No description available.

La protéine NCp7 du VIH-1 est une cible thérapeutique de choix car, en plus d’être conservée, elle intervient lors de nombreuses étapes du cycle rétroviral. A ce jour, peu de données existent concernant l’interaction possible de NCp7 avec les membranes lipidiques. Or il a récemment été montré que, lors de l’assemblage des virions, le précurseur Gag adopte une conformation repliée qui permettrait à son domaine NC d’interagir avec la membrane plasmique. Dans le but de tester cette hypothèse nous avons utilisé NCp7 sous sa forme mature. Nos résultats indiquent que NCp7, libre ou liée à des acides nucléiques, se lie aux membranes lipidiques chargées négativement, possède la capacité de recruter les lipides chargés négativement de manière à optimiser sa liaison sur la membrane et peut également déstabiliser ces membranes. Finalement, en utilisant un système modèle, nous avons mis au point les conditions de travail pour la poursuite de cette étude à l’aide de la microscopie super-résolutive. / The NCp7 protein is an interesting antiviral target since it is conserved and plays a numerous key roles in the HIV-1 replication cycle. Interestingly, while only few data are currently available on the possible interaction of NCp7 with lipid membranes, it has been recently shown that during assembly, the Gag precursor can adopt a bent conformation where the NC domain may interact with the plasma membrane. In order to check this hypothesis we used the mature NCp7. Our data indicated that the NCp7 protein, free or bound with nucleic acids, binds to negatively charged lipid membrane with high affinity, can recruit negatively charged lipids to optimize its binding to lipid membranes and has the ability of to destabilize negatively charged lipid membranes at high concentrations. Finally, by using a receptor/ligand model system we established the working conditions to investigate Gag/membrane interactions by high resolution microscopy.

This diploma thesis is focused on long term monitoring of thermodynamic stability of ettringite under selected conditions and its possible destabilization or transformation into another AFt phase, especially thaumasite. Ettringite samples were made in two ways, by hydrating the yeelimite in the system with the alite and by addition of aluminium sulphate and calcium hydroxide.

Le traitement des infections intracellulaires est compliqué par la capacité des bactéries à «se cacher» à l’intérieur des cellules de l’hôte, en particulier celles du système immunitaire, entravant ainsi l’action de nombreux agents antimicrobiens. La diffusion croissante de souches résistantes est très inquiétante. Dans ce cadre, les nanoparticules (NPs) constituent une stratégie prometteuse pour administrer de manière optimisée des agents antimicrobiens.Ce travail de thèse, réalisé dans le cadre du projet européen ITN Cyclon Hit, visait à développer et caractériser des NPs biodégradables et biocompatibles chargées en antibiotiques, composés d’acide polylactique (PLA), d’acide poly (lactique-co-glycolique) (PLGA) et de polycaprolactone (PCL) ou de cyclodextrines polymérisées (pCD).Les deux premiers chapitres sont consacrés aux verrous technologiques liés à l'encapsulation de certains médicaments puissants dans les NPs polymériques. Tout d'abord, ces vecteurs ont été utilisés pour la délivrance simultanée d'une combinaison de molécules actives récemment découverte, l'éthionamide (ETH) et son Booster, pour le traitement de la tuberculose. Deuxièmement, ils ont été employés pour relever les défis liés à l'incorporation d'une quinolone de première génération, l'acide pipémidique (PIP), dans le but d'optimiser sa distribution intracellulaire dans des infections telles que la salmonellose.La co-incorporation efficace de l'ETH et du booster a dû surmonter de nombreuses difficultés liées à des problèmes de solubilité, de cristallisation et de biodisponibilité. Nos NPs en PLA et en pCD ont montré leur capacité de co-encapsuler efficacement les deux molécules et tout particulièrement celles en pCD. Elles incorporent les médicaments à la fois dans les cavités des CD et dans des microdomaines hydrophobes. Les NPs en pCD, non toxiques après administration pulmonaire répétée de fortes doses, ont été administrés in vivo par voie endotrachéale directement au site d'infection. Elles ont permis une diminution de 3-log de la charge bactérienne pulmonaire des animaux infectés après seulement 6 administrations. De même, l'incorporation de PIP a été confrontée à des défis liés à la cristallisation de PIP et à sa libération incontrôlée. Malheureusement, le PIP présentait une faible affinité pour tous les matériaux polymériques étudiés et son encapsulation physique était infructueuse. Ainsi, une approche alternative a été développée en couplant le PIP au PCL via une réaction sans catalyseur initiée par le médicament. Le conjugué PCL-PIP se auto-assemble en forme de NPs avec une charge en PIP de 27%. Cependant, le PCL-PIP n'a pas pu être dégradé in vitro, mais l’approche de synthèse de conjugués est séduisante pour obtenir de particules stables et avec un contenu important en PIP.La compréhension approfondie de la structure et de la composition du noyau et de la couronne des nanostructures contenant une ou deux molécules actives est cruciale pour leur optimisation. Les deux derniers chapitres sont donc consacrés à l'application innovante de l'AFM-IR, une méthode nanospectroscopique originale combinant la microscopie à force atomique (AFM) avec la spectroscopie infrarouge (IR), à l'analyse chimique des NPs en PLGA ou à leur détection sans marquage après internalisation dans les cellules.L’AFM-IR est capable de fournir une caractérisation chimique à l'échelle nanométrique (résolution ~10 nm). Une avancée majeure du travail est l'application du mode tapping permettant l'investigation individuelle de chaque NP. Le signal IR spécifique des composants des NPs a été utilisé pour appréhender la composition chimique de leur cœur et couronne ainsi que pour localiser précisément le médicament. De plus, l'AFM-IR en mode contact a permis pour la première fois la localisation sans marquage et l'identification chimique des NP à l'intérieur des cellules. Ce travail ouvre la voie à d'innombrables applications de cette technique dans le domaine de la nanomedecine. / The treatment of intracellular infections is very challenging given the ability of bacteria to “hide” inside the cells of the host, especially the ones of the immune system, thus hampering the action of many antimicrobial agents. The battle against these bacteria has been further exacerbated by the increasing diffusion of antimicrobial resistant strains. In this frame, nanoparticles (NPs) are a very promising strategy to overcome the limitations of free antimicrobial agents by administering them in an optimized manner.This PhD work, performed as part of the European Project ITN Cyclon Hit, aimed at the development and advanced characterisation of antibiotic-loaded biodegradable and biocompatible NPs made of poly (lactic acid) (PLA), poly (lactic-co-glycolic) (PLGA) and polycaprolactone (PCL) or of polymerised cyclodextrins (pCDs).The first two chapters are dedicated to the encapsulation of powerful but challenging drugs in polymeric NPs. Firstly, these carriers were employed for the simultaneous delivery of a potent drug combination recently discovered, ethionamide (ETH) and its booster, for tuberculosis therapy. Secondly, they were used to address the challenges related to the incorporation of a first-generation quinolone, pipemidic acid (PIP), with the aim of optimising its intracellular delivery in infections such as salmonellosis.The efficient co-incorporation of ETH and booster had to overcome several technological barriers. These drugs presented solubility, crystallisation and bioavailability-related problems which were overcome thanks to the developed NPs. Our engineered PLA and pCD NPs were both able to efficiently co-encapsulate the two molecules. Among the in depth-characterised formulations, pCDs NPs displayed the best physico-chemical properties and were shown to host the drugs both in the CD cavities and in confined spaces inside NPs crosslinked polymer. The pCD NPs were administered in vivo by endotracheal route directly to the infection site. Empty NPs were shown non-toxic after repeated pulmonary administration of high doses. Moreover, loaded pCD NPs led to a 3-log decrease in the pulmonary bacterial load of infected animals after only 6 administrations. Similarly, the incorporation of PIP faced challenges mainly related to PIP crystallization and burst release. Unfortunately, PIP displayed poor affinity for all the studied polymeric materials and its physical encapsulation was unsuccessful. Thus, an alternative approach was developed by coupling PIP to PCL by using an original catalyst-free drug-initiated reaction. The PCL-PIP conjugate self-assembled in NPs with up to 27 wt% PIP which were thoroughly characterised. However, the conjugate couldn’t be enzymatically degraded. With the design of novel PCL-PIP conjugates, this self-assembly approach could represent a promising strategy.The deep understanding of the structure and composition of complex core-corona nanocarriers containing one or two active molecules is crucial for their optimisation. The last two chapters are devoted to the innovative application of AFM-IR, an original nanospectroscopic method combining atomic force microscopy (AFM) with infrared (IR) spectroscopy, to the chemical analysis of PLGA NPs or to their label-free detection after cell internalisation.AFM-IR is able to provide chemical characterisation at the nanometer scale (resolution ~10nm). One main breakthrough here is the application of the recently developed tapping mode allowing the investigation of single polymeric NPs. The specific IR signal of NPs constituents was used to unravel the chemical composition of their core and corona as well as to precisely locate the drug. Moreover, the AFM-IR in contact mode enabled for the first time the label-free localisation and unambiguous chemical identification of NPs inside cells using the polymer IR specific response as a fingerprint. This work paves the way for countless application of this technique in the field of drug delivery.

The cellular membrane is a complex biological entity, far from being an inert assembly of protein and lipids which separates cells from the surrounding environment. A multitude of biological processes, ranging from active transport of ions into and out of the cell, to the immune response, are regulated at the level of the plasma membrane. The understanding of their molecular basis is among the central goals of modern biological research. In order to dissect the complexity of actual cell membranes, which involves a very complex network of intermolecular interactions, a “divide and conquer” strategy proves very useful. To this end, researchers try to isolate molecules from complex biological contexts to understand their function in simple model systems under controlled conditions. A variety of model membranes have thus been developed in order to gain insight into membrane processes. This approach has resulted in a deeper knowledge on how lipids and proteins interact and how these interactions govern the function of cellular membranes. In the recent past in fact, a connection has been established between the lateral structure of the plasma membrane and its biological function. Furthermore, a large range of biophysical techniques have been used to characterize protein-lipid microdomains. For example, atomic force microscopy (AFM) is a powerful technique which allows a highly detailed topographical characterization of lipid domains in physiological conditions. While AFM imaging offers an extremely high spatial resolution, up to the nanometer scale, the limited image acquisition speed (minutes) can pose a severe drawback in adequately studying fast dynamic processes. On the other hand, fluorescence based imaging techniques are much faster (10-3-100 s), but certainly lack the high spatial resolution that AFM offers. FCS in particular can also provide information about dynamic processes, like diffusion of fluorescent membrane components. For these reasons, implementing a combination of the above mentioned techniques on the same sample (e.g. cell membrane models) would prove extremely beneficial in the complete dynamic and structural characterization of molecular interactions. . The work described in this thesis can be summarized in two main points: i) the development of a novel combined approach of atomic force microscopy (AFM), laser scanning imaging (LSM), and fluorescence correlation spectroscopy (FCS) and ii) the study of the effects of ceramide in the lateral organization of model plasma membranes. We described one of the first simultaneous applications of AFM and FCS on biologically relevant systems. More specifically, model membranes showing complex phase separation were investigated with a combined approach of AFM, confocal fluorescence imaging, force measurements and FCS, based on commercially available instruments. AFM conveys information about the structural and mechanical properties of the different lipid phases. Different membrane domains can be distinguished based on height difference, elastic properties and line tension as measured by the AFM tip. Simultaneous optical measurements offer the correlation of these data in real time with the partition behavior and diffusion of fluorescent lipids and proteins. We established a clear link between the local membrane viscosity, probed by FCS, and the lipid-lipid interactions involved in line tension, probed by AFM force measurements. An example of a significant drawback circumvented by the AFM-FCS approach involves the use of AFM micromanipulation to eliminate unwanted interactions between lipid particles — similar to intra-cellular vesicles found in vivo experiments — and the membrane, which usually result in distorted FCS autocorrelation curves. Finally, the combined application of AFM and FCS on membrane-anchored proteins reconstituted in lipid bilayers has been instrumental in clarifying inconsistencies that arose in work that focused solely on either AFM or fluorescence techniques. We have shown that, in the case of proteins diffusing in the plane of the membrane, AFM can unambiguously detect only a small immobile fraction. Furthermore, since AFM detection of proteins might be facilitated by high local membrane viscosity (e.g. in ordered lipid phases), the measurement of protein partition between disordered and ordered membrane domains might be biased toward the latter. In this case, the use of FCS as a complementary technique allows a more thorough investigation and deeper understanding of the system of interest. The second part of this thesis dealt with the study of complex lipid mixtures which are used to model the putative lipid/proteins domains in cells, called “rafts”. Firstly, we proved how the combined fluorescence imaging/AFM approach is useful in general for studying supported lipid membranes and the role of lipid domains in biological contexts. We investigated the effect of environmental stress on biological membranes and the protective effects of several substances. Our experimental approach was shown to be a new valuable method to visualize the dehydration damage and its effects on the lateral organization of lipid domains. Our results demonstrated that disaccharides like trehalose or sucrose are effective in protecting lipid membranes, not only on a macroscopic scale — preserving the overall integrity of the bilayer — but also on a microscopic scale, preventing the clustering of microdomains. These phenomena are interesting in the context of biological damage to living cells which need to be stored for long time, like organs to be transplanted or blood platelets. Finally, a large section of this thesis focused on the effects of a specific lipid called “ceramide” on the lateral organization of proteins and lipids in the plasma membrane. Ceramide is produced by cells in several situations, like bacterial infections or apoptosis. As consequence of ceramide production in vivo, the local concentration and the dynamic behavior of lipids and membrane receptors are supposed to exhibit strong variations. In order to understand the molecular mechanisms responsible for these effects, we applied a combination of AFM, FCS and fluorescence imaging on simple model membranes containing ceramide. We could show for the first time that, in presence of raft-like Lo/Ld phase separation, physiological quantities of ceramide induced the formation of a highly ordered gel phase, constituted of ceramide and sphingomyelin. The enzymatic production of ceramide was monitored both in supported and in free-standing bilayers. In the second case, ceramide production was connected to selective vesicle budding from the raft-like phase. Since short-chain analogues are often used in both medical applications and biochemical research to mimic the effect of long-chain ceramides, we investigated the effect of chain-length on ceramide-induced membrane reorganization. We could show that only long-chain ceramides (C18 and C16) form highly ordered domains. Interestingly, FCS measurements indicated that the physical properties of the Lo raft-like domains are hardly affected by the presence of ceramide domains. Furthermore, the increased thickness of the Ld phase — as measured by AFM — and its higher viscosity — as measured by FCS — strongly support the hypothesis of ceramide-induced cholesterol displacement from rafts. On the other hand, short-chain ceramides showed completely different biophysical properties that lead to a destabilization of the raft domains, possibly acting as surfactants between the different lipid phases. Our findings contribute to the explanation of in vivo experiments where short-chain ceramides inhibit cell signaling by disrupting the lipid order in the plasma membrane. We have so far demonstrated that ceramide plays a fundamental role in lipid-lipid interactions. In a physiological context, it is also known to produce dramatic effects in living cells. Since a majority of the processes in vivo are thought to be governed by the activity of proteins, it is highly likely that ceramide not only affects lipid organization but also modifies protein-protein and protein-lipid interactions to produce its effects. To test this hypothesis, we reconstituted several membrane proteins in lipid bilayers containing Ld, Lo, and ceramide-rich domains. We were able to show that some membrane proteins are sorted into ceramide-rich domains. More specifically, the raft-associated proteins we tested were enriched in the highly ordered ceramide-rich domains, while the Ld-associated components were excluded from them. Furthermore, the inclusion of any membrane component in ceramide-rich domains is directly connected to a dramatic reduction of its in-plane diffusion. In an in vivo context, such a reorganization of membrane receptors might be used by the cell to alter the signaling process, for example, by i) separating raft receptors from inhibitors with lower raft affinity, ii) bringing both raft-associated receptors and raft-associated signaling molecules into contact, or iii) stabilizing the interactions between a receptor and its ligand by decreasing their diffusion coefficients. In conclusion, this thesis describes a novel combination of AFM, LSM, and FCS for the investigation of the lateral organization of biological membranes. Our results show that this approach applied on model membranes of increasing complexity is an effective tool for understanding the molecular mechanisms behind the organization of biological membranes. This report opens up new possibilities for further investigation in living cell membranes using the same methodology we have described.

No description available.

Most animal cells adopt an approximately spherical shape when entering mitosis. This process has been termed mitotic rounding. It ensures the correct morphogenesis of the mitotic spindle and, in turn, successful cell division. When cells acquire a round shape at the entry of mitosis, they need to mechanically deform the surrounding tissue to do so. Previous studies suggest that the forces necessary for this deformation emerge from the contractility of the mitotic actin cortex. In fact, at the onset of mitosis, cortical contractility was found to be upregulated giving rise to an increased cell surface tension which drives the mitotic cell into a spherical shape. In a growing tumor, an increasing cell density generates a compressive mechanical stress which would likely lead to an increasing mechanical obstacle for mitotic rounding. Indeed, mechanical confinement or external pressure have been shown to hamper cell proliferation in tumor spheroids. Thus, it has been hypothesized that the actin cortex of cancer cells exhibits oncogenic adaptations that allow for ongoing mitotic rounding and division inside tumors. In fact, it was shown that the human oncogene Ect2 contributes to mitotic rounding through RhoA activation and that Ras overexpression promotes mitotic rounding. Epithelial-mesenchymal transition (EMT) is a cellular transformation in which epithelial cells loose epithelial polarity and intercellular adhesiveness gaining migratory potential. EMT, a hallmark in cancer progression, is commonly linked to early steps in metastasis promoting cancer cell invasiveness. Moreover, EMT was connected to cancer stem cells and the outgrowth of secondary tumors, suggesting that EMT may also be important for cell proliferation in a tumor. In this work, I investigated the role of EMT in actin cortex mechanics and mitotic rounding. To assess cortex mechanics, I measured the mechanical properties of the actin cortex in mitosis, in particular cortical stiffness and contractility before and after EMT. Furthermore, I also determined the mechanical changes of the actin cortex of interphase cells upon EMT; mechanics of interphase cells may critically influence mitotic rounding as interphase cells are a major constituent of the surrounding of a mitotic cell which needs to be deformed in the process of rounding. For our cortex-mechanical measurements, I used an established dynamic cell confinement assay based on atomic force microscopy. I show striking cortex- mechanical changes upon EMT that are opposite in interphase and mitosis. They are accompanied by a strong change in the activity of the actomyosin master regulators Rac1 and RhoA. Concomitantly, I characterize cortex-mechanical changes induced by Rac1 and RhoA signaling. In particular, I show that Rac1 inhibition restores epithelial cortex mechanics in post-EMT cells. Furthermore, I give evidence that EMT, as well as Rac1 activity changes induce actual changes in mitotic rounding in spheroids embedded in mechanically confining, covalently crosslinked hydrogels. Overall, I give evidence that EMT-induced changes results in a softer and less contractile cortex in interphase and a stiffer and more contractile cortex in mitotic cells, and it correlates with increased proliferation in confined environment.:Summary Zusammenfassung Acknowledgements 1-Introduction 1.1-The actin cortex 1.1.1-Regulation of actin cortex polymerization 1.1.2-Rho-GTPases in actin cortex regulation 1.1.3-The actin cortex in cell shape regulation and mitotic rounding 1.1.4-Experimental approaches to measure actin cortex mechanics 1.1.5-AFM cell confinement assay – a new tool for actin cortex-mechanical measurements 1.2-Epithelial-mesenchymal transition in cancer progression and metastasis 1.2.1-EMT effects on cell proliferation 1.2.2-EMT effects on Rho-GTPases activities 1.2.3-EMT effects on transcription factors 1.3-Outline of the thesis 2-Pharmacological induction of EMT 3-Mechanical changes of actin cortex mechanics upon EMT 3.1-Cell volume change during AFM confinement 3.2-Interphase and mitotic actin cortex mechanical changes upon EMT 3.3- Rho-GTPases activity changes upon EMT 4- Molecular perturbations of the cortex and their impact on cortex mechanics 5-Mitotic rounding in confined cell spheroids before and after EMT 5.1-The effect of cortex regulators on confined spheroids upon EMT 6-Time-dependence of actin cortex mechanics in breast epithelial cells 6.1-Rheology of actin cortex as a thin active film 6.2-Viscoelasticity of the actin cortex in relation to malignancy 7-Discussion 8-Outlook 8.1-Mitosis duration and quiescence in confined spheroids 8.2-Signalling cascades that trigger EMT-induced cortex-mechanical phenotype 8.2-Membrane tension upon EMT 9-Bibliography 10-Appendix 10.1-Abbreviations 10.2-Symbols / Die meisten tierischen Zellen nehmen beim Eintritt in die Mitose eine annähernd kugelförmige Form an. Dieser Vorgang wird als mitotische Aufrundung bezeichnet. Sie sorgt für die korrekte Morphogenese der mitotischen Spindel und damit für eine erfolgreiche Zellteilung. Wenn Zellen beim Eintritt der Mitose eine runde Form annehmen, müssen sie das umgebende Gewebe mechanisch verformen. Frühere Studien legen nahe, dass die für diese Verformung erforderlichen Kräfte aus der Kontraktilität des mitotischen Aktin-Cortexes resultieren; zu Beginn der Mitose führt ein Anstieg der kortikalen Kontraktilität zu einer erhöhten Zelloberflächenspannung, die die mitotische Zelle in eine kugelförmige Form treibt. Bei einem wachsenden Tumor erzeugt eine zunehmende Zelldichte einen Kompressionsdruck, der vermutlich ein zunehmendes mechanisches Hindernis für die mitotische Aufrundung darstellt. Es wurde gezeigt, dass mechanische Begrenzung oder äußerer Druck die Zellproliferation in Tumorsphäroiden hemmen. Es wurde daher die Hypothese aufgestellt, dass der Aktinkortex von Krebszellen onkogene Anpassungen aufweist, die eine fortlaufende mitotische Aufrundung und Zellteilung innerhalb von Tumoren ermöglichen. Weiterhin wurde gezeigt, dass das humane Onkogen Ect2 durch RhoA-Aktivierung zur mitotischen Aufrundung beiträgt und dass die Überexpression von Ras die mitotische Aufrundung fördert. Die epithelial-mesenchymale Transition (EMT) ist eine zelluläre Transformation, bei der Epithelzellen die epitheliale Polarität und die interzelluläre Adhäsivität verlieren und Migrationspotential gewinnen. EMT, ein Kennzeichen für das Fortschreiten von Krebs, ist häufig mit frühen Schritten der Metastasierung und einer Steigerung der Invasivität von Krebszellen verbunden. Darüber hinaus wird die EMT mit Krebsstammzellen und der Entstehung von Sekundärtumoren in Verbindung gebracht, was darauf hindeutet, dass die EMT auch für die Zellproliferation in einem Tumor wichtig sein könnte. In dieser Arbeit wurde die Bedeutung der EMT für die Mechanik des Aktinkortex und die mitotische Aufrundung untersucht. Die mechanischen Eigenschaften des Zellkortexes, insbesondere die kortikale Steifheit und Kontraktilität, wurden in mitotischen und nicht-adhärenten Interphasezellen gemessen vor und nach der EMT. Die Mechanik von Interphasenzellen kann die mitotische Aufrundung entscheidend beeinflussen, da Interphasenzellen ein Hauptbestandteil der Umgebung einer mitotischen Zelle sind, die während des Aufrundungsprozesses deformiert werden muss. Für meine kortexmechanischen Messungen verwendete ich einen etablierten Assay, der auf Rasterkraftmikroskopie basiert. Ich konnte ausgeprägte kortexmechanische Veränderungen durch die EMT feststellen, die in Interphase und Mitose entgegengesetzt sind. Diese kortikalen Veränderungen gehen mit einer starken Modifikation der Aktivitäten der Actomyosin-Hauptregulatoren Rac1 und RhoA einher. Weiterhin konnte ich kortexmechanische Veränderungen charakterisieren, die durch Rac1- und RhoA- Signale induziert werden. Insbesondere zeige ich, dass die Rac1-Hemmung die epitheliale Kortexmechanik in Post-EMT-Zellen wiederherstellt. Darüber hinaus fand ich Hinweise darauf, dass EMT- und Rac1-Aktivitätsänderungen zu einer Änderung der mitotischen Aufrundung in eingebetteten Sphäroiden führen. Insgesamt zeigen die Daten in dieser Arbeit klare Hinweise darauf, dass EMT-induzierte Veränderungen zu einem weicheren und weniger kontraktilen Kortex in der Interphase und einem steiferen und kontraktileren Kortex in mitotischen Zellen führen und mit einer erhöhten Proliferation in mechanisch begrenzten Zellumgebungen korrelieren.:Summary Zusammenfassung Acknowledgements 1-Introduction 1.1-The actin cortex 1.1.1-Regulation of actin cortex polymerization 1.1.2-Rho-GTPases in actin cortex regulation 1.1.3-The actin cortex in cell shape regulation and mitotic rounding 1.1.4-Experimental approaches to measure actin cortex mechanics 1.1.5-AFM cell confinement assay – a new tool for actin cortex-mechanical measurements 1.2-Epithelial-mesenchymal transition in cancer progression and metastasis 1.2.1-EMT effects on cell proliferation 1.2.2-EMT effects on Rho-GTPases activities 1.2.3-EMT effects on transcription factors 1.3-Outline of the thesis 2-Pharmacological induction of EMT 3-Mechanical changes of actin cortex mechanics upon EMT 3.1-Cell volume change during AFM confinement 3.2-Interphase and mitotic actin cortex mechanical changes upon EMT 3.3- Rho-GTPases activity changes upon EMT 4- Molecular perturbations of the cortex and their impact on cortex mechanics 5-Mitotic rounding in confined cell spheroids before and after EMT 5.1-The effect of cortex regulators on confined spheroids upon EMT 6-Time-dependence of actin cortex mechanics in breast epithelial cells 6.1-Rheology of actin cortex as a thin active film 6.2-Viscoelasticity of the actin cortex in relation to malignancy 7-Discussion 8-Outlook 8.1-Mitosis duration and quiescence in confined spheroids 8.2-Signalling cascades that trigger EMT-induced cortex-mechanical phenotype 8.2-Membrane tension upon EMT 9-Bibliography 10-Appendix 10.1-Abbreviations 10.2-Symbols

Cell motility has been found to play a role in many important body functions as well as the embryogenenis of mulitcellular organisms like vertebrates. From a physics point of view the interesting questions behind every motion are: Why is it moving? Where do the forces come from? Today we know that the motility of many cells is dependent on an active polymer network. Actin, one of the most abundant proteins in the body, is constantly polymerized, being moved around and depolymerized in motile cells. Until now, only forces outside the cell like traction forces could be measured. The direct measurement of the force generated by polymerizing actin filaments has only been measured by our lab and the lab of M. Radmacher. In these measurements fish keratocytes were used. Whereas I did these experiments, for the first time, on mammalian cells. To measure forward forces on neuronal growth cones I stabilized the SFM, as measurement times went up from minutes to hours. Furthermore measurements had to be performed at 37°C instead of room temerature, this induced drifts of the substrate. I incorporated an optical trap into the microscope to track the motion of the substrate. A feedback loop moved the SFM cantilever to minimize relative motion of substrate and cantilever. For keratocytes I directly measured the forces produced by actin polymerization and, to my knowledge for the first time, the forces associated with the retrograde actin flow using a SFM. The result was that both actin and myosin play important but different roles in motility. For actin it turned out that considering just the polymerization was not enough. Actin depolymerization and the resulting entropic forces are a completely new physical effect in actin based cell motility. With this new force in the force balance I can explain all effects observed in my experiments without introducing any new biochemical feedback loops. Finally I showed that neuronal growth cones are very soft and weak structures. They are at least one order of magnitude softer and weaker as for example fibroblasts or cells forming the blood vessel walls. As neurons are usually located in soft environments this does not impede their normal outgrowth. It could even serve as a safety mechanism that prevents cell from growing into wrong areas like breaching the blood-brain-barrier, on a physical level. For a neuron the wall of a blood vessel feels like a brick wall for us.

Mechanoresponsive Systeme antworten auf mechanische Reize mit einer Eigenschaftsänderung. Diese Dissertation umfasst die Arbeiten mit zwei mechanoresponsiven Systemen, die optisch auf mechanische Reize antworten. Sie basieren auf polymeren Strukturen, einer Polymerbürste und einem Hydrogelnetzwerk. Ihr optischer Antwortmechanismus ermöglicht die Beobachtung wirkender Kräfte als ein Ansatz zur in situ-Kraftmessung. Im ersten Teil wird ein existierendes, mechanoresponsives System zur Anwendung gebracht, das auf einer mit Fluoreszenzfarbstoff markierten Polyelektrolytbürste basiert. Die Ladungen des Polyelektrolyts können die Fluoreszenz des Farbstoffs unterdrücken, sodass lokale Kompression und Zugspannung über die Fluoreszenzintensität unterschieden werden können. Die mechanoresponsive Polymerbürste wurde als mechanosensitive Oberflächenbeschichtung angewandt, um Unterschiede in der Kontaktspannungsverteilung von Gecko-inspirierten adhäsiven Mikrostempelstrukturen aufzuklären. Die erarbeiteten Ergebnisse und daraus abgeleiteten Ablösemechanismen der Mikrostempeltypen deckten sich qualitativ mit Vorhersagen aus theoretischen Ansätzen. Aufgrund geometrischer Einschränkungen einer planaren Oberflächenbeschichtung zielt der zweite Teil darauf ab, dieses mechanoresponsive Prinzip in ein dreidimensionales Netzwerk zu überführen und ein mechanoresponsives Hydrogelnetzwerk als Plattform zur Kraftmessung zu entwickeln. Konzeptionell besitzt ein homogenes Netzwerk vorhersagbare mechanische Eigenschaften, sodass lokale optische Antworten auf mechanische Kräfte ermöglichen könnten, die wirkenden Kräfte zu lokalisieren und quantifizieren. Basierend auf einer Gestaltung nach der Flory-Rehner-Theorie wurden Präkursoren mit vordefinierter Größe und Architektur für die Hydrogelherstellung eingesetzt, um auf ein homogenes Netzwerk abzuzielen. Zu diesem Zweck wurde das Mischungsvolumen durch Tropfenmikrofluidik reduziert. Für den optischen Antwortmechanismus wurden die Hydrogelnetzwerk-Präkursoren mit zwei verschiedenen Fluorophoren markiert, die sich durch abstandsabhängige Emission über Förster-Resonanzenergietransfer auszeichnen. Die Funktionalität des optischen Antwortmechanismus wurde auf globaler Ebene durch Kollabieren und kontrolliertes Quellen des Netzwerks, dann auf lokalisierter Ebene durch definierte mechanische Belastung mit Rasterkraftmikroskopie gezeigt. Durch ihre Anpassbarkeit könnte die Hydrogelplattform zukünftig verschiedenste Anwendungen im Bereich intrisischer Kraftmessung weicher Materie bedienen. / Mechanoresponsive systems respond to mechanical triggers by changes in a certain property. This thesis covers the work conducted with two mechanoresponsive systems that respond optically to mechanical triggers. These two systems are based on polymer structures, a polymer brush and a hydrogel network. Thus, the optical response mechanism allows observing acting forces as an approach to force sensing in situ. In the first part, an existing mechanoresponsive system based on a polyelectrolyte brush labeled with a fluorescent dye is engaged in application. The charges of the polyelectrolyte are able to quench the fluorescence of the dye so that local compression or tension can be distinguished from the local fluorescence intensity. The mechanoresponsive polymer brush was applied as mechanosensitive surface coating to elucidate differences in the contact stress distributions of gecko-inspired adhesive micropillar structures. The determined results and the derived detachment mechanisms of the micropillar types were in qualitative accordance with predictions from theoretical approaches. Overcoming the geometrical limitations of a planar surface coating, the second part aims at translating the mechanoresponse principle to a three-dimensional network and developing a mechanoresponsive hydrogel as a platform for force sensing. Conceptually, a homogeneous network allows to predict mechanical properties so that localized optical mechanoresponses could enable locating and quantifying acting forces. Based on network design principles from the Flory-Rehner theory, precursors with predefined size and architecture were utilized in hydrogel preparation, aiming for a homogeneous network. Further in this regard, the mixing volume was reduced by employing droplet microfluidics. As optical response mechanism, the hydrogel network precursors were labeled with two kinds of fluorophore, featuring distance-dependent emission from Förster Resonance Energy Transfer. The functionality of the optical response mechanism was demonstrated on global level by collapsing and controlled swelling of the network, and on a localized level by defined mechanical stress, applied with Atomic Force Microscopy. Owing to its adjustability, the hydrogel platform might be employed in various applications that require intrinsic force sensing of soft matter in future.

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