Дисертації з теми "Cellular Migration"

Migration of myogenic cells is an important step in myogenesis and skeletal muscle repair. Migration is required for the cells to reach the site of damage, for their alignment and subsequent fusion. Limited migration is also one of the limitations of proposed therapies of diseases, such as Duchenne Muscular Dystrophy (DMD). Therefore, revealing the regulators of myogenic cell migration is important for improving our knowledge of myogenesis, but could also be applied in therapies for conditions, associated with loss of muscle mass and muscle weakness. In this thesis, extracellular and intracellular regulation of C2C12 myoblast migration was investigated. It was demonstrated that medium conditioned by myotube cultures in vitro, is capable of inducing the migration and chemotaxis of myoblasts. A model of serially passaged myoblasts was used to reveal potential changes in the migratory behaviour of these cells, in the context of skeletal muscle ageing. PI3K/AKT and MAPK/ERK pathways were investigated and their requirement for the process of myoblast migration was revealed. Further activation of these pathways with phospho-tyrsoine phosphatase and PTEN inhibitor Bpv(Hopic) was associated with larger increases in myoblast migration. Silencing of either PI3K/AKT or MAPK/ERK signalling pathways, in a situation where the other pathway remained activated, resulted in a significant inhibition of myoblast migration. Similarly, inhibition of FAK signalling, using the PF-228 inhibitor did not significantly affect PI3K/AKT and MAPK/ERK pathways, but resulted in reduced myoblast migration, suggesting the indispensability of individual signalling pathways for myoblast migration in response to myotube CM, regardless of the activity of other signalling pathways. Finally, considering the link between myoblast fusion and migration and in an attempt to propose genetic targets for future research, an investigation was made on the expression of Spire and Formin genes, involved in actin polymerisation and intracellular trafficking, in myoblasts undergoing differentiation and fusion. The expression of these genes was revealed in C2C12 myoblasts and it was demonstrated that the expression levels of two of these genes (Spire1 and Formin1) are altered following inhibition of myoblast differentiation/fusion by both 10μM Bpv(Hopic) and serial passaging, suggesting their potential association with these processes. Further investigations to reveal the function of Spire and Formin genes and their protein products in skeletal muscle are proposed.

2011/2012
ABSTRACT - The ability to control the cellular microenvironment, such as cell-substrate and cell-cell interactions at the micro- and nanoscale, is important for advances in several fields such as medicine and immunology, biochemistry, biomaterials, and tissue engineering. In order to undergo fundamental biological processes, most mammalian cells must adhere to the underlying extracellular matrix (ECM), eliciting cell adhesion and migration processes that are critical to embryogenesis, angiogenesis, wound healing, tissue repair, and immunity response, to cite few. For instance, upon receiving and responding to complex molecular signals, cells migrate from the epithelial layers to target locations, where they differentiate to form specialised cells that make up various organs and tissues. However, improper cell adhesion and migration have been implicated in disease states such as tumour invasion and cancer cell metastasis. In the past few years, several tailored surfaces that aim to mimic cell-ECM interactions have been developed, including biodevices based on proteins and shorter peptide chains, DNA, RNA, and lipids. Among the different nanomaterials employed in such studies, those resulting from self-assembled monolayers (SAMs) of alkanethiols on gold (Au) probably represent the most useful and flexible model systems of surface engineering for cell biology evaluations. These platforms are promising for tuning surface properties or to introduce novel biofunctionalities via coupling reactions with various alkanethiols tail groups that can be exposed to the solution phase. Deeply involved in this research field, the aim of this doctoral work was to gain a basic understanding and develop chemical strategies towards the controlled multidirectional (i. e. bidirectional) cellular migration on tailored Au surfaces. As already described, several artificial substrates were prepared in the last years to better understand the cellular responses to different mechanical and biochemical surface properties. To date, however, no reports concerning the bidirectional movement of the cells along a defined substrate have been published. The controlled multidirectional migration offers several advantages respect to the monodirectional approach, since the cellular functions can be obtained and, in principle, recycled with spatio-temporal control. In fact, once the cells reach the target position along the surface and perform specific biochemical or physiological cellular functions (repair, growth, movement, immunity, communication, and phago/endocytosis), the reversible movement could allow to recall them back to the starting position. By this way, also studies of dynamic cell-cell interactions can also be exploited allowing for a deeper knowledge about the fundamentals of the cell biology and biochemistry. The multidirectional migration can be determined through the production of dynamic haptotactic chemical gradients along Au surfaces. Specifically, the long-term idea of this project is to use SAMs of thiolated DNA chains (DNA-SH) adsorbed onto Au surfaces as a template for the hybridisation with complementary peptidic nucleic acid (PNA) strands functionalised with peptidic motifs able to stimulate cellular motility. By this way, supramolecular chemical gradients of motogenic motifs can be bound in a directional manner onto Au surfaces and dictate a dynamic bidirectional cell migration. Framed in such research project, this doctoral thesis focused on the production of a static, monodirectional and motogenic gradient along Au surfaces, to prove the efficacy of a specific peptidic motif, and generate modified PNA strands necessary for the production of the corresponding dynamic gradients. Chapter 1 deals with a careful description of the biochemical mechanisms involved in the cellular migration process, focusing on the chemotaxis and haptotaxis phenomena. Through a comprehensive overview on the state of the art concerning the biomimetic approaches for studying the cellular migration, the main strategies towards the engineering of different surfaces, have been thoroughly reviewed by means of key examples reported in the literature. Chapter 2 is centred on the results obtained by producing and using the thiolated peptide isoleucine-glycine-aspartic acid-glutammine-lysine-1-thiol decanoic acid (IGDQK-SH) as a motogenic motif for both cells found in physiologic environment (fibroblasts) and phatological conditions (MDA-MB-231 cancer cells). Upon synthesising IGDQK-SH (1), a systematic approach for the generation of the motogenic chemical gradient along Au surfaces has been developed. Evidences of the success of the preparation of such static chemical gradient were obtaining by engaging specific characterisation methodologies, such as water contact angle (WCA), Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analysis, along with computational analysis of peptide’s conformations once bound to the different Au surfaces. This allowed determining the biophysical properties, morphology, chemical composition and possible structure of the resulting Au surfaces, respectively. IGDQK-SH chemical gradient was able to induce and control the cellular migration of the two different cell lines showing interesting differences related to the surface properties and peptide’s conformations after the formation of SAMs in the presence of filler molecules with different hydrophobicity. In particular, the experimental findings suggested a pronounced migration attitude of the cancer cells upon their exposition to the IGDQK-SH-bearing surfaces, compared to the fibroblasts. This result might suggest a role of the IGD motif in the stimulation of the cancer cells towards their enhanced motility and metastatic progression in vivo, and is currently under investigation. Once proved the efficiency of the motogenic peptide, we moved towards the final goal of the project synthesising two functionalised single-stranded PNA dodecamers (ssPNA 12-mers) 30 and 31 bearing the Rhodamine B and the tetrapeptide IGDQ for characterising the chemical gradient through microscopy-based investigations and stimulate cell motility, respectively. Chapter 3 indeed provides a general overview on the different methodologies available for the solid phase peptide synthesis (SPPS) describing the synthetic attempts to produce the desired PNAs. Attention will be focused on the Fmoc/Cbz protecting group strategy, which allowed us to isolate the target PNA oligomers.
RIASSUNTO - Lo studio e il controllo dei microambienti cellulari, quali interazioni cellula-superficie e cellula-cellula, assumono particolare rilevanza in diversi campi scientifici come medicina e immunologia, biochimica, ingegneria dei tessuti e dei biomateriali. Al fine di svolgere le funzioni biologiche fondamentali, le cellule dei mammiferi devono poter aderire alla matrice extra-cellulare (ECM) sottostante, provocando adesione e migrazione cellulare che risultano essenziali, ad esempio, nei processi di embriogenesi, angiogenesi e riparazione dei tessuti. Infatti, stimolate da complessi segnali molecolari, le cellule migrano dagli strati epiteliali verso il loro target, raggiunto il quale si differenziano e specializzano formando organi e tessuti. Al contrario, anomalie nell’adesione e migrazione cellulare possono dar luogo al sorgere di diverse malattie, quali tumori e metastasi cancerose. Negli ultimi anni sono state progettate e sviluppate diverse superfici, compresi biodispositivi basati su proteine, DNA, RNA e lipidi, con lo scopo di mimare le interazioni cellula-ECM. Tra i nanomateriali impiegati in questi studi, quelli derivanti dalla formazione di self-assembled monolayers (SAMs) di tioli alchilici su oro (Au) rappresentano probabilmente il modello più adatto e flessibile di superfici ingegnerizzate al fine di valutare i fenomeni biologici. Questi sistemi permettono di modulare le proprietà delle superfici o di introdurre nuovi gruppi funzionali attraverso reazioni di coupling, sfruttando la presenza dei gruppi terminali dei tioli che risultano esposti al solvente. Lo scopo di questo lavoro di dottorato è quello di acquisire le conoscenze di base e di sviluppare metodologie chimiche al fine di indurre e controllare la migrazione cellulare multidirezionale (i.e. bidirezionale) su superfici di Au funzionalizzate. Come già descritto, negli anni sono stati impiegati diversi substrati artificiali con lo scopo di meglio comprendere le reazioni cellulari alle differenti proprietà meccaniche e biochimiche di tali superfici. Tuttavia, ad oggi, non sono stati ancora pubblicati studi riguardanti il movimento bidirezionale di cellule lungo un substrato. Rispetto all’approccio monodirezionale, la migrazione multidirezionale controllata offre diversi vantaggi, poiché in questo modo le funzioni cellulari possono essere indotte e, in principio, replicate attraverso un controllo spazio-temporale. Infatti, una volta raggiunto l’obiettivo sulla superficie e svolte le funzioni cellulari specifiche (riparazione, crescita, movimento, immunità, comunicazione, fagocitosi), il movimento reversibile permette di richiamare le cellule alla posizione iniziale. Pertanto, anche lo studio delle interazioni dinamiche cellula-cellula potrà fornire una più approfondita conoscenza della biologia e della biochimica cellulare. La migrazione multidirezionale può essere determinata attraverso la produzione di gradienti chimici dinamici aptotattici su superfici di Au. Nel dettaglio, l’idea alla base di questo progetto è quella di utilizzare SAMs di catene di DNA aventi un tiolo terminale (ssDNA-SH) per la funzionalizzazione di superfici di Au, e usarle come template nell’ibridizzazione con catene complementari di acido nucleico peptidico (PNA) aventi un peptide in grado di stimolare la migrazione cellulare. In questo modo è possibile generare un gradiente chimico supramolecolare direzionale lungo le superfici di Au al fine di ottenere al migrazione cellulare bidirezionale. Questa tesi di dottarato è focalizzata sulla produzione di un gradiente statico, monodirezionale e motogenico su superfici di Au, per provare l’efficacia di un motivo peptidico specifico, e generare filamenti di PNA modificati, necessari per la produzione di corrispondenti gradienti dinamici. Il Capitolo 1 riporta un’accurata descrizione dei meccanismi biochimici coinvolti nei processi di migrazione cellulare, concentrandosi sui fenomeni di chemiotassi e aptotassi. Dopo un’esauriente studio dello stato dell’arte, le principali strategie di funzionalizzazione di diverse superfici sono state dettagliatamente riviste attraverso gli esempi chiave riportati in letteratura. Il Capitolo 2 è centrato sui risultati ottenuti producendo e utilizzando il pentapeptide composto da isoleucina-glicina-acido aspartico-glutammina-lisina-acido decanoico-1-tiolo (IGDQK-SH) come motivo motogenico per le cellule presenti in ambienti fisiologici (fibroblasti) e in condizioni patologiche (MDA-MB-231 cellule cangerogene). Una volta sintetizzato l’IGDQK-SH(1) è stato sviluppato un approccio sistematico per la produzione del gradiente motogenico sulle superfici di Au. Al fine di verificare l’effettiva presenza di tale gradiente sono state utilizzate differenti tecniche di caratterizzazione, quali water contact angle (WCA), Atomic Force Microscopy (AFM) e X-ray photoelectron spectroscopy (XPS) analysis, oltre all’analisi computazionale per stabilire la conformazione del peptide una volta legato alla superficie di Au. Questo ha permesso di determinare le proprietà biofisiche, la morfologia, la composizione chimica e la possibile struttura delle superfici finali di Au funzionalizzate. Il gradiente chimico di IGDQK-SH ha permesso di indurre e controllare la migrazione di due differenti linee cellulari, mostrando interessanti differenze relative alle proprietà della superficie e alla conformazione del peptide dopo la formazione del SAMs in presenza di molecole filler aventi diversa idrofobicità. In particolare, i risultati sperimentali suggeriscono una maggiore attitudine alla migrazione da parte delle cellule cancerogene su superfici di Au funzionalizzate con il peptide IGDQK-SH rispetto ai fibroblasti. Questo risultato potrebbe suggerire un ruolo del motivo IGD nella stimolazione della mobilità e della progressione metastatica in vivo delle cellule cancerogene, ed è attualmente oggetto di ricerca. Una volta provata l’efficienza del peptide motogenico, obiettivo finale di questo lavoro è stata la sintesi di due singoli filamenti di dodecamero di PNA 30 e 31 funzionalizzati rispettivamente con la Rodammina B e il tetrapeptide IGDKQ al fine di caratterizzare il gradiente chimico utilizzando tecniche microscopiche e stimolare la migrazione cellulare. Il Capitolo 3 offre una visione generale sulle differenti metodologie impiegate nella sintesi peptidica in fase solida (SPPS), descrivendo le strategie sintetiche utilizzate per produrre gli oligomeri di PNA necessari, con particolare attenzione per la strategia dei gruppi protettivi Fmoc/Cbz.
RéSUMé - La possibilité de contrôler le microenvironnement cellulaire, telles que les interactions cellule-substrat et cellule-cellule à l’échelle micro et nano, est importante pour les avancées dans certains domaines tels que la médecine et l’immunologie, la biochimie, les biomatériaux, et l’ingénierie tissulaire. Afin d’être soumis aux processus biologiques fondamentaux, la plupart des cellules mammifères doivent adhérer à la matrice extracellulaire sous-jacente (ECM), en induisant des procédés d’adhésion et de migration cellulaires qui sont critiques à l’embryogenèse, l’angiogenèse, la cicatrisation des blessures, la réparation des tissus, et la réponse immunitaire, pour n’en citer que quelques-uns. Par exemple, lorsque les cellules reçoivent et répondent à des signaux moléculaires complexes, elles migrent des couches épithéliales aux emplacements cibles, où elles se différencient afin de former des cellules spécialisées qui constituent divers organes et tissus. Cependant, une adhésion et une migration cellulaire incorrecte ont été impliquées dans des états de maladie tels que l’invasion de tumeur et les métastases de cellules cancéreuses. Au cours des dernières années, plusieurs surfaces confectionnées dans le but d’imiter les interactions cellule-ECM ont été développées, incluant des bio dispositifs basés sur des protéines et des chaines peptidiques courtes, sur l’ADN, l’ARN, et sur des lipides. Parmi les différents nanomatériaux employés dans de telles études, ceux résultants de monocouches auto-assemblées (SAMs) d’alcanethiols sur l’or (Au) représentent probablement les systèmes modèles les plus utiles et flexibles d’ingénierie de surface pour des évaluations biologiques cellulaires. Ces plateformes sont prometteuses pour moduler des propriétés de surface ou pour introduire de nouvelles biofonctionnalités via des réactions de couplage avec divers groupements alcanethiols qui peuvent être exposés à la phase liquide. Fortement impliqué dans ce domaine de recherche, l’objectif de ce travail de doctorat était d’acquérir une compréhension basique et de développer des stratégies chimiques à l’égard de la migration cellulaire multidirectionnelle contrôlée (i.e. bidirectionnelle) sur des surfaces d’Au fonctionnalisées. Comme cela a déjà été décrit, plusieurs substrats artificiels ont été préparés au cours des dernières années afin de mieux comprendre les réponses cellulaires à différentes propriétés mécaniques et biochimiques de surface. Cependant, jusqu’à présent, aucun rapport sur le mouvement bidirectionnel de cellules le long d’un substrat défini n’a été publié. La migration multidirectionnelle contrôlée offre plusieurs avantages par rapport à l’approche monodirectionnelle, puisque les fonctions cellulaires peuvent être obtenues et, en principe, recyclées avec un contrôle spatio-temporel. En fait, une fois que les cellules atteignent la position cible le long de la surface et réalisent des fonctions cellulaires biochimiques ou physiologiques spécifiques (réparation, croissance, mouvement, immunité, communication, et phago/endocytose), le mouvement réversible pourrait permettre de les rappeler à la position de départ. De cette façon, des études d’interactions cellule-cellule dynamiques peuvent également être exploitées, menant à une connaissance plus approfondie des fondamentaux de la biologie et biochimie des cellules. La migration multidirectionnelle peut être établie par la production de gradients dynamiques chimiques haptotactiques le long de surfaces d’Au. Plus précisément, l’idée à long terme de ce projet est d’utiliser des SAMs de chaînes d’ADN thiolées (ADN-SH) adsorbées sur des surfaces d’Au comme modèles pour l’hybridation avec des brins d’acides nucléiques peptidiques (ANP) complémentaires, fonctionnalisés avec des motifs peptidiques capables de stimuler la motilité cellulaire. De cette façon, les gradients chimiques supramoléculaires de motifs motogéniques peuvent être liés d’une manière directionnelle sur des surfaces d’Au et peuvent dicter une migration cellulaire bidirectionnelle dynamique. Cette thèse de doctorat, incluse dans un tel projet de recherche, s’est concentrée sur la production d’un gradient statique, directionnel et motogénique le long de surfaces d’Au, afin de prouver l’efficacité d’un motif peptidique spécifique, et de générer des brins d’ANP modifiés nécessaires à la production des gradients dynamiques correspondant. Le Chapitre 1 donne une description minutieuse des mécanismes biochimiques impliqués dans le procédé de migration cellulaire, se concentrant sur les phénomènes de chimitaxie et haptotaxie. A travers une vue d’ensemble complète sur l’état de l’art des approches biomimétiques pour l’étude de la migration cellulaire, les stratégies principales menant à l’ingénierie de différentes surfaces, ont été revues en détails à l’aide d’exemples clés reportés dans la littérature. Le Chapitre 2 est centré sur les résultats obtenus par la formation et l’utilisation du peptide thiolé isoleucine-glycine-aspartic acid-glutammine-lysine-1-thiol decanoic acid (IGDQK-SH) en tant que motif motogénique pour les cellules à la fois trouvées dans un environnement physiologique (fibroblastes) et dans des conditions pathologiques (cellules cancéreuses MDA-MB-231). Après avoir synthétisé IGDQK-SH (1), une approche systématique pour la génération du gradient chimique motogénique le long de surfaces d’Au a été développée. Des preuves du succès de la préparation de tels gradients chimiques statiques ont été obtenus par des méthodologies de caractérisation spécifiques, telles que des analyses d’angle de contact (WCA), par microscopie à force atomique (AFM) et par spectrométrie photoélectronique X (XPS), accompagné d’analyses informatiques des conformations du peptide une fois lié aux différentes surfaces d’Au. Ceci a permis de déterminer les propriétés biophysiques, la morphologie, la composition chimique et la structure possible des surfaces d’Au résultantes, respectivement. Le gradient chimique de IGDQK-SH a pu induire et contrôler la migration cellulaire de deux différentes lignes cellulaires montrant des différences intéressantes liées aux propriétés de surface et aux conformations du peptide après la formation des SAMs en présence de molécules de remplissage présentant différentes hydrophobicités. En particulier, les résultats expérimentaux ont suggéré une attitude de migration prononcée des cellules cancéreuses, après leur exposition aux surfaces portant l’IGDQK-SH, comparé aux fibroblastes. Ce résultat peut suggérer un rôle du motif IGD dans la stimulation des cellules cancéreuses à l’égard de leur mobilité accrue et progression métastatique in vivo, et est actuellement analysé. Une fois que l’efficacité du peptide motogénique fut prouvée, nous nous sommes penchés sur l’objectif final du projet, en synthétisant deux dodécamères d’ANPs simples brins fonctionnalisés 30 et 31, portant la Rhodamine B et le tétrapeptide IGDQ pour caractériser le gradient chimique par des analyses de microscopie et pour stimuler la motilité de la cellule, respectivement. En effet, le Chapitre 3 donne une vue d’ensemble sur les différentes méthodologies disponibles pour la SPPS décrivant les essais synthétiques afin de synthétiser les ANPs désirés. L’attention sera concentrée sur la stratégie impliquant les groupements protecteurs Fmoc/Cbz, qui nous a permis d’isoler les oligomères d’ANP cibles.
XXV Ciclo
1984

La motilité cellulaire directionnelle au cours du développement de l'organisme et des tissus, l'homéostasie et la maladie nécessite une rupture de symétrie. Ce processus repose sur la capacité des cellules individuelles à établir une polarité avant-arrière, et peut se produire en l'absence de signaux externes. L'initiation de la migration a été attribuée à la polarisation spontanée des composants du cytosquelette, tandis que l'évolution spatio-temporelle des forces du cytosquelette résultant de l'interaction mécanique cellule-substrat continue n'a pas encore été résolue. Ici, nous établissons un test de migration microfabriqué unidimensionnel qui imite un environnement fibrillaire complexe in vivo tout en étant compatible avec les mesures de force à haute résolution, la microscopie quantitative et l'optogénétique. La quantification des paramètres morphométriques et mécaniques révèle un comportement de stick-slip générique initié par un détachement stochastique des contacts adhésifs d'un côté de la cellule dépendant de la contractilité, qui est suffisant pour conduire la motilité cellulaire directionnelle en absence de polarité du cytosquelette préétablie ou de gradients morphogènes. Un modèle théorique valide le rôle crucial de la dynamique d'adhésion au cours de la rupture de symétrie spontanée, en proposant que le phénomène examiné puisse émerger indépendamment d'un système auto-polarisant complexe
Directional cell motility during organism and tissue development, homeostasis and disease requires symmetry breaking. This process relies on the ability of single cells to establish a front-rear polarity, and can occur in absence of external cues. The initiation of migration has been attributed to the spontaneous polarization of cytoskeleton components, while the spatio- temporal evolution of cytoskeletal forces arising from continuous mechanical cell-substrate interaction has yet to be resolved. Here, we establish a one- dimensional microfabricated migration assay that mimics complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to drive directional cell motility in absence of pre-established cytoskeleton polarity or morphogen gradients. A theoretical model validates the crucial role of adhesion dynamics during spontaneous symmetry breaking, proposing that the examined phenomenon can emerge independently of a complex self-polarizing system

Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration. Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo. The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA. The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface. The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed. In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.

Dictyostelium discoideum is a simple model widely used to study many cellular functions, including differentiation, gene regulation, cellular trafficking and directional migration. Adaptation mechanisms are essential in the regulation of these cellular processes. The misregulation of adaptation components often results in persistent activation of signaling pathways and aberrant cellular responses. Studying adaptation mechanisms regulating cellular migration will be crucial in the treatment of many pathological conditions in which motility plays a central role, such as tumor metastasis and acute inflammation. I will describe two adaptation mechanisms regulating directional migration in Dictyostelium cells. The Extracellular signal Regulated Kinase 2 (ERK2) plays an essential role in Dictyostelium cellular migration. ERK2 stimulates intracellular cAMP accumulation in chemotaxing cells. Aberrant ERK2 regulation results in aberrant cAMP levels and defective directional migration. The MAP Phosphatase with Leucine-rich repeats (MPL1) is crucial for ERK2 adaptation. Cells lacking, MPL1 (mpl1- cells) displayed higher pre-stimulus and persistent post-stimulus ERK2 phosphorylation, defective cAMP production and reduced cellular migration. Reintroduction of a full length Mpl1 into mpl1- cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild type cells (Wt). These results suggest Mpl1 is essential for proper regulation of ERK2 phosphorylation and optimal motility in Dictyostelium cells. Cellular polarization in Dictyostelium cells in part is regulated by the activation of the AGC-related kinase Protein Kinase Related B1 (PKBR1). The PP2A regulatory subunit, B56, and the Glycogen Synthase Kinase 3 (GSK3) are necessary for PKBR1 adaptation in Dictyostelium cells. Cells lacking B56, psrA-cells, exhibited high basal and post-stimulus persistent phosphorylation of PKBR1, increased phosphorylation of PKBR1 substrates, and aberrant motility. PKBR1 adaptation is also regulated by the GSK3. When the levels of active GSK3 are reduced in Wt and psrA- cells, high basal levels of phosphorylated PKBR1 were observed, in a Ras dependent, but B56 independent mechanism. Altogether, PKBR1 adaptation is regulated by at least two independent mechanisms: one by GSK3 and another by PP2A/B56.

La capacité des cellules à générer spontanément de l'ordre a l’échelle supra cellulaire repose sur l'interaction de signaux mécaniques et biochimiques. Si le consensus général est que la signalisation chimique est le régulateur principal du comportement cellulaire, il est aujourd’hui bien établi que l'impact des facteurs mécaniques est primordial sur des processus fondamentaux de la physiologie cellulaire tel que la différenciation, la prolifération, la motilité et qu’une dérégulation des paramètres mécaniques du microenvironnement des cellules sont impliqués dans un grand nombre de pathologies allant du cancer aux myopathies. Dans ce contexte, plusieurs études ont récemment mis en évidence l'existence d’ondes mécaniques se propageant à l’échelle supra-cellulaire.Nous étudions dans le cadre de cette thèse l'origine de ces ondes de vitesse dans les tissus et discutons leur origine biologique. En pratique, nous confinons des monocouches de cellules épithéliales à des géométries quasi unidimensionnelles, pour forcer l'établissement presque omniprésent d'ondes au niveau tissulaire. En accordant la longueur des tissus, nous découvrons l'existence d'une transition de phase entre les oscillations globales et multi-nodales, et prouvons que dans ce dernier régime, longueur d'onde et période sont indépendantes de la longueur de confinement. Ces résultats démontrent que l’origine de ces oscillations est intrinsèque au système biologique, ce mécanisme apparait comme un candidat pertinent permettant aux cellules de mesurer avec précision des distances au niveau supra-cellulaire et potentiellement de structurer spatialement un tissu. Des simulations numériques basées sur un modèle de type Self-propelled Voronoi reproduisent la transition de phase que nous avons observé expérimentalement et aident à guider nos recherches sur l'origine de ces phénomènes ondulatoires et leur rôle potentiel dans l'apparition spontanée des follicules pileux dans les explants cutanés des souris
The ability of organisms to spontaneously generate order relies on the intricate interplay of mechanical and bio-chemical signals. If the general consensus is that chemical signaling governs the behavior of cells, an increasing amount of evidence points towards the impact of mechanical factors into differentiation, proliferation, motility and cancer progression. In this context, several studies recently highlighted the existence of long-range mechanical excitations (i.e. waves) at the supra-cellular level.Here, we investigate the origins of those velocity waves in tissues and their correlation with the presence of boundaries. Practically, we confine epithelial cell mono-layers to quasi-one dimensional geometries, to force the almost ubiquitous establishment of tissue-level waves. By tuning the length of the tissues, we uncover the existence of a phase transition between global and multi-nodal oscillations, and prove that in the latter regime, wavelength and period are independent of the confinement length. Together, these results demonstrate the intrinsic origin of tissue oscillations, which could provide cells with a mechanism to accurately measure distances at the supra-cellular level and ultimately lead to spatial patterning. Numerical simulations based on a Self-propelled Voronoi model reproduce the phase transition we measured experimentally and help in guiding our preliminary investigations on the origin of these wave-like phenomena, and their potential role for the spontaneous appearance of hair follicles in mouse skin explants

Glioblastoma multiforme (GBM) is the most common and deadliest brain cancer in adults. Despite considerable efforts at both bench and bedside, the average survival for GBM patients is only 14-15 months. This dismal prognosis stems from challenges in treatment and a malignant tumour biology. A key need in addressing GBM is to better understand and therapeutically target GBM cell invasion into the surrounding healthy brain tissue. Cytoskeletal remodelling and dynamics, mediated by ROCK effector proteins, play an important role in the ability of GBM cells to migrate. ROCK inhibition is being considered as potential cancer therapy; however, there is insufficient data examining a chemical pan-ROCK inhibition effect in the cellular context of GBM. I address this gap in the context of undifferentiated patient-derived brain tumour stem cell (BTSC) models. My results show that chemical ROCK pathway inhibition with several different compounds led to a reversible neurite-like outgrowth phenotype across three different patient-derived cell models. This phenotype was accompanied by a decrease in BTSCs motility, which enabled the cells to form an interactive multicellular network. Interestingly, ROCK inhibition did not alter the self-renewal ability or proliferation capacity of BTSCs. To further investigate this diffusive nature of GBM cells, I developed an in vitro 3D model that allows the study of GBM infiltration in real-time. My work demonstrates the ability of GBM spheres to spontaneously fuse with, and infiltrate, neural-like early-stage cerebral organoids (eCOs) with the use of stem cell culture-based organoid methodology. In addition, this 'hybrid' GBM tumour organoid possessed an invasive tumour compartment, which was specific to GBM cells. Thus, this self-assembly GBM tumour organoid may be used to identify anti-GBM invasion treatment approaches.

Le bon fonctionnement des réseaux neuronaux dépend des interactions entre les neurones et les cellules gliales. Alors que de nombreux efforts ont été faits pour comprendre les interactions entre les neurones, moins est connu sur la nature des interactions entre les cellules gliales ; ceci est due à la complexité du système nerveux des vertébrés, qui comprend plus de cellules gliales que de neurones. Cependant, le système nerveux de la drosophile à un rapport neurones-cellules gliales faible, ce qui fait de cet animal simple un modèle idéal pour évaluer ce concept. J’ai utilisé des approches génétiques à résolution cellulaire pour disséquer les mécanismes cellulaires et moléculaires de la migration collective des cellules gliales in vivo. En résumé, mes données révèlent les bases du mécanisme contrôlant la migration cellulaire collective : 1) les cellules du front de migration interagissent entre elles en amont et en aval et 2) N-cad est nécessaire pour une migration optimal de la glie
The functionality of the complex neural network depends on the interactions between neurons and glia. While many efforts have been made to understand the neuron-neuron interactions, less is known about those amongst glial cells. Due to the complexity of the vertebrate nervous system, which comprises manifold more glia than neurons, it is hard to tackle the role of glia-glia interactions. The nervous system of Drosophila, however, has a lower glia-neuron ratio, which makes this simple animal an ideal model. I use genetic approaches at cellular resolution to dissect the cellular and molecular mechanisms of glial collective migration in vivo. In Sum, I have shown some basic mechanism controlling collective cell migration: 1) cells at the front of the collective interact with each other through anterograde and retrograde bidirectional interaction. 2) N-cad appears necessary for timely movement of glial community

The eukaryotic cytoskeleton is composed of varying proteinaceous filaments and is responsible for intracellular transport, cell proliferation, cell morphogenesis, and cell motility. Microtubules are one of three cytoskeletal components and have a unique polymer structure. The hollow cylinders undergo rapid polymerization and depolymerization events (i.e. dynamic instability) to promote assembly at the leading edge of the cell and disassembly in the rear of the cell to drive the cell front forward and facilitate directional migration. High-resolution light microscopy and automated tracking allow visualization and quantification of microtubule dynamics (i.e. growth speeds and growth lifetimes) during time-lapse imaging. These techniques were used to understand how the physical environment influences molecular control of endothelial cell morphology. The ultimate goal of this work is to test hypotheses relevant to vascular development and diseases associated with endothelial cell angiogenesis – defined as the development of new blood vessels from pre-existing vessels. Angiogenesis is of particular relevance because it is a commonality underlying many diseases affecting over one billion people worldwide, including all cancers, cardiovascular disease, blindness, arthritis, and Alzheimer's disease.

Breast cancer is the most common malignancy in women, affecting one out of eight women worldwide. Even if most of the breast tumors are efficiently treated using targeted therapies, there is still a heterogeneous breast cancer subpopulation known as "triple-negative", which is highly metastatic and, due to the absence of targeted therapies, of poor prognosis. The elucidation of the processes involved in tumor progression and metastasis remains an important challenge in the search for new therapies against this subtype of breast cancer. Previous results from the laboratory have shown that ATIP3, a major product of the candidate tumor suppressor gene MTUS1, is a microtubule associated protein (MAP), whose expression is decreased in 85% of high grade, 83% of triple negative and 62% of metastatic breast carcinomas. Re-expression of ATIP3 in breast cancer cells significantly reduces cell proliferation in vitro, and tumor growth in vivo. Based on these results, my PhD project aimed at evaluating the role of ATIP3 in tumor cell migration and cancer metastasis. In the first part of my thesis, I will present data showing that ATIP3 is a novel prognostic marker for breast cancer patients' survival and a new anti-metastatic molecule. By means of DNA microarray analysis, we showed that low ATIP3 expression levels correlate with reduced overall survival of metastatic breast cancer patients. Using an in vivo model for cancer metastasis, we then showed that re-expression of ATIP3 reduces metastatic progression and lowers the number and size of metastatic foci. At the functional level, ATIP3 reduces breast cancer cell migration by reducing cell velocity and directionality. At the molecular level we further showed, using nocodazole washout experiments and MT growing ends tracking, that ATIP3 slows MT regrowth and decreases MT dynamics. Altogether, these studies indicate that ATIP3 is a novel MT stabilizing protein that controls the ability of MT tips to reach the cell cortex during migration, a mechanism that may account for reduced cell migration and metastasis. In the second part of my thesis, I will present data investigating the mechanisms by which ATIP3 regulates MT dynamics. To this end, we searched for new ATIP3-interacting partners. Interestingly, EB1, the core component of plus-end tracking proteins, was found to interact with ATIP3 not at the growing end of the MTs (as most EB1-interacting proteins), but mostly in the cytosol and at the MT lattice. The identification of the EB1-interacting domain of ATIP3 (termed CN) and further characterization of deletion mutants revealed that ATIP3-EB1 interaction is involved in impaired accumulation of EB1 at the plus-end. Based on these results and on FRAP analysis of EB1-GFP fluorescence recovery, a model was proposed in which the interaction between ATIP3 and EB1 may slower EB1 turnover at the MT plus-end, possibly by limiting EB1 association with its recognition site. In line with this model, in ATIP3-depleted cells dynamic EB1 molecules are more prone to accumulate at the growing end to increase MT dynamics. Relevance of this model in human pathology was then tested by evaluating ATIP3-EB1 expression levels in breast tumors, indicating that combined relative expression levels of both proteins may be considered as a prognostic marker of patient survival. Finally, in a third part of my thesis, I will present some preliminary data showing that ATIP3 may interact with the depolymerizing kinesin MCAK and the tumor suppressor APC, both of which are also well-known partners of EB1. The characterization and the implication of these interactions on ATIP3 functions (MT dynamics for MCAK interaction and cell polarity for APC interaction) remains to be investigated.

Cellular behaviour depends ultimately on the transcription of genes. If we know how transcription is controlled we have a better chance of understanding cellular processes. This thesis presents six studies, all concerning cellular regulatory mechanisms. One study is purely experimental and five are computational studies. A large part of the research concerns the Epstein-Barr virus (EBV). We investigate the latency programme switching of EBV, with an equilibrium statistical mechanics model that describes the transcription activities of two central viral promoters. We demonstrate that this system is bistable and predict promoter activities that correlate well with experimental data. Further we study the switching efficiency of one of the promoters, highlighting how competitive binding of transcription factors generates a more efficient geneticswitch. The EBV protein EBNA1 is known to affect cellular gene expression. With a dinucleotide position weight matrix we search the complete human genome for regions with multiple EBNA1 binding sites. 40 potential binding regions are identified, with several of particular interest in relation to EBV infections. The final study on EBV is purely experimental, in which we demonstrate an interaction between the Syk kinase and integrin β4. Moreover, we show how reduced levels of these proteins affect migration of epithelial LMP2a positive cells, and hypothesise that these effects are due to the Syk-β4 interaction. The two remaining studies presented in this thesis concern other cellular systems. Dynamic properties of two different regulatory feedback mechanisms for transport and metabolism of small molecules are investigated. The synergetic effect of adding a regulatory loop is exemplified with the iron metabolism in bacteria. The final project concerns the λ phage. With the equilibrium statistical mechanics method for describing promoter activities we characterise the equilibrium properties of λ mutants and compare with experimental findings. We argue that the observed differences between model and experiment are due to a larger perturbation of the genetic circuit than presumed. The research presented in this thesis shed light on the properties of several regulatory mechanisms. As computational studies they add perspective to the experimental research in this field and provide new hypothesis for further research.
QC20100720

Une caractéristique fondamentale de nombreux types de cellules dans le corps, c'est leur capacité à se déplacer. L'aile de la drosophile en développement fournit un excellent modèle pour suivre le comportement de la migration des cellules gliales en chaînes in vitro et in vivo par microscopie time-lapse. Les cellules gliales sont organisées d'une manière tête-à-queue et se déplacent le long des axones. Avec la technique «UV-ablation», j’ai démontré que les cellules pionnières au début de la chaîne sont nécessaires pour la migration de la chaîne avant qu’elles commencent à bouger. Par l'ablation des cellules dans différentes combinaisons au début de la chaîne, j'ai montré que la première cellule isolée de ses voisins immédiats ne bouge pas. En augmentent le nombre de cellules isolées, la fonctionnalité de la migration en chaîne devient de plus en plus proche de contrôle. Ces données indiquent que l'intégrité dans le mouvement glial collectif pourraient être réalisés grâce à «l'effet communauté». Pendant la migration collective, les cellules sont toujours collées les unes aux autres par l'adhésion cellulaire. J'ai montré pour la première fois que N-cadhérin (N-cad) est présent dans les glies périphériques de pupe chez la drosophile. En surexprimant (GOF) ou repriment N-cad (LOF) spécifiquement dans les cellules gliales, j'ai observé, respectivement, un retard ou une accélération de l'achèvement de la migration. La répression de N-cad dans les glies cause une baisse modérée du nombre des jonctions adhérentes entre les cellules gliales. Toutefois, sur GOF de N-cad, Armadillo est recruté à la membrane cellulaire des glies
A basic characteristic of many cell types in the body is their ability to move. The developing wing of the Drosophila provides an excellent model system to follow the behavior of the chain-migrating glial cells in vitro and in vivo by time-lapse microscopy. The glial cells are organized in a head-to-tail manner and move along the axons. With UV-ablation technique, I show that the pioneer cells at the migration front are required to promote chain migration from early stages on. By ablating cells in different combination at the front of the chain, I show that the first cell isolated from its neighbors cannot move and eventually dies. By increasing the number of cells in isolation, the functionality of the group of cells gradually becomes alike that of a control chain. These data indicate that integrity in collective glial movement might be realized through “community effect”, where a certain number of cells are required to assemble and form a migratory unit. During collective movement, cells are attached to each other via cell-cell adhesion. I show for the first time that N-cad is present in the peripheral glia of the Drosophila embryo and pupa. By overexpressing or downregulating N-cad specifically in the glial cells, I observe delay or acceleration, respectively, in completion of migration but the integrity of the chain remains intact. This suggests a role for N-cad in regulating the timing of glial movement. The downregulation of N-cad in the glia causes a mild decrease in the number of glia-glia adherens junctions, however, upon N-cad overexpression, Armadillo is recruited to the cell membrane

En cas d'infection, les cellules dendritiques matures (CDm) migrent des tissus périphériques vers les ganglions lymphatiques où ils déclenchent la réponse immunitaire adaptative. Ce déplacement impose une série de contraintes physiques sur les CDm. Au niveau cellulaire, la migration des CDm repose sur la contractilité du cytosquelette d’actine et de myosine. Toutefois, la réponse mécanique spécifique qui permet aux CDm d'adapter leur mode de migration aux contraintes physiques n'a pas été entièrement caractérisée. Dans ce travail, nous avons combiné une série d'approches, des outils microfluidiques aux modèles ex vivo, pour disséquer les réarrangements du cytosquelette nécessaires à l’adaptation du mode de migration des cellules dendritiques aux propriétés physiques de leur microenvironnement. Nous avons montré que les CDm sont capables de maintenir une vitesse constante tout en migrant à différents niveaux de confinement. Cela révèle la capacité des CDm à adapter leur mode de migration en réponse aux changements dans la géométrie de leur microenvironnement. Au niveau cellulaire, le confinement dans les microcanaux induit un remodelage rapide et spécifique du cytosquelette d’actine et de myosine. Il est essentiel à la plasticité migratoire des CDm et optimise le déplacement de ces cellules dans des environnements 3D complexes. Ces travaux conduisent à une meilleure compréhension des mécanismes permettant aux CDm d’adapter leur motilité face à des structures tissulaires spécifiques. Ils permettront de mieux appréhender le contrôle de la migration des leucocytes dans des espaces confinés et pouvoir ainsi la moduler avec précision afin de favoriser ou de prévenir les réponses immunitaire
Upon infection, mature dendritic cells (mDCs) migrate from peripheral tissue to lymph nodes and initiate the adaptive immune response. This fast and tightly regulated process imposes a series of physical constraints and is tuned by different microenvironmental factors, such as the physical properties of the tissue. Mechanistically, mDCs migration relies on actomyosin flow and contractility, which are dependent on non‐muscular Myosin IIA activity. However, the specific mechanoresponse that allows mDCs to adapt their migration machinery to irregular 3D landscapes has not been fully characterized. In this work, we combined a series of approaches, from micro‐fabricated devices to ex vivo skin models, to dissect the cytoskeleton rearrangements used by mDCs to overcome the physical barriers imposed by the tissue. We have shown mDCs are able to maintain a constant speed while migrating at different levels of confinement. This reveals the extreme capacity of mDCs to adapt their migration machinery in response to changes in the geometry of their microenvironment. At the cellular level, confinement in microchannels induces a fast and specific actomyosin remodelling in mDCs. This reveals a complete actomyosin rearrangement triggered by confinement, which is essential for mDCs migratory plasticity that allows these cells to move in intricate 3D geometries. The full understanding of how mDCs and other leukocytes adapt their motility to specific tissue structures will provide better knowledge on how cell migration is controlled in confined spaces and new insight to finely tune their migration to promote or prevent immune responses

The directed migration of neurons is influenced by multiple guidance cues, that may include soluble attractive chemotactic factors and cell-cell contact mediated collective migration. The nature of these neuron-neuron interactions and their integration with chemotaxis remains unclear. Contact inhibition of locomotion (CIL), a process whereby cells undergoing a collision cease their migration towards the colliding cell, has been identified as a driving force behind the collective migration of several cell populations in vivo, but has not been described for neurons in the central nervous system. We have established that Cadherin2 (Cdh2), a cell adhesion molecule, mediates the physical interactions between facial branchiomotor neurons (FBMNs) that promote the collective mode of migration. Using live imaging, we observed transient cell-cell contact between the somas of FBMNs during migration. Following neuron-neuron collisions, we observed two directional outcomes: i) both neurons remain travelling posteriorly, or ii) the neurons migrate in opposite directions (one anterior and one posterior). This latter observation is a hallmark of CIL behavior. These CIL events occur in approximately 50% of soma-soma collisions. Consistent with the repulsive nature of CIL events, live imaging of Tg(isl1:GFP-CAAX)fish show that CIL events are characterized by a collapse of protrusions upon collision. Our data indicate that CIL-based neuron-neuron interactions influence the directionality of FBMN movement and may underlie the collective nature of FBMN migration. To determine whether chemotaxis could influence FBMN directionality after cell-cell collisions, we examined the interplay between Cdh2-mediated collective migration and SDF1a-mediated chemotaxis. We found partial FBMN migration defects under conditions when Cdh2 function is partially inactivated or when the chemokine SDF1a is knocked down. Strikingly, we find an almost complete migration block when both SDF1a is depleted and Cdh2 function is inactivated. These findings suggest that FBMNs integrate multiple inputs arising from cell-cell contact induced polarity changes and SDF1a-mediated chemotaxis to achieve sustained directed migration.

Le récepteur ERRα (Estrogen Receptor-Related Receptor alpha) appartient à la superfamille des récepteurs nucléaires. Une forte expression de ERRα est corrélée à un mauvais pronostic, suggérant l’implication de ce récepteur dans le processus métastatique. Mon projet est d'analyser le rôle de ERRα dans les mouvements cellulaires. J’ai montré qu’inhiber ERRα perturbe la migration cellulaire. L’étude des mouvements montre que l’absence de ERRα induit une perturbation de l’orientation cellulaire, du nombre des fibres de stress et des protrusions membranaires. Les cellules migrent de façon désorientée. J’ai démontré l’existence d’une cascade de régulation où ERRα stimule transcriptionnellement l'expression de la protéine BACURD2/TNFAIP1, elle-même régulant la stabilité de RhoA. Inhiber ERRα induit également une surexpression de RhoA, sa suractivation et une perturbation de la migration orientée. Cette cascade a été confirmée par des expériences de complémentation. J’ai vérifié ces résultats par des expériences in vivo et ex vivo, chez les souris KO pour ERRα. L’absence de ce récepteur induit une diminution de l’expression de TNFAIP1 inhibant la dégradation de RhoA et entrainant finalement une perturbation des mouvements cellulaires. Ainsi ERRα régule positivement la migration cellulaire conduisant à une forte potentialité métastatique dans les tumeurs surexprimant ce récepteur.L’ensemble de mes résultats pourrait faire de ERRα une nouvelle cible en vue de nouvelles thérapies anticancéreuses et nous pourrions proposer BACURD2/ TNFAIP1 comme un nouveau marqueur de pronostic dans les cancers
High expression of the orphan nuclear receptor ERRα is strongly correlated with poor prognosis in various types of tumors, including those of the breast. The fact that high ERRα expression in tumors is also correlated with elevated invasiveness suggests that this nuclear receptor positively regulates cell migration and invasiveness.This possibility was investigated using MDA-MB231 breast cancer cell line as a model. Inactivating ERRα impairs cell migration. Using time-lapse-based cell tracking analysis and Golgi positioning, we show that this impairment is not due to reduced migration speed but rather to cell disorientation. The enhanced number of cell protrusions present in migrating cells and disorganized actin fibers confirm this. In summary cells do migrate but do not sustain persistent linear movement. We observed that upon ERRα inactivation, RhoA, which is instrumental in oriented movement, is overexpressed at the protein level. Further analysis showed that the stability and proteasome-dependent degradation of the protein is affected. To analyze the relationship between ERRα (as a transcription factor) and RhoA protein stability we performed a transcriptomic analysis comparing (by RNA-Seq) wt cells to ERRα-depleted ones. We identified genes regulated by ERRα that are involved in both cell migration (as a biological process) and in protein stability and degradation, more specifically that of RhoA protein (as a molecular process). TNFAIP1/Bacurd2 is stimulated by ERRα and fits these criteria: this protein mediates the Culin3-based, proteasome-dependent of RhoA and its inactivation leads to defects in cell migration.TNFAIP1/RhoA cascade is a major downstream effector of ERRα in cell migration

Angiogenesis, the formation of new blood vessels, requires the reorganization of microtubules (MT), which are polar cytoskeletal structures, consisting of a free-plus end and a minus-end that is free or anchored. CAMSAP2 and CAMSAP3 have been shown to bind and stabilize MT minus-ends; yet, this activity's contribution to MT organization and directional migration is unknown. To investigate this contribution, we performed live-cell imaging of polarized HUVECs expressing CAMSAP2 or CAMSAP3. Our results show that CAMSAP2 and CAMSAP3 localized to the trailing edge of cells. Pharmacologic disassembly of MTs resulted in CAMSAP reorganization to the leading edge. MCAK expression is not sufficient for CAMSAP reorganization, but may recruit CAMSAP to the MT minus-end. MT growth dynamics analysis revealed that CAMSAP2 and CAMSAP3 promoted dynamic MT growth. These results suggest that CAMSAP2 and CAMSAP3 protect MTs against MCAK]mediated disassembly and also function to nucleate new, dynamic MTs at the leading edge.

Directional cell migration, chemotaxis, requires a polarized cell morphology in which the cell extends pseudopodia at the front and contracts the rear to move towards a stimulus. PI(4,5)P2 levels set up a threshold for the activity of signaling molecules at the rear and the leading-edge of a cell. To further demonstrate the importance of plasma membrane (PM) PI(4,5)P2 in maintaining cell morphology during chemotaxis, we used a mutant strain of the eukaryotic model system, Dictyostelium discoideum. This mutant strain lacks the type I PIP5 kinase, which is the main enzyme synthesizing PI(4,5)P2. These cells, designated pikI-, have highly reduced PI(4,5)P2 levels and higher Ras GTPase activity compared to wildtype cells. Leading-edge biosensors diffuse to the cytosol when the pikI- round-up and translocate back to the PM when the cells spread. These observations propose that PI(4,5)P2 levels elevate as cells round-up and decrease as cells spread. This interesting phenotype resembles the front and rear of a migratory cells. Interestingly, pikI- resemble similar cell morphology and biosensors dynamics observed when we use an inducible system to deplete PM PI(4,5)P2 levels. We, also, observed the dynamics of a biosensor for an F-actin polymerization protein called formin A (ForA). ForA has been shown to localize at a polarized cell’s rear and in the cleavage furrow of dividing cells. In addition, ForA have a PI(4,5)P2 binding motif and binds to PI(4,5)P2 preferentially in vitro. Our results support a role for PI(4,5)P2 in regulating ForA with the plasma membrane. Taken together, we proposed that local levels of PI(4,5)P2 contribute to the electrostatic interactions of regulatory proteins controlling actin dynamics and membrane protrusions. PM PI(4,5)P2 below a threshold activate regulatory proteins that excite the signaling that promotes protrusions, while below threshold levels would inhibit those proteins activity. The change in PI(4,5)P2 levels would be predicted to affect the membrane’s charge, which in turn changes the interaction and disassociation of many anionic regulatory proteins involved in the signaling pathway and cytoskeletal rearrangements. Additionally, we show for the first time, a correlation between the PM PI(4,5)P2 threshold and rates of phosphatidylserine (PS) exposure in cancer therapeutics. Receptor-mediated cell stimulation triggers PS exposure to the outer leaflet of the plasma membrane. Interestingly, pik1- and cells using an inducible system to deplete PM PI(4,5)P2 levels depicted the same responses. In addition to PI(4,5)P2, PS exposure affects the membrane’s charge which impacts the signaling molecules activity in the pathway. Altogether, PI(4,5)P2 and PS are proposed to be novel therapeutic targets in cancer treatments. Chemotaxis is a feature of metastatic cancer cells and is regulated by various regulators including actin cytoskeleton. Actin cytoskeleton reorganization during chemotaxis is regulated by actin-binding proteins including those that interact with the PM PI(4,5)P2. Energy production regulates cell migration as well, through glycolysis pathway. A previous study proposed that actin reorganization releases Aldo A enzyme which enters glycolysis, through activating PI3K signaling pathway. However, the mechanism of action remains unclear. We speculate that local PI(4,5)P2 levels regulate Aldo Activity through regulating actin-severing proteins activity including cofilin and gelsolin, and actin polymerizing protein including ForA. PI(4,5)P2 levels below a threshold release actin-severing proteins to the cytosol triggering the severing of actin and the release of Aldo A. while, above PI(4,5)P2 threshold activates and localizes for a on the PM promoting the F-actin polymerization and the sequester of Aldo A into F-actin. The goal of this work is to discover a new model for actin cytoskeleton regulation during migration, as its linkage to glycolysis and metabolism has important implications for cancer.

Binary image processing algorithms have been implemented in this study to create a background subtraction mask for the segmentation of cellular time lapse images. The complexity in the development of the background subtraction mask stems from the inherent difficulties in contrast resolution at the cellular boundaries. Coupling the background subtraction mask with the path reconstruction method via superposition of overlapping binary segmented objects in sequential time lapse images produces a semi-automatic method for cellular tracking. In addition to the traditional center of mass or centroid approximation, a novel quasi-center of mass (QCM) derived from the local maxima of the distance transformation (DT) has also been proposed in this study. Furthermore, image isolation and separation between spreading/motile and mitotic cells allows the extraction of both migratory and divisional cellular information. DT application to isolated mitotic cells permits the ability to identify distinct morphologic phases of cellular division. Application of standard bivariate statistics allows the characterization of cellular migration and growth. Determination of Hotelling???s confidence ellipse from cellular trajectory data elucidates the biased or unbiased migration of cellular populations. We investigated whether it was possible to describe the trajectory as a simple binomial process, where trajectory directions are classified into a sequence of (8) discrete states. A significant proportion of trajectories did not follow the binomial model. Additionally, a preliminary relationship between the image background area, approximate number of counted cells in an image frame, and imaging time is proposed from the segmentation of confluent monolayer cellular cultures.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by a very high mortality rate and is the 4th most common cause of cancer death (Siegel et al., 2012). The disease initially develops asymptomatically, and at the time of diagnosis patients usually have multiple metastases (Rhim et al., 2012). It would therefore be highly desirable to develop treatments which specifically impede the ability of PDAC cells to metastasise by interfering with the cellular processes responsible for efficient cellular migration. Intracellular signalling cascades, which utilise various signalling proteins, ultimately lead to the appropriate cell coordination and enable efficient cellular motility. One such signalling pathway that participates in the regulation of migration is controlled by the second messenger cyclic adenosine monophosphate (cAMP) (Howe, 2004). Several effectors of cAMP have been found which include protein kinase A (PKA) (Tasken & Aandahl, 2004), exchange factors activated by cAMP (EPAC) (Bos, 2006), and cyclic nucleotide-regulated cation channels (Biel, 2009). PKA has been intimately linked with several cellular processes which contribute towards cell motility. In most cases, the various specific effects of PKA signalling require selective targeting of the kinase into microdomains through interaction with A-kinase-anchoring proteins (AKAPs) (Pidoux & Tasken, 2010). Other cAMP effectors such as EPAC have defined roles in controlling various aspects of migration, such as cellular adhesion to the extracellular matrix (Bos, 2005). The effect of modulating cAMP signalling on the rate of migration has been investigated in several cancer types. Interestingly the results obtained were rather varied; both inhibition and stimulation of migration was observed (Chen et al., 2008; Baljinnyam et al., 2009; Grandoch et al., 2009; Shaikh et al., 2012). However, the effect of cAMP, and its effectors, on the rate of migration has not been investigated in PDAC; this was the main aim of this study. Classical cAMP elevating agents such as forskolin and 3-Isobutyl-1-methylxanthine (IBMX), as well as the cAMP analogue 8-Bromoadenosine 3’5’-cyclic monophosphate (8Br-cAMP), were found to inhibit migration of the PANC-1 cells. The role of cAMP signalling was further supported by the results of experiments utilising cAMP FRET sensors, which were imaged in live single cells. Further characterisation of cAMP effects in 4 other diverse PDAC cell lines yielded similar results, indicating that the mechanism of inhibition was common to all PDAC cell types tested. PANC-1 cell invasion was also inhibited by cAMP elevation. I went on to investigate events such as cell ruffling and focal adhesion assembly, which are processes closely associated with cellular motility. Dual transfection with a cAMP sensor and GFP tagged paxillin revealed a relationship between cAMP elevation and the loss of paxillin from focal adhesions, which was quickly reversible upon cAMP returning back to basal levels. Using a similar approach, peripheral cell ruffling was found to be inhibited by intracellular cAMP elevation. These results indicated that the inhibition of migration upon cAMP elevation was likely to occur as a result of immediate signalling events (and not due to cAMP-dependent changes in gene expression). The final part of the project concentrated on the individual contribution of the downstream effectors of cAMP, with particular emphasis on selective PKA and EPAC modulation. Utilising both PKA and EPAC sensors, I determined the appropriate concentrations of N6-benzoyl-cAMP (6Bnz) and 8-pCPT-2’OMe-cAMP (8pCPT) required to achieve selective PKA and EPAC activation respectively. Interestingly, I found that the two effectors had opposing actions; EPAC activation was found to induce migration, while PKA was found to suppress migration. Further investigation utilised a potent and selective PKA inhibitor peptide (PKI), which upon expression was found to prevent inhibition of ruffling, paxillin loss from focal adhesions, and inhibition of migration in response to cAMP elevation. Furthermore, it was found that suppression of basal PKA activity had a tendency to induce migration. I also utilised a cell permeable peptide (st-Ht31) which inhibits PKA interaction with AKAPs, thus effectively reducing its function by uncoupling the kinase from its specific signalling microdomains. The resulting effect was found to be a large potentiation of PANC-1 migration, which further highlighted the importance of PKA activity in the control of migration.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment for blood disorders and autoimmune diseases. Following HSCT, these cells must successfully migrate to the marrow niche and replenish the blood system of the recipient. This process requires both non-cell and cell-autonomous regulation of hematopoietic stem and progenitor cells (HSPCs). A transgenic reporter line in zebrafish allowed the investigation of factors that regulate HSPC migration and function. To directly observe cells in their endogenous microenvironment, confocal live imaging was used to track runx1:GFP+ HSPCs as they arrive and lodge in the niche. A novel cellular interaction was observed that involves triggered remodeling of perivascular endothelial cells during niche formation. A chemical screen identified the TGF-beta pathway as a regulator of HSPC and niche interactions. Chemical manipulation of HSPCs was used to improve engraftment and repopulation capability following transplantation. Runx1:GFP fish treated with prostaglandin E2 (PGE2) during embryogenesis exhibit increased runx1+ cells in the AGM and CHT, consistent with previous in situ data. This increase in HSPCs is maintained into adulthood, even in the absence of prolonged PGE2 exposure. Kidney marrow from these treated fish can outcompete control marrow in transplantation assays. The ability of PGE2 to confer a long-term advantage on sorted mouse marrow populations in competitive transplantation assays was tested. I found that PGE2-treated short-term (ST)-HSCs, but not long-term (LT)-HSCs show enhanced transplantability in recipients compared to control animals. My studies demonstrate that the effects of PGE2 on HSC function persist over substantial time despite transient exposure. A population of short-term HSCs can engraft and give rise to long-term multilineage reconstitution following PGE2 treatment. Collectively, our studies have led to novel insights regarding the pathways involved in HSC migration, homing, and repopulation.

Thesis (M.S. )--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on July 17, 2008). Research advisor: Xin Zhang, Ph.D. Document formatted into pages (xv, 197 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 159-197).

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 20, 2010). Thesis advisor: Dr. Anand Chandrasekhar. Includes bibliographical references.

The investigation of complex biological processes, such as wound healing, cell migration, cancer cell invasion, and gene regulatory networks can be benefited tremendously by novel biosensing techniques with high stability and spatiotemporal resolution. In particular, molecular probes with qualities including high stability, sensitivity, and specificity are highly sought-after for long-term monitoring of gene expression in individual cells. Among different single-cell analysis techniques oligonucleotide optical probes is a promising detection method to monitor the dynamics of cellular responses. Herein, the design and optimization of double-stranded LNA probes are first investigated. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is highly stable in living cells and is capable of detecting changes in gene expression induced by external stimuli. Using dsLNA probes we then demonstrate the novel approaches to monitor the spatiotemporal gene expression response during cell injury. Our results also suggest a potential autoregulatory role of Nrf2 in injury induced EMT. We also show that the signaling level of dsLNA probe can serve as a molecular signature for the leader cells near the wound which allows us to track the behaviors of leader cells during collective cell migration. Finally multimodal GNR-LNA approach is proposed to map spatiotemporal gene expression profile and reveal dynamic characteristics of heat shock response in photothermal operations.

Neuronal migration is an essential process in the development of the nervous system. Neurons are born in one location and migrate sizable distances to their final location. In many other developmental processes, cells migrate as collectives, where the migration of one cell influences the migration of another cell; this process has yet to be shown in the developing central nervous system. Using the conserved tangential migration of facial branchiomotor neurons (FBMNs), I aim to determine the nature of the collective migration in the developing nervous system. Here, two models of FBMN collective migration are tested: the “Pioneer” model, where following FBMNs migrate intimately on the axon of the first neuron to migrate and the “Contact inhibition of locomotion (CIL)” model, where transient cell-cell contacts are the driving influence of the proper caudal migration of FBMNs. Using fixed tissue imaging, it was found that early born FBMNs do not contact the axon. In contrast, they are more likely to make soma-soma contact and display morphology typical of CIL. FBMNs that do contact the axon do not display an elongated morphology that is predicted of a cell using the leader axon as a substrate for migration. Further, wild-type FBMNs are able to rescue PCP-deficient FBMNs. Therefore, blastula-stage transplantation of PCP-deficient neurons into wild-type hosts allows us to live image the method of collective migration. CIL events were observed between PCP-deficient neurons and wild-type neurons, indicating that PCP is not required for CIL. In addition, PCP-deficient neurons making sustained contact with wildtype axons were not rescued, arguing against the Pioneer model. Taken together, these observations are more consistent with the “CIL” model of FBMN collective migration in which transient soma-soma interactions are required for the coordinated movement of neurons as they migrate in the developing nervous system.

Mechanical properties of cells, mainly defined by their cytoskeleton, are closely related to cell function and can be measured with a dual-beam laser trap (optical stretcher). Functional changes, which go hand in hand with changes of the cytoskeleton, also occur during differentiation of stem cells. This suggests monitoring differentiation by the changing compliance of the cells. During the course of my PhD I measured the compliance of three different types of stem cells before and after differentiation and was able to detect differences in some of the cell types. In order to relate rheological experiments to cell migration as a further example of functional change I investigated the migration behavior of cells that showed different compliance and found differences in migration. I was additionally able to show an altered migration behavior after I actively changed the mechanical behavior of one cell type using cytoskeletal drugs. These migration experiments have been carried out in 2D and 3D migration assays. Furthermore, the influence of the stiffness of the surrounding material on the migration behavior has been investigated. After relating functional changes to changes in compliance, I studied which mechanisms can be used to actually influence cell compliance and investigated the effect of cytoskeletal stabilizers or destabilizers as well as drugs acting on molecular motors. The effect of the surrounding temperature has been considered as well. Finally, I developed a new version of the optical stretcher measurement tool, which enables cell sorting and drug screening using a monolithic glass chip. With the results presented in this thesis I relate mechanical compliance to the cytoskeleton and specific cellular functions. I deliver insights how mechanical changes in cells can be used to identify and follow functional changes and how this knowledge can help to interfere with such functions, specifically in pathologies correlated to these functions. My modified optical stretcher would be developed to screen the effects of drugs on cell compliance and to sort cells with different mechanical properties. Such drug screening and cell sorting will offer diagnostic treatment options for various pathologies.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2007.
Includes bibliographical references.
The fundamental question posed in this thesis is: how does a cell 'decide' to behave in a particular way? The human body is comprised of [approx.] 1014 cells that interpret extracellular information and respond with such behavior as migration, proliferation, apoptosis, or differentiation. Thirty years of research in the related fields of biochemistry, molecular biology, and genetics have demonstrated that, in most cases, the cellular decision-making process cannot be described or predicted by regulation of only one gene or one protein alone. Instead, it has become clear that cellular behavior is a function of information flow through multiple intracellular molecules. Furthermore, the molecules responsible for the control of cell behavior comprise a surprisingly short list, indicating that factors such as signaling dynamics and intensity coupled with combinatorial control are essential to produce the wide array of observed cell behavior. The identification of protein kinases as transducers of large amounts of intracellular information led us to pose the hypothesis that the quantitative regulation of key kinases governs cellular behavior. The goal of this thesis was to identify rules governing multi-kinase behavioral control and to then, on the basis of these rules, predict changes in cell function in response to changes in receptor expression, ligand treatment, and pharmacological intervention.
(cont.) A human mammary epithelial cell (HMEC) system with varying levels of the human epidermal growth factor receptor 2 (HER2) was chosen to explore cell decision processes. HER2 overexpression is found in 30% of breast cancers and correlates with poor prognosis and increased metastasis. In particular, we investigated the effects of HER2 overexpression on signaling networks and resultant cell proliferation and migration in the presence of epidermal growth factor (EGF) or heregulin (HRG), two EGFR-family ligands that promote HER2 heterodimerization. To investigate HER2-mediated signaling and cell behavior we developed and applied high-throughput experimental techniques to measure kinase activity and phosphorylation as well as cell proliferation and migration. Measurement of -~100 different kinases downstream of HER2 resulted in the identification of network signaling mechanisms. Application of a novel high-throughput migration assay enabled the identification of HER2-mediated increases in cell migration due to increases in the directional persistence of movement. Linear mapping techniques related to partial least squares regression (PLSR) defined and predicted cell behavior in response to HER2 overexpression.
(cont.) Combining quantitative datasets of both biological signals and behavior using PLSR, we identified subsets of kinase phosphorylation events that most critically regulate HER2-mediated migration and proliferation. Importantly, we demonstrated that our models provide predictive ability through a priori predictions of cell behavior in HER2-overexpressing cells. Application of linear models in response to pharmacological inhibition resulted in the a priori prediction of cell migration, and identified an EGFR kinase inhibitor Gefitinib as a potent inhibitor of HER2-mediated migration. In conclusion, the application of computational linear modeling to quantitative biological signaling and behavior datasets captured systems-level regulation of cell behavior and, based on this, predicted cell migration and proliferation in response to HER2 overexpression and pharmacological inhibition. Further application of quantitative measurement together with linear modeling should enable the identification of salient cell signal-cell response elements to understand how cells make decisions and to predict how those decisions can be therapeutically manipulated.
by Neil Kumar.
Ph.D.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 26, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Neurobiology." Discipline: Neurobiology; Department/School: Medicine.

Cellular microenvironments are an important area of study, and their implications with regard to development, tissue function, and disease, mean that they have particular relevance in tissue engineering. The development of tissue engineered therapeutics is underpinned by the understanding of how the cells exist in their natural environment. A fundamental lack of insight into the signalling mechanisms within microenvironments, due to in part a lack of appropriate technologies, has meant that the therapeutic potential of tissue engineering is limited. To this end, the development of a micropatterning technology that enables control over solute signalling dynamics on the micron scale has been investigated. A bespoke holographic optical tweezers (HOTs) system was used to precisely position cells and controlled release vehicles into three-dimensional arrangements that resemble basic cellular micro-architectures. Via optical manipulation, release vehicles could be patterned to create solute release patterns to mimic signalling events in vitro. A proof of concept was established to demonstrate fluorophore release from microparticles positioned with high precision, into previously unobtainable micron-scale patterns. Such developments required optimisation of the system and protocols, for use with cell and microparticle manipulation and, creating a tool-set suitable for address unsolved biological questions. Biological investigations were completed to demonstrate how the HOTs can be used to control zonal cell differentiation and migration. These processes are paramount to cell microenvironment function, and this study has shown that the HOTs patterning setup is capable of achieving such signalling models in vitro. Herein is presented compelling evidence that optically manipulated release sources can achieve new levels of precision over signalling dynamics, over the length scales suitable for even the smallest cell microenvironments. It is hoped that through the better in vitro modelling of such cellular microenvironments and other signalling events, investigators will be able to elucidate new mechanisms through which cells proliferate and function.

Cell migration is a fundamental biological process involved in tissue development, wound repair, and diseases such as cancer metastasis. It is a biomechanical process involving the adhesion of a cell to a substratum, usually an elastic extracellular matrix, as well as the physical contraction of the cell driven by intracellular actomyosin network. In the migration of cells as a group, known as collective migration, the cells are also physically linked to one another through cell-cell adhesions. How mechanical interactions with cell substratum and with neighboring cells regulate movements during collective migration, nevertheless, is poorly understood. To address this question, the effects of substrate stiffness on sheet migration of MCF10A epithelial cells were systematically analyzed. Speed, persistence, directionality and coordination of individual cells within the migrating sheet were all found to increase with substrate stiffening. Substrate stiffening also enhanced the propagation of coordinated movement from the sheet edge into the monolayer, which correlated with an upregulation of myosin-II activity in sheet edge cells. This mechano-response was dependent on cadherin-mediated cell-cell adhesions, which are required for the transmission of directional cue. Importantly, myosin-II contractility modulated cadherin- dependent cell-cell coordination, suggesting that contractile forces at cadherin adhesions regulate collective migration. To measure forces transmitted through cell-cell adhesions, a quantitative approach was developed in which cell-cell forces were deduced from cell-substrate traction forces, based on force balance principles and simple cell mechanics modeling. This method enabled the analysis of cell-cell mechanical interactions in small cell clusters of complex topology. The dynamic fluctuations of cell-cell forces over time revealed that force transmission between non-adjacent cells is typically limited, but is enhanced when the cell across which forces are being transmitted has reduced myosin-IIA or talin-1. This suggests that cells in a group may differentially regulate their levels of myosin-II contractility and cell-matrix mechanotransduction to promote longer-range force transmission during collective migration. Together, the results in this dissertation led to a working model of collective cell migration as regulated by cell-matrix mechanical properties and cell-cell mechanical interactions. This model, as well as the quantitative techniques developed here, will drive future studies on the mechanisms underlying collective migration.

Organ formation is an important step during development of an organism that combines different scales from the molecular to the tissue level. Many organogenesis phenomena involve epithelial morphogenesis, where sheets of cells undergo rearrangements to form complex architectures – organ precursors, which subsequently develop into mature organs. Timely development of the characteristic architectures of the organ precursors is crucial for successful organogenesis and is determined by the choice of epithelial rearrangements that organise the constituent cells in space and time. However, for many organogenesis events the cellular dynamics underlying such epithelial rearrangements remain elusive. In the work presented here, I investigated the morphogenesis of the hemispherical retinal neuroepithelium (RNE), that serves as an organ precursor of the neural retina. Formation of RNE is an important event in vertebrates that shapes the optic cup and sets the stage for subsequent eye development. I investigated RNE morphogenesis in the developing zebrafish embryo by visualising and investigating the cellular dynamics of the process in vivo. My findings show that the zebrafish RNE is shaped by the combined action of two different epithelial rearrangements – basal shrinkage of the neuroepithelial cells and involution of cells at the rim of the developing optic cup. The basal shrinkage of the neuroepithelial cells bends the neuroepithelial sheet and starts the process of invagination. However, my results show that the major player in RNE morphogenesis is rim involution. Rim involution translocates prospective RNE cells to their designated location in the invaginating layer and contributes to RNE invagination. My work unravelled the so far unknown mechanism of rim involution. I show that the rim cells involute by collective epithelial migration using directed membrane protrusions and dynamic cell-matrix contacts. If rim migration is perturbed, the prospective RNE cells cannot reach the invaginating layer. As a result, these migration-defective cells attain the RNE fate at an ectopic location and disrupt the tissue architecture. Therefore, rim migration coordinates the cellular location with the timing of RNE fate determination and orchestrates RNE morphogenesis in space and time. Overall, my work highlights how morphogenetic processes shape the organ precursor architecture and ensure timely organ formation. These findings provide important insights not only for eye development but also for epithelial morphogenesis and organogenesis in many other systems
Für die Entwicklung eines Organismus ist die Bildung von Organen (Organogenese) von zentraler Bedeutung. Organogenese umfasst Prozesse auf allen Ebenen der Längenskala: von der molekularen Ebene, der Gewebeebene, bis hin zur Ebene des ganzen Organismus. Viele Phänomene der Organogenese beinhalten dabei Veränderungen von Epithelien, bei der sich Schichten von Zellen zu komplexen Strukturen - Organvorläufern - umwandeln. Diese entwickeln sich später zu vollständigen Organen. Die rechtzeitige Entwicklung der charakteristischen Architektur der Organvorläufer ist entscheidend für eine erfolgreiche Organogenese und wird durch die Wahl der epithelialen Umwandlungsprozessen bestimmt, welche die Zellen in Raum und Zeit koordinieren müssen. Für viele dieser Prozesse ist jedoch genau diese zugrundeliegende Zelldynamik unklar. In der hier vorgestellten Arbeit untersuchte ich die Bildung des hemisphärischen retinalen Neuropepithels (RNE). Das RNE ist der Organvorläufer der neuralen Retina, weshalb dessen korrekte Bildung die Voraussetzung für die korrekte Entwicklung der Augen ist. Ich untersuchte die RNE-Morphogenese in sich entwickelnden Zebrafisch-Embryos durch Visualisierung und Untersuchung der zellulären Dynamik der beteiligten Prozesse in vivo. Meine Ergebnisse zeigen, dass das RNE in Zebrafischen durch die kombinierte Umwandlung von zwei verschiedenen Epithelien geformt wird. Zum einen findet eine Verkleinerung des basalen Prozesses der neuroepithelialen Zellen statt, zum anderen die Involution von Randzellen. Die basale Verkleinerung der neuroepithelialen Zellen verbiegt die neuroepitheliale Schicht und führt zur Einstülpung des RNE. Meine Ergebnisse zeigten allerdings, dass Involution von Randzellen noch bedeutsamer für die RNE-Morphogenese ist. Die involution von Randzellen transportiert potenzielle RNE-Zellen in das Neuroepithel und trägt zur RNE-Einstülpung bei. Die Bedeutung meiner Arbeit liegt darin, den bisher unbekannten Mechanismus der Randzell-Involution entdeckt zu haben. Ich zeigte, dass die Randzellen sich aktiv durch kollektive epitheliale Migration bewegen indem sie gerichtete Membranforsätze und dynamische Zell zu Matrix Kontakte etablieren. Wird die Migration der Randzellen inhibiert, so führt dies dazu, dass diese Zellen die eingestülpte RNE Schicht nicht erreichen. Sie landen dann an den falschen Positionen, wo sie die Gewerbearchitektur stören können. Daher koordiniert die Randzellmigration die Position der Zellen und orchestriert die RNE-Morphogenese in Raum und Zeit. Insgesamt zeigt meine Arbeit, wie morphogenetische Prozesse die Organvorläuferarchitektur prägen und eine rechtzeitige Organbildung sicherstellen. Diese Erkenntnisse sind sowohl für das Verständnis der Augenentwicklung, als auch für das der epithelialen Morphogenese und Organogenese in anderen Systemen von großer Bedeutung

Prostate cancer metastasis is a complex process, involving multiple pathways in its orchestration. Malignant cells are influenced by different growth factors from the extracellular environment which promote or inhibit cell movement and metastasis. HGF has been implicated in progression and metastasis of prostate cancer. A cell interacts with the environment through surface molecules like integrins. These interactions are further translated in to different responses through various intracellular machineries. Furthermore organization of the actin cytoskeleton is vital for many cellular functions. WAVEs are member of WASP family of proteins, which have important role in regulation of actin dynamics through regulation of actin related protein (ARP 2/3). The role of individual members of WASP family has been investigated in development and progression of different cancers. We documented the expression of different WAVE family members in various prostate cancer cell lines. Expression of WAVE-3 was effectively knocked down with the use of hammer head ribozymes. Loss of WAVE-3 expression resulted in reduced cell movement and invasion in the PC-3 cell line. These cells failed to show any significant increase in cellular movement and invasive potential following treatment with HGF. Further experiments to investigate the underlying mechanism of this phenotypic change revealed that optimum levels of phosphorylated paxillin play an important role in this change. Our study also indicates that reduced potential of invasive capability following WAVE-3 knock down, may be related to reduced availability of MMP-2 in the cellular environment.

A cell's ability to sense and respond to mechanical signals highlights the significance of physical forces in biology; however, to date most biomedical research has focused on genetics and biochemical signaling. We sought to further understand the physical mechanisms that guide the cellular migrations that occur in a number of biological processes, such as tissue development and regeneration, bacterial infections and cancer metastasis. We investigated the migration of single cells and determined whether the biomechanics of these cells could be used to elucidate multi-cellular mechanisms. We first studied Borrelia burgdorferi (Bb), the bacterium that causes Lyme disease. We created a mathematical model based on the mechanical interactions between the flagella and cell body that explained the rotation and undulation of the cell body that occurs as the bacterium swims. This model further predicts how the swimming dynamics could be affected by alterations in flagellar or cell wall stiffnesses. Fitting the model to experimental data allowed us to calculate the flagellar torque and drag for Bb, and showed that Treponema pallidum (Tp), the syphilis pathogen, is biomechanically similar to Bb. Next, we used experimentally-determined parameters of Bb's motility to develop a population-level model that accounts for the morphology and spreading of the "bulls-eye" rash that is typically the first indicator of Lyme disease. This work supported clinical findings on the efficacy of antibiotic treatment regimes. Finally, we investigated the dynamics of epithelial monolayers. We found that intracellular contractile stress is the primary driving force behind collective dynamics in epithelial layers, a result previously predicted from a biophysical model. Taken together, these findings identify the relevance of physics in cellular migration and a role of mechanical signaling in biomedical science.

Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Doctor of Philosophy(PhD)
Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins. This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures. With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells. In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor. Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient.
(Faculty of Medicine)

Abstract While most of the in vitro cultures are carried out on bi-dimensional (2D) substrates, most of the in vivo extracellular matrices are threedimensional (3D). Consequently cells behave differently on 2D substrates as a way to self-adaptation to a non-physiological environment. This fact has encouraged the development of more relevant culture conditions seeking to provide more representative models for biomedicine (e.g. cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. Different 3D culture systems have been established though their variability and complexity hinder their standardisation in common cell culture procedures. So, this thesis deals with the dimensionality issue in cell/material interactions and introduces sandwich-like microenvironments as a versatile tool to study cell behaviour. Cells cultured within this system use both dorsal and ventral receptors to adhere and spread, undergoing important changes with respect to the 2D cultures and approaching to 3D conditions. Stimulation of dorsal receptors has been previously addressed by overlaying a protein gel on cells already attached on a 2D surface. Here we propose a sandwich-like system that consists of two 2D surfaces so that wider spectra of conditions can be investigated by changing the nature of the substrate (material, topography…) and the protein coatings of both ventral and dorsal sides. Since sandwich culture provides an altered cellular adhesion compared to the traditional 2D substrates by the excitation of the dorsal receptors, changes in the intracellular signalling are expected, which might alter important processes such as proliferation, morphology, migration and differentiation. Hence this thesis evaluates the effect of different sandwich culture parameters in cell behaviour. First, cell fate upon adhesion was evaluated in terms of morphology, proliferation and adhesion. Different conditions were studied such as materials with different properties or protein coatings (dorsal and ventral substrates), as well as the effect of sandwiching cells just after seeding or after been allowed to adhere to the ventral substrate. Interesting results were obtained such as the relationship between the ability of cells to reorganise the ECM with cell morphology, proliferation and adhesion, similarly as observed in 3D hydrogels (degradable vs nondegradable systems). Then, cell migration within sandwich culture was studied by live imaging of a wound healing assay. Results revealed the key effect of both ventral and dorsal substrates in determining the migration rate as well as the migration mode used by cells. Moreover cells within the sandwich culture migrating in the wound healing assay adopted an elongated cell morphology that resembled cells migrating in other 3D systems. Beyond differences in cell morphology and migration, dorsal stimulation promoted cell remodelling of the extra-cellular matrix (ECM) over simple ventral receptor activation in traditional 2D cultures. Finally the effect of sandwich culture on cell differentiation was evaluated. First we showed an increase in C2C12 myogenic differentiation when cultured within the sandwich system. This enhancement was shown to be dorsal stimulation dependent and related to an alteration of the signalling pathway and the growth factor release. To determine if sandwich culture leads only to myogenic differentiation or whether it allows differentiation to other lineages, 4 different human mesenchymal stem cells (hMSCs) lines were cultured under the same conditions. Results showed the same sandwich environment triggered different cell differentiation. This points out the importance of the microenvironment cell niche in vivo, which highly influence cell fate, and thus the need of mimicking it properly in vitro. Overall, sandwich-like microenvironments switch cell behaviour towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo.
Ballester Beltrán, J. (2014). Sandwich-like systems to engineer the cellular microenvironment [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48166
TESIS

The Met receptor tyrosine kinase (RTK) and its ligand, hepatocyte growth factor (HGF) are potent regulators of epithelial remodeling, dispersal, and invasion and their deregulation is frequently observed in human cancers. These processes are coordinated through signal transduction pathways activated downstream of Met. In turn, it is now understood that an important aspect of signalling outcome is modulated through RTK downregulation via receptor internalization, degradation and also RTK recycling to spatially restricted subcellular domains. Although a strong relationship between RTK endocytosis and signalling had been established prior to this thesis, the precise mechanism(s) of how this is achieved is still poorly understood and little was known for the Met RTK.To address this specifically for the Met RTK, I have characterized the function of two endocytic proteins in Met RTK trafficking and the consequence for biological response. Work in this thesis shows that following HGF-mediated Met activation, the endocytic adaptor protein, Eps15, is recruited to the Met receptor, and becomes both tyrosine-phosphorylated and ubiquitinated. Recruitment of Eps15 requires Met receptor kinase activity and involves two distinct Eps15 domains. Unlike previous reports for other receptors, recruitment of Eps15 to Met involves a distinct mode of recruitment involving the coiled-coil domain of Eps15 and the signaling adaptor molecule, Grb2. This identified a new mechanism of recruitment for Eps15 downstream of the Met receptor.In the third part of my thesis, I establish that the endocytic adaptor molecule, Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), interacts selectively with Met when stimulated by HGF, to sort Met for recycling through a Rab4 positive compartment rather than degradation. I provide evidence supporting a molecular mechanism through which GGA3 regulates Met recycling and demonstrate that this is essential for both prolonged signaling and HGF-mediated biological response. This data defines an active recycling pathway involving GGA3, and supports a broader role for GGA3-mediated cargo selection in targeting receptors destined for recycling.Finally, in the fourth chapter of this thesis, evidence for microtubule interactions in Met-mediated Rab4 vesicle recycling is presented. We characterize a mechanism reliant on the microtubule +TIP, CLIP-170, linking Met/Rab4-positive recycling vesicles to microtubules for transport to the lamellipodia. We propose that the specific targeting of the Met receptor to the leading edge functions as a specialized signalling microenvironment, required for maintaining normal migration dynamics. These studies identify Met RTK endocytosis as a crucial regulatory process for RTK signalling and biology and stress the need for further studies examining the interplay of RTK endocytosis and signalling in the context of normal development and cancer settings.
Le récepteur tyrosine kinase (RTK) Met, ainsi que son ligand, le facteur de croissance des hepathocytes (HGF), sont de puissants régulateurs du remodelage, de la dispersion et de l'invasion des cellules épithéliales, et leur dérèglement est fréquemment observé dans divers types de cancers chez l'humain. Ces processus sont coordonnés par l'activation de voies de signalisation induites par Met. Il est maintenant connu que l'internalisation, la dégradation ainsi que le recyclage des RTKs vers des domaines spécifiques de la cellule constituent des facteurs importants influençant les conséquences de l'activation de ces voies de signalisation. Bien que le lien entre l'endocytose et la signalisation des RTKs ait été démontré avant ce mémoire, les mécanismes spécifiques de cette association restent encore mal compris. Pour aborder ce problème spécifiquement concernant le récepteur Met, j'ai caractérisé les rôles de deux protéines endocytiques impliquées dans le trafic de Met, et leur implication dans les réponses biologiques induites par Met. Les travaux présentés dans ce mémoire montrent qu'en réponse à l'activation de Met par HGF, l'adaptateur endocytique Eps15 est recruté par le récepteur et devient tyrosine phosphorylé et ubiquiné. Le recrutement d'Eps15 requiert l'activation du récepteur tyrosine kinase Met, et implique deux domaines distincts d'Eps15. Contrairement à ce qui a été rapporté pour d'autres récepteurs, le recrutement d'Eps15 par Met met en jeu un mode de recrutement distinct impliquant le faisceau d'hélices (coiled-coil domain) d'Eps15 et l'adapteur signalétique Grb2. Ceci a permis d'identifié un nouveau mécanisme de recrutement d'Eps15 par le récepteur Met.Dans la troisième partie de ce mémoire, j'ai établi que l'adaptateur endocytique Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3) interagit spécifiquement avec Met lorsque celui-ci est stimulé par HGF. En conséquence, GGA3 trie Met pour le recyclage via un compartiment contenant Rab4, plutôt que de le trier pour être dégradé. J'y démontre un nouveau mécanisme moléculaire par lequel GGA3 contrôle le recyclage de Met, ce qui est essentiel pour la signalisation prolongée de Met et des réponses biologiques induites par HGF. Ces données définissent un nouveau mécanisme de recyclage actif impliquant GGA3 et supportent un rôle plus large joué par GGA3 dans la sélection des récepteurs destinés au recyclage.Finalement, le rôle des microtubules dans le recyclage des vésicules contenant Rab4 induit par Met est présenté dans le quatrième chapitre de ce mémoire. Nous avons caractérisé un mécanisme dépendant d'une protéine de l'extrémité (+TIP), CLIP-170, qui lie les vésicules de recyclage contenant Met et Rab4 aux microtubules, contrôlant ainsi leur transport vers les lamellipodes. Nous proposons que le ciblage spécifique du récepteur Met vers le front de migration fonctionne comme un microenvironnement signalétique spécifique requis pour le maintient de la dynamique migratoire. Ces travaux ont permis d'identifié l'endocytose du récepteur tyrosine kinase Met comme étant un processus de régulation cruciale pour la signalisation des RTK et pour les réponses biologiques associées à cette signalisation. Ces travaux démontrent qu'il y a un besoin important d'examiner la relation entre l'endocytose des RTKs et leur signalisation aussi bien dans le contexte du développement normal que dans le cadre du développement cancéreux.