintient l'oscillation de la myosine ? 1) Contrairement à ce qui est déjà connu, Je me suis intéressé à l'effet des adhésions cellule-cellule et cellule-matrice dans le contrôle des voies de signalisation gouvernant l'oscillation de la myosine basale. Les adhésions cellule-matrice, mais pas les adhésions cellule-cellule, sont positivement corrélées avec l'intensité et la polarité dorso-ventrale de la myosine, indiquant que les adhésions cellule-matrice pourraient être les facteurs en amont de la voie Rho1-myosine. Les adhésions cellule-matrice régulent positivement l'activité de Rho1 près des jonctions et gouvernent les flux de ROCK et myosine à l'intérieur du domaine median, contrôlant ainsi l'élongation du tissu. D'une autre manière, les adhésions cellule-cellule affectent indirectement les flux de ROCK and myosine en contrôlant la distribution subcellulaire de ROCK et du réseau d'actomyosine. L'inhibition des adhésions cellule-cellule, qui a un effet mineur sur l'élongation du tissu, provoque la redistribution des adhésions cellule-matrice et des filaments F-actin entrainant le chargement de la myosine à différentes positions. 2) J'ai montré que l'oscillation de la myosine basale dépend peu de la tension corticale de l'actomyosine : l'inhibition du chargement de la myosine sur les filaments d'actine n'affecte pas le flux de myosine alors qu'il bloque fortement le cycle périodique des contractions/relaxations de la cellule indiquant que l'oscillation est principalement due à une réaction biochimique plutôt qu'à une tension corticale. Au cours de l'oscillation de la myosine, les protéines Rho1 et leur activité sont principalement distribuées et enrichies au niveau et près des jonctions basales, et le contrôle majeur de cette oscillation est le flux des signaux ROCK qui diffusent des jonctions basales au cortex medio-basal. Ce mouvement de ROCK est initié grâce à une interaction transitoire entre ROCK et Rho1 actif au niveau et près des jonctions basales, conduisant ainsi à l'ouverture et activation de la kinase ROCK. Au cours de ce mouvement, l'activation de ROCK permet l'accumulation et l'amplification des signaux ROCK; Cette amplification entraîne la phosphorylation de la myosine, qui ensuite génère la redistribution dynamique de la phosphatase MBS. Enfin, l'enrichissement des signaux MBS arrête les signaux ROCK et myosine. Dans ces deux études, nous avons construit un outil optogénétique confirmant les différentes étapes de l'oscillation de la myosine basale. L'ensemble de ces résultats démontrent que le mécanisme contrôlant l'oscillation de la myosine basale nécessite une réaction biochimique, et met en évidence deux contrôles diffèrent de cette oscillation par les adhésions cellule-cellule et les adhésions cellule-matrice. / Tissue morphogenesis in multicellular organisms is very important in both development and human disease. Tissue morphogenesis is driven by bio-mechanic force that is normally generated by molecular motors such as myosin and transmitted via cytoskeleton and adhesion structures within and between cells. Myosin contractility, often as an oscillatory pattern, has been studied mainly in apical but less in basal domains of epithelial cells during development. Basal myosin oscillation is important in control of tissue elongation during Drosophila oogenesis. Although a signal cascade (Rho1-ROCK-myosin-MBS) has been known to regulate myosin activity, the detailed controlling mechanism is unclear. My project is aimed to address two questions: first, what is the upstream factor of this signal cascade? Second, how does this signal cascade create and maintain basal myosin oscillation? For this first question, I am interested in the effect of cell-cell and cell-matrix adhesion in control of this signal cascade governing basal myosin oscillation. Cell-matrix adhesion (Integrin and Talin), but not cell-cell adhesion (E-cadherin), is positively correlated with the intensity and Dorsal-ventral (DV) axis polarity of basal myosin oscillation, indicating that cell-matrix adhesion might be the upstream control of Rho1-myosin signal cascade. Cell-matrix adhesion positively regulates the Rho1 activity near junction and governs the pulsed ROCK and myosin signals within basal-medial domain, thus strongly controlling tissue elongation. Differently, cell-cell adhesion indirectly affects the ROCK and myosin pulses through controlling the subcellular distribution of ROCK and actomyosin network. Inhibition of cell-cell adhesion results in the redistribution of cell-matrix adhesion and F-actin filaments leading to different position of myosin loading, which plays minor effect on tissue elongation. For the second question, I unraveled that basal myosin oscillation is barely dependent on actomyosin cortical tension: inhibition of myosin loading to F-actin filament seems not to affect basal pulsatile myosin flows, while it strongly blocks the periodic cycle of cell contraction and relaxation at basal surface, thus indicating that oscillation is mainly from biochemical reaction rather than cortical tension. This observation highlighted that biochemical reaction is the main control of oscillation occurrence. During basal myosin oscillation, Rho1 proteins and Rho1 activity are mainly distributed and enriched at and near basal junction and the major control of basal myosin oscillation is the flow movement of oscillatory ROCK signals from basal junction to medio-basal cortex. This ROCK flow movement is initiated from the transient interaction of ROCK with active Rho1 at and near basal junction, thus leading to the opening and activation of ROCK kinase capability. During the membrane-medial flow movement, ROCK kinase activity mediates the accumulation and thus the amplification of ROCK signals; this positive signal amplification turns on the phosphorylation of myosin regulatory light chain (MRLC), which governs the dynamic redistribution of MBS. Finally, enriched MBS signals shut off both ROCK and myosin signals. In both study, an optogenetic tool named as LARIAT was built up in vivo to confirm the various status of basal myosin oscillation. Altogether, these results demonstrated two different controls of basal actomyosin signals by cell-matrix adhesion and cell-cell adhesion, and further demonstrated the underlying mechanism of basal myosin oscillation at the biochemical levels.