Auswahl der wissenschaftlichen Literatur zum Thema „Cellular Migration“

Computational modelling of cells can reveal insight into the mechanisms of the important processes of tissue development. However, current cell models have limitations and are challenged to model detailed changes in cellular shapes and physical mechanics when thousands of migrating and interacting cells need to be modelled. Here we describe a novel dynamic cellular finite-element model (DyCelFEM), which accounts for changes in cellular shapes and mechanics. It also models the full range of cell motion, from movements of individual cells to collective cell migrations. The transmission of mechanical forces regulated by intercellular adhesions and their ruptures are also accounted for. Intra-cellular protein signalling networks controlling cell behaviours are embedded in individual cells. We employ DyCelFEM to examine specific effects of biochemical and mechanical cues in regulating cell migration and proliferation, and in controlling tissue patterning using a simplified re-epithelialization model of wound tissue. Our results suggest that biochemical cues are better at guiding cell migration with improved directionality and persistence, while mechanical cues are better at coordinating collective cell migration. Overall, DyCelFEM can be used to study developmental processes when a large population of migrating cells under mechanical and biochemical controls experience complex changes in cell shapes and mechanics.

Cell migration has a profound effect on the generation of traction forces and the phosphorylation of focal adhesion proteins. The mechanism may involve the dynamic turnover of focal adhesions during cell migration and mechanical interactions between nascent and preexisting focal adhesions.

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.

Cell migration plays crucial role in regeneration, morphogenesis and cancer metastasis. We present a novel hardware-software method for multiplexed (3-cameras) holographic lensless label-free full-culture live-cell quantitative migration sensing with single-cell sensitivity and sub-cellular motion precision.

Abstract Hematopoietic stem cell [HSC] therapy has its origins as hematological rescue following marrow ablative high-dose therapy for refractory cancers and myelodysplastic syndromes. In its simplest form, bone marrow is collected from a tissue-matched related normal donor and intravenously infused into the patient who has usually received high-dose chemo/radiotherapy for his or her disease. The stem cells migrate to the marrow spaces, where they multiply and differentiate to restore blood cell-forming activity and immune defenses in the recipient Restrictions in the availability of tissue-matched marrow donors have prompted investigations into new sources of stem cells. These include use of the autologous marrow, which is collected from the patient and cryopreserved prior to therapy. The risk of tumor contamination in these grafts has resulted in the development of numerous methods to purge cancer cells from marrow ex vivo, or to enrich stem cells from the graft. This provided one of the earliest clinical applications for monoclonal antibodies and immunomagnetic cell separation. Another approach has been to use tissue-matched unrelated volunteer marrow donors, or grafts from mismatched family members. In both cases, ex vivo removal of T lymphocytes from the graft is advisable to prevent these cells initiating a severe or fatal reaction in the recipient - graft-versus-host disease. Recent studies have shown that recombinant human growth factors can be used to stimulate migration of HSC from the marrow into the blood, from which they are collected by automated cell separators, obviating the need for harvesting marrow under general anesthesia. The same growth factors have been used to expand and differentiate HSC ex vivo, with the hope that clinical grafts could ultimately be grown in the laboratory from a few milliliters of blood or marrow. Transplants have also been performed using placental/umbilical cord blood as the HSC source, and there are some indications that these cells may be more suitable for mismatched transplants. Genetic manipulation of stem cells can be used to correct a number of inherited diseases, and ultimately may allow specific beneficial properties to be introduced into blood-derived cells e.g. drug resistance into stem cells - to allow higher doses of chemotherapy to be used without destroying the bone marrow. The rapid expansion in this field of medicine, combined with advances in molecular and cellular biology offer the promise of stem cell engineering as an effective therapy in a variety of diseases.

Improved imaging of morphological changes has the potential of offering new insight into the complex process of embryonic development. Optical coherence tomography (OCT), is a new imaging technique for performing in vivo cross-sectional imaging of architectural morphology by measuring backscattered infrared light. This study investigates the application of OCT for imaging developing structure in Xenopus laevis (African frog) and Brachydanio rerio (zebra fish), two developmental biology animal models. Images are compared to corresponding histological preparations. Cross sectional imaging can be performed and structural morphology identified at greater imaging depths than possible with confocal and light microscopy. Repeated OCT imaging may be performed in vivo in order to track structural changes throughout development. Imaging in vivo microscopic embryonic morphology with OCT is a fundamental biological research application for the study of genetic disease.

Understanding cellular dynamics is fundamental to increasing the healing and regenerative capacity of biomedical scaffolds. The ability to investigate environmental cues and cell-cell interactions in vitro with successful translation to in vivo therapies will enhance many tissue engineering technologies. Understanding the dynamics of a cell in response to external mechanical stimuli can help achieve directed cellular migration by varying cellular environment geometries. Customized scaffolds can then be designed to achieve desired cellular migration rates, cell-cell interaction pathways, increased proliferation and directed cellular differentiation platforms to achieve tissue engineering specific goals. In this study, a unique fiber manufacturing platform known as STEP (Spinneret-based Tunable Engineered Parameters) is used to create and manipulate geometrical cues for cellular migration. The cell’s reaction to these geometric cues provides valuable insight into cellular behavior, which can be used to determine the optimal engineered microenvironment. We envision that studying cellular behavior on STEP enabled customized scaffolds will aid in the design and fabrication of accurate mechanistic environments for different cell types which can then be coupled with chemical cues to achieve desired results.

The wavelength dependence of light transport parameters of human tissue are estimated using Mie calculations of scattering from a simple model of a distribution of single sized spherical scatterers in an otherwise homogeneous background medium. The results are consistent with a dominant contribution to tissue scattering from small cellular or intercellular constituents such as mitochondria. The early arriving transmitted fraction of short optical pulses is calculated using both a diffusion approximation, and a Monte Carlo solution of the transport problem. Both predict enhancement of this transmission as the incident wavelength is changed from 700 nm to 1300 nm. However, the Monte Carlo solution predicts a much larger enhancement, showing strong disagreement with the diffusion result for early arriving photons at all tissue depths greater than a few millimeters.

This paper describes the relationship between the ultrastructure of biological tissues and the observed macroscopic optical scattering properties. A summary of the tissue absorption spectrum is also presented. The scattering of soft tissues (liver, prostate, etc.) and fibrous tissues such as dermis are considered. The scattering of soft tissues is attribued to membranous structures and modeled as Mie scattering from spheres in the 0.2-2-μm diameter range, where membrane lipids occupy about 1-20% of the cellular volume and the refractive index mismatch is 1.46/1.35. The scattering of dermis is modeled as scattering from collagen fiber bundles in the 2.8-μm diameter range occupying 21% of the dermal volume and the refractive index mismatch is 1.38/1.35. The effects of a component of small-scale particle scattering in the Rayleigh limit is also considered. The models are compared with tissue values from the literature for the reduced scattering coefficient, μs(1-g), and the anisotropy, g. The models roughly match the absolute value and wavelength dependence of scattering in the 300-1100 nm wavelength range.