ERRα est un récepteur nucléaire dont l’activité est controlée par des co-régulateurs transcriptionnels. La forte expression de ERRα dans les cancers est corrélée à un mauvais pronostic. Les mécanismes par lesquels ERRα régule la migration des cellules cancéreuses sont mal compris, tout comme les co-régulateurs impliqués. Nous avons identifié deux enzymes modificatrices d’histone, LSD1 et SET7, agissant comme régulateurs positifs de ERRα.I. ERRα modifie les activités biochimiques de la déméthylase LSD1 vers la déméthylation (activatrice) de H3K9me2. L’activation des cibles de ERRa-LSD1 (identifiées par RNA-Seq) requiert le recrutement de ce complexe aux sites d’initiation de la transcription (TSSs), réalisé par le facteur de transcription NRF1 qui, lui, ne régule pas l’activité enzymatique de LSD1.II. Un autre groupe de cibles de ERRα (identifié par RNA-Seq) est sous le contrôle de l’histone méthyltransférase SET7 qui mono-méthyle H3K4. Le recrutement de SET7 aux TSSs est contrôlé par le facteur de transcription ETS1, qui promeut les interactions entre SET7 et ERRα, conduisant à l’activation de l’expression des gènes en aval.Des analyses par Gene Ontology ont montré que les cibles communes de ERRα/LSD1 et de ERRα/SET7 sont fortement enrichies en termes d’invasion cellulaire. De manière cohérente, la déplétion individuelle de chacun de ces facteurs (et également celle de NRF1 ou ETS1) réduit les capacités d’invasion, observée en tests in vitro (transwell) ou in vivo par xénogreffe sur embryons de poisson-zèbre.En résumé, nos résultats montrent deux réseaux de régulation impliquant des modifications d’histone induites par ERRα, conduisant à l’invasion cellulaire. / ERRα is a nuclear receptor whose activity mainly depends on its interaction with transcription co-regulators. High levels of ERRα are found in various cancer types and correlate with poor prognosis. However, the mechanisms linking ERRα to cancer cell migration as well as the coregulators involved are unclear. In our study, we found two histone-modifying enzymes, LSD1 and SET7, acting as positive regulators of ERRα.I. ERRα impacts the biochemical activities of the LSD1 demethylase. Activation of ERRα -LSD1 targets (identified by RNA-Seq) requires the recruitment of this complex at Transcriptional Start Sites (TSSs), which is achieved by the NRF1 transcription factor. In our study, we have shown several points: NRF1, but not ERRα , is involved in positioning LSD1 to TSS, whereas ERRα , but not NRF1, regulates LSD1 enzymatic activities towards demethylating H3K9me2.II. A distinct group of ERRa target genes (identified by RNA-Seq) is under the control of the histone methyltransferase SET7 which mono-methylates H3K4. Appropriate recruitment of SET7 at TSSs is controlled by the ETS1 transcription factor, promoting the interaction between SET7-ERRa, leading to target gene expression.Gene Ontology analysis revealed that ERRa-LSD1 co-targets, as well as ERRa-SET7 co-targets, are enriched in terms of cell invasion. Consistently, depletion of each of these factors, as well as depletion of NRF1 or ETS1, leads to reduced cell invasion capacities as observed in transwell assays or in vivo, using xenotransplantation in the zebrafish embryo.Altogether, our results show two regulatory networks involving histone modifications induced by nuclear receptors, leading to increased cell invasion.

La motilité des cellules est un phénomène fondamental en biologie souvent étudié sur des surfaces planes, conditions peu physiologiques. Nous avons analysé la migration cellulaire dans une matrice cellulaire 3D contenant de la fibronectine fluorescente. Nous démontrons que les cellules y sont confinées, et déforment leur environnement de manière cyclique avec une période de ~14 min avec deux centres de contractions à l’avant et à l’arrière de la cellule qui contractent avec un déphasage de ~3.5 min. Une perturbation de ces cycles entraîne une réduction de la motilité. Par l’utilisation d’inhibiteurs spécifiques, nous avons identifié l’acto-myosine comme étant l’acteur principal de ce phénomène. En imposant des contractions-relaxations locales par ablations laser, nous avons déclenché la motilité cellulaire ce qui confirme notre hypothèse. L’ensemble de cette étude met en évidence un nouveau mécanisme fondamental de dynamique cellulaire impliqué dans le mouvement des cellules. / Cell motility is an important process in Biology. It is mainly studied on 2D planar surfaces, whereas cells experience a confining 3D environment in vivo. We prepared a 3D Cell Derived Matrix (CDM) labeled with fluorescently labeled fibronectin, and strikingly cells managed to deform the matrix with specific patterns : contractions occur cyclically with two contraction centers at the front and at the back of the cell, with a period of ~14 min and a phase shift of ~3.5 min. These cycles enable cells to optimally migrate through the CDM, as perturbation of cycles led to reduced motility. Acto-myosin was established to be the driving actor of these cycles, by using specific inhibitors. We were able to trigger cell motility externally with local laser ablations, which supports this framework of two alternating contractions involved in motion. Altogether, this study reveals a new mechanism of dynamic cellular behaviour linked to cell motility.

Chan, Ka Ki Alex. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 245-263). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General introduction --- p.1 / Chapter 1.1 --- Structure and function of the enteric nervous system --- p.1 / Chapter 1.2 --- Neural crest cells (NCC) --- p.5 / Chapter 1.2.1 --- Vagal neural crest cells --- p.7 / Chapter 1.2.2 --- Sacral neural crest cells --- p.10 / Chapter 1.3 --- Prespecialization of the neural crest cells to form ENS --- p.15 / Chapter 1.4 --- Signaling pathways involved in ENS development --- p.19 / Chapter 1.4.1 --- Endothelin signaling pathway --- p.20 / Chapter 1.4.2 --- Ret signaling pathway: GDNF/Ret/GFRa1 --- p.22 / Chapter 1.4.3 --- Ret signaling pathway: NRTN/Ret/GFRa2 --- p.26 / Chapter 1.4.4 --- Phox2b --- p.28 / Chapter 1.4.5 --- Sox10 --- p.29 / Chapter 1.5 --- Hirschsprung's Disease (HSCR) --- p.31 / Chapter 1.6 --- Objective of studies --- p.32 / Figures and legends --- p.35 / Chapter Chapter Two --- The early migratory pathways of mouse sacral neural crest cells --- p.39 / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.2 --- Materials and Methods --- p.46 / Chapter 2.2.1 --- Animals --- p.46 / Chapter 2.2.2 --- Isolation of the mouse embryos at E95 --- p.46 / Chapter 2.2.3 --- Preparation ofWGA-Au --- p.47 / Chapter 2.2.4 --- Preparation of Dil --- p.48 / Chapter 2.2.5 --- Microinjection ofWGA-Au or Dil --- p.48 / Chapter 2.2.6 --- Preparation of rat serum --- p.49 / Chapter 2.2.7 --- Preparation of culture medium --- p.50 / Chapter 2.2.8 --- in vitro whole embryo culture system --- p.50 / Chapter 2.2.9 --- Examination of embryo after culture --- p.51 / Chapter 2.2.10 --- Histological preparation of WGA-Au labelled embryos --- p.51 / Chapter 2.2.11 --- Silver enhancement staining on sections of WGA-Au labelled embryo --- p.52 / Chapter 2.2.12 --- Histological preparation of Dil labelled embryos --- p.53 / Chapter 2.2.13 --- Reconstruction of the mouse embryos --- p.53 / Chapter 2.2.14 --- Cell counting on labelled sacral NCC between the anterior and posterior halves of the somite --- p.54 / Chapter 2.2.15 --- Cell counting on migrating labelled sacral NCC for each somite at different developmental stages --- p.55 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Development of E9.5 mouse embryo in vitro and in vivo --- p.57 / Chapter 2.3.2 --- Labelling of sacral neural crest cells by means of different cell markers --- p.58 / Chapter 2.3.3 --- Migration of sacral neural crest cells at different developmental stages --- p.59 / Chapter 2.3.3.1 --- Distribution of sacral NCC at the 26th somite stage --- p.60 / Chapter 2.3.3.2 --- Distribution of sacral NCC at the 28th somite stage --- p.61 / Chapter 2.3.3.3 --- Distribution of sacral NCC at the 30th somite stage --- p.61 / Chapter 2.3.3.4 --- Distribution of sacral NCC at the 32nd somite stage --- p.63 / Chapter 2.3.3.5 --- Distribution of sacral NCC at the 34th somite stage --- p.64 / Chapter 2.3.4 --- Defined migration pathways of the sacral neural crest cells --- p.65 / Chapter 2.3.5 --- Quantification of migrating sacral NCC at different somite axial levels at different developmental stages --- p.66 / Chapter 2.4 --- Discussion --- p.68 / Chapter 2.4.1 --- E9.5 mouse embryo grew normally in vitro using whole embryo culture --- p.69 / Chapter 2.4.2 --- Migration of sacral neural crest cells at 26th somite stage --- p.70 / Chapter 2.4.3 --- Migration of sacral neural crest cells at 28th somite stage --- p.72 / Chapter 2.4.4 --- Migration or sacral neural crest cells at 30th somite stage --- p.73 / Chapter 2.4.5 --- Migration of sacral neural crest cells at 32nd somite --- p.75 / Chapter 2.4.6 --- Migration of sacral neural crest cells at 34th somite stage --- p.77 / Chapter 2.4.7 --- Majority of sacral neural crest cells migrate along the dorsomedial pathway --- p.80 / Figures and Legends --- p.82 / Tables --- p.136 / Chapter Chapter Three --- The early migratory pathways of Dom mouse sacral neural crest cells --- p.139 / Chapter 3.1 --- Introduction --- p.139 / Chapter 3.2 --- Materials and Methods --- p.145 / Chapter 3.2.1 --- Animals --- p.145 / Chapter 3.2.2 --- In vitro culture of Dom mouse embryos --- p.145 / Chapter 3.2.3 --- Genotyping by polymerase chain reaction (PCR) --- p.146 / Chapter 3.2.4 --- Treatment of the harvested Dom mouse embryos --- p.147 / Chapter 3.2.5 --- Reconstruction of images and cell counting --- p.148 / Chapter 3.2.6 --- Percentage of migrating sacral neural crest cells reduction in Dom mouse embryo --- p.148 / Chapter 3.3 --- Results --- p.150 / Chapter 3.3.1 --- Migration of sacral neural crest cells in Dom mouse embryos at different developmental stages --- p.150 / Chapter 3.3.1.1 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 26th somite stage --- p.150 / Chapter 3.3.1.2 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 28th somite stage --- p.151 / Chapter 3.3.1.3 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 30th somite stage --- p.152 / Chapter 3.3.1.4 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 32nd somite stage --- p.154 / Chapter 3.3.1.5 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 34th somite stage --- p.156 / Chapter 3.3.2 --- Number of migrating sacral NCC of different genotypes of Dom mouse embryos at different developmental stage --- p.158 / Chapter 3.4 --- Discussion --- p.160 / Chapter 3.4.1 --- The use of Dom mouse model to study the etiology of Hirschsprung's disease (HSCR) --- p.161 / Chapter 3.4.2 --- Migration of sacral NCC in Dom mouse embryos --- p.164 / Figures and legends --- p.169 / Tables --- p.230 / Chapter Chapter Four --- General discussion and conclusions --- p.236 / Appendix --- p.241 / References --- p.245

La neurofibromatose de type 2 (NF2) est une maladie autosomique causée soit par l'inactivation du gène NF2, soit par la perte de la protéine issue due ce gène, Merline. Cela entraîne à son tour la formation de plusieurs tumeurs nerveuse bénignes (non invasives) comme les schwannomes, méningiomes et les épendymomes. De plus, une diminution de l'expression de Merline est observée dans les cancers du sein invasifs, toutefois le rôle de Merline dans ces tumeurs invasives est peu étudié. Merline est la seule protéine ayant un rôle de suppresseur de tumeur dans la famille des ERM (Ezrin / Radixin / Moesin). Nous, ainsi que d'autres groupes, avons montré que la partie C-terminale de Merline est importante pour sa fonction inhibitrice de la croissance cellulaire. Par conséquent, j'ai cherché à mettre en évidence de nouveaux partenaires d'interaction non décrits à ce jour, ainsi que de nouveaux sites de phosphorylation sur l'extrémité C-terminale de Merline qui pourrait expliquer la fonction de suppresseur de tumeur de Merlin. L'utilisation d’expériences d'immunoprécipitation couplées à la spectrométrie de masse nous a permis d’identifier de nouveaux interacteurs ainsi que de nouveaux sites de phosphorylation sur ce domaine C-terminal de Merline. Nous avons analysé l'importance d'un nouvel interacteur, AmotL1, ainsi que d'un nouveau site de phosphorylation sur la threonine 581 (T581), dans la fonction suppresseur de tumeur de Merline. La protéine AmotL1 appartient à la famille des motines, qui sont connues pour être impliquées dans la régulation de la migration cellulaire. A cet égard, nous avons montré qu’AmotL1 est un nouveau partenaire d'interaction de Merline. Nous avons étudié l'importance de cette interaction entre Merline et AmotL1 dans la migration cellulaire et nos données suggèrent fortement que Merlin pourrait inhiber la migration cellulaire médiée par AmotL1 dans les cellules du cancer du sein, via notamment la régulation de son expression et de sa localisation. Enfin, nous avons également identifié plusieurs nouveaux interacteurs de Merline, qui pourraient expliquer comment Merlin pourrait agir comme une protéine d'échafaudage à la membrane plasmique, en interagissant avec des composants essentiels de la voie Hippo, comme AmotL1, Kibra, Lats et YAP, pour réguler la prolifération et la migration cellulaire. Dans la deuxième partie, nous avons identifié un nouveau site de phosphorylation spécifique à l'isoforme 1 de Merline, la T581, et nous avons démontré que la phosphorylation de cette threonine est importante pour la progression en mitose au moment approprié. De plus, dans cette étude, nous avons montré que Merlin est un substrat potentiel de la kinase Aurora A, un oncogène majeur, au cours de la mitose et de l'interphase, dans des lignées cellulaires de cancer du sein. Enfin, nous avons fourni des données préliminaires sur la façon dont Aurora A régule la signalisation Hippo et la fonction de DCAF1 en phosphorylant Merline. En résumé, cette thèse met en évidence deux fonctions importantes de Merline : premièrement comment Merline régule la migration/invasion cellulaire dans des tumeurs non-nerveuses telles que les cancers du sein et deuxièmement, comment Merline est régulé au cours de la mitose et de l'interphase dans des lignées de cancer du sein, en agissant comme un substrat pour la kinase Aurora A qui est surexprimée dans plusieurs cancers comme celui du sein, du côlon et l'HCC. Prise dans son ensemble, notre étude montre le rôle potentiel de Merline dans les tumeurs invasives telles que celles rencontrées dans les cancers du sein. / Neurofibromatosis type 2 (NF2) is an autosomal disorder caused by inactivation of NF2 gene or loss of the NF2 product, Merlin. This in turn results in formation of multiple benign (noninvasive) nerve tumors such as schwannomas, meningiomas and ependymomas. Additionally reduced expression of Merlin is observed in invasive breast cancers however the role of Merlin in these invasive tumors is poorly investigated. Merlin is the only tumor suppressor protein in Ezrin/Radixin/Moesin (ERM) family proteins. Previously we and others have shown that C-terminus of Merlin is important for its growth suppressive function. In this regard, I set out to investigate whether there were undiscovered interacting partners and novel phosphorylation sites on the C-terminus of Merlin that could account for tumor suppressor function of Merlin. Using immunoprecipitation coupled to mass spectrometry we have identified new interactors as well as novel phosphorylation on this C-terminus domain of Merlin. We analyzed importance of new interactor, AmotL1, as well as novel phosphorylation site on T581 in the tumor suppressor function of Merlin. AmotL1 belongs to AMOT family proteins which are known to involve in the regulation of cell migration. In this regard, we have shown that AmotL1 is novel interacting partner of Merlin. We have investigated the importance of Merlin and AmotL1 interactions in cell migration and our data strongly suggest that Merlin might inhibit AmotL1 mediated cell migration in breast cancer cells by regulating its expression and localization. Finally, we have also found several new interactors of Merlin and that could explain how Merlin might acts as scaffolding protein at the plasma membrane by interacting with Hippo core components such as AmotL1, Kibra, Lats and YAP to regulate cell proliferation and migration. In the second part, we have identified a novel phosphorylation site at T581 which is specific to Merlin isoform 1 and demonstrated that phosphorylation of Merlin on T581 is important for the timely mitotic progression. Further in this study, we have shown that Merlin is a potential substrate for major oncogene Aurora kinase A in mitosis as well as in interphasic breast cancer cell lines. Finally we have provided initial clues how Aurora A regulates Hippo signaling and DCAF1 function by phosphorylating Merlin. In the summary, this thesis highlights two important functions of Merlin: firstly how Merlin regulates the cell migration/invasion in non-nerve tumors such as breast cancers and secondly how Merlin is regulated in mitosis and interphasic breast cancer cells by acting as a substrate to Aurora Kinase A which is over expressed in several cancers such as breast, colon and HCC. All together our study indicates the potential role for Merlin in invasive tumors such as breast cancers.

Atherosclerosis, characterized by the build-up of cholesterol, immune cells and cellular debris within arterial walls, is accelerated following myocardial infarction by poorly understood mechanisms. Ubiquitin, a small, well-studied intracellular protein involved in protein turnover via the proteasome pathway, has recently been shown to exert extracellular effects on cardiac myocytes, in vitro, and in mice undergoing myocardial remodeling. This study investigates the potential role of extracellular ubiquitin in atherosclerosis by determining its effects on two critical atherosclerotic processes: the migration of vascular smooth muscles cells and the uptake of modified LDL by monocyte/macrophages in foam cell formation. In the presence of ubiquitin, smooth muscle cell migration was accelerated and foam cell formation was enhanced, suggesting that ubiquitin has an active role in atherosclerosis.

La toxine pertussique (PTX) est une exotoxine produite uniquement par Bordetella pertussis, un pathogène de la coqueluche. Les effets de la toxine au cours d'une infection bactérienne sont bien connus, et sont pour la plupart liés à son activité ADP-ribosyltransférase qui cible les GPCRs. Or, la PTX est un antigène majeur permettant d’établir une réponse immunitaire contre B. pertussis ce qui en fait donc un composant principal de tous les vaccins anti-coqueluche actuels. De nombreux travaux sur la PTX concernent ses mécanismes moléculaires et son rôle durant la phase d'infection. Mais, il y a un manque d'information sur le rôle immunogène de la PTX.En utilisant un modèle d'infection intranasale par B. pertussis, nous avons constaté que la génération de lymphocytes T CD4 mémoires résidant (Trm) dans les poumons dépendait de l'exposition à la PTX. La toxine pertussique est couramment utilisée pour inhiber la réponse aux chimiokines, dans l'étude de la migration des cellules T. Etant donné que la plupart des récepteurs aux chimiokines sont des GPCRs, la mobilité de nombreuses cellules immunitaires, y compris les cellules T, est facilement affectée par la PTX. La migration des cellules T est un phénomène sophistiqué régulé spatio-temporellement. Nos résultats démontrent que la PTX n’affecte pas les étapes de la migration dépendantes des intégrines lorsque les cellules T sont activées.Ce travail s’intéresse à l'impact de la PTX sur la biologie des cellules T en étudiant son rôle dans la réponse immunitaire adaptative in vivo, dans un modèle animal d'infection et son impact sur la migration des lymphocytes T in vitro. / Pertussis toxin (PTX) is an exotoxin uniquely produced from Bordetella pertussis, a human respiratory tract pathogen causing pertussis disease, also known as whooping cough. The toxin is well described its virulence effects during bacterial infection. Most of these effects are due to ADP-ribosyltransferase activity of the molecule that targets G-protein coupled receptors (GPCR). On the other hand, PTX is an important antigen that provides protection against pertussis disease and a major component of all current pertussis vaccines. There are numerous literatures on PTX about its molecular mechanisms and its role during infection phase. Instead, lack of information on how PTX contributes host’s adaptive immunity has incurred confusion in understanding the immunogenic role of PTX. With intranasal infection model of B. pertussis, we detected the generation of CD4 lung-resident memory T cells (Trm) were depending on PTX exposure. For T cell migration study, PTX is being used to inhibit chemokine response. Because most of chemokine receptors are GPCR, the motility of many immune cells including T cells is easily affected by PTX. T cell migration is a sophisticate phenomenon regulated space-temporally. The results demonstrated, once T cells become activated and effector, are less influenced than inactivated T cells.This thesis reports the impact of PTX on T cells in two parts; 1) Role of PTX in adaptive immune response by in vivo infection system and 2) Influence of PTX on T cell motility by in vitro assays.

La matrice extracellulaire est un réseau enchevêtré de macromolécules variées, en étroite relation avec les cellules qu'elle environne. Les interactions bidirectionnelles qui s'établissent entre les cellules et leur microenvironnement matriciel régulent mutuellement leur comportement et devenir. La diversité biochimique des constituants moléculaires de la matrice, leurs propriétés biophysiques, leur architecture tout comme leur dynamique représentent autant de signaux régulateurs. Parmi les constituants de la matrice, la fibronectine (FN) est une glycoprotéine structurale et fonctionnelle majeure intervenant dans de nombreux processus physiologiques et pathologiques. Ces fonctions diverses sont directement liées à la dynamique structurale de cette protéine et à sa capacité à interagir avec les autres molécules matricielles, dont elle-même. Retrouvée sous forme soluble dans les fluides biologiques, la FN est incorporée dans les matrices insolubles sous forme d'assemblages supramoléculaires principalement fibrillaires mais aussi sous forme d'agrégats. Ces assemblages sembleraient être impliqués dans des processus physiologiques et pathologiques distincts.Si l'étude des assemblages de FN est rendue possible par l'élaboration de modèles in vitro, les mécanismes de polymérisation et l'effet d'assemblages de structures définies sur le comportement cellulaire restent cependant à mieux élucider et constituent le cœur de ce travail.Les travaux ont donc consisté à élaborer des assemblages de FN, à caractériser les mécanismes et structures impliqués dans leur polymérisation, et à étudier leur influence sur un modèle de cellules cancéreuses ovariennes. D'autre part, des études préliminaires comparatives ont été menées avec un analogue végétal de la FN.L'irréversibilité de la dénaturation thermique de la FN entraîne la formation d'agrégats de type amyloïde. Deux populations d'agrégats coexistent en solution. Cette agrégation est corrélée à une diminution de l'accessibilité des sites de liaison à la gélatine et des sites RGD, et à une diminution de l'incorporation dans les réseaux matriciels. De plus, si la FN sous sa forme agrégée n'est pas cytotoxique pour les cellules étudiées, la modification de la conformation de la FN favorise leur migration isolée et aléatoire.Ces résultats soulèvent la question de l'implication de ces agrégats de FN dans des processus pathologiques tels que le développement tumoral. / Extracellular matrix is a complex meshwork of various macromolecules that have a tight relationship with the surrounding cells. Bidirectional interactions between cells and the microenvironment control their respective behaviors and fate. The biochemical diversity of matrix molecular components, their biophysical properties, their architecture but also their dynamic represent as many regulator signals. Among the components of the matrix, fibronectin (FN) is a major structural and functional glycoprotein involved in numerous physiological and pathological processes. These various functions are directly linked to the structural dynamic of this protein and its ability to interact with others matrix components, in particular with itself. Found as a soluble protein in biological fluids, FN is also incorporated in insoluble matrix as supramolecular assemblies, mainly fibrils but also aggregates. These assemblies could be involved in distinct physiological and pathological processes.If the study of the assembly of the FN is possible with the help of in vitro models, the mechanism of polymerization and the effects of defined assemblies on the cell behavior still have to be better defined.Therefore, this work consisted in elaborating FN assemblies, in characterizing the mechanisms and structures involved in their polymerization and in studying their influence on behaviors of a model of ovarian cancer cells. Besides, preliminary comparative studies have been performed with a plant analogous of FN.We show that irreversible thermal unfolding of FN triggers amyloid-like aggregation. Two states of aggregates could coexist in solution. FN aggregation correlates with a decrease of gelatin-binding domain and RGD sequence accessibility, and a decrease of the incorporation in the matrix network. Moreover, if aggregates are not cytotoxic for the studied cells, conformation change of FN promotes their single-cell and random migration.These results raise questions about the role of FN aggregates in pathological processes like tumor development.

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.

The cell infiltration into the myocardial infarction (MI) site was studied using magnetic resonance imaging (MRI) with micrometer-sized iron oxide particles (MPIO) as cell labeling probes. MI is a leading cause of global death and disability. However, the roles of inflammatory cells and stem cells during the post-MI remodeling and repair processes are yet to be discovered. This study was to develop noninvasive MRI techniques to monitor and quantify the cellular infiltration into the MI site. MPIO can produce pronounced signal attenuation at regions of interest in MRI. Therefore, cells labeled with these particles can be detected after they are activated and home to the MI site. In the first project, MPIO of various doses were injected into the mouse blood stream 7 days before the MI surgery. Serial MRI was performed at various time points post-MI to monitor the inflammatory cell infiltration into the MI site. Significant signal attenuation caused by labeled cells, in particular macrophages, was observed at the MI site. The study suggests an optimal imaging window should be from 7 to 14 days post-MI, during which the MR signal was inversely proportional to the MPIO dose. The study also suggests an optimal MPIO dose should be between 9.1 and 14.5 µg Fe/g body weight. In the second project, mesenchymal stem cells labeled with MPIO were transplanted into the mouse bone marrow 14 days before the MI surgery. Serial MRI was performed at various time points post-MI to monitor the labeled cells, which mobilized from the bone marrow and homed to the MI site. All the MRI findings were further confirmed by histology. In addition to revealing the characteristics of cell infiltration during MI, this study also provides noninvasive MRI techniques to monitor and potentially quantify labeled cells at the pathological site. The technique can also be used to investigate the function of cells engaged in MI and to test the effect on cell infiltration caused by any treatment strategies.

Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo. The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them. In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration. Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification. It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively. These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.