Dans les tissus, les cellules génèrent et sont soumises en permanence à des forces mécaniques. Les perturbations biochimiques à l'intérieur des cellules, ainsi que les altérations de leur environnement mécanique peuvent modifier l'équilibre physiologique et mener à des pathologies, comme le cancer. Bien que les propriétés mécaniques puissent être modifiées à l'échelle du tissus, la compréhension de la mécanique au niveau de la cellule unique demeure importante. En particulier, la différenciation, la migration des cellules immunitaires et le caractère invasif d'un cancer dépendent fortement des propriétés mécaniques des cellules uniques. Les déformations mécaniques peuvent induire un changement de la surface et du volume cellulaires. Nous nous intéressons particulièrement à la régulation du volume cellulaire chez les cellules mammifères dans le contexte de déformations à différentes échelles de temps. Jusqu'à présent, la régulation du volume dans ce contexte n'a été que très peu étudiée, en raison de la difficulté d'obtention de mesures précises, et du fait que le volume de la cellule est généralement considéré comme constant. Nous avons développé une méthode de mesure du volume cellulaire reposant sur l'exclusion de fluorescence, qui nous permet d'effectuer des mesures de volume précise au niveau de la cellule unique. Dans cette étude, nous nous sommes concentrés sur la régulation du volume cellulaire au cours de l'étalement dynamique sur un substrat (échelle de temps : minutes). Nous avons démontré qu'il existe différents régimes de régulation du volume lors de l'étalement : les cellules réduisent, augmentent ou ne modifient pas leur volume, en fonction de l'état du cortex d'actomyosine et de la vitesse d'étalement. Nous avons constaté que les cellules s'étalant plus vite ont tendance à perdre davantage de volume. Notre hypothèse est que lors d'une extension rapide de lamellipode dépendante d'Arp2/3, l'actine tire sur la membrane et génère une tension et l'activation de transport ionique, s'accompagnant d'une perte de volume compensatoire. L'inhibition de la polymérisation de l'actine ou de sa ramification dépendante d'Arp2/3 réduit la vitesse d'étalement et ainsi la perte de volume. Nous avons ensuite montré que l'inhibition de la contractilité augmente la vitesse d'étalement et la perte de volume. Cependant, l'inhibition d'Arp2/3 dans des cellules à faible contractilité conduit à un étalement rapide sans perte de volume. En effet, l'inhibition d'Arp2/3 induit des bulles de membranes, une déformation rapide n'induirait donc pas de perte de volume car la cellule peut relâcher la tension en dépliant la membrane. Nous avons également montré que la régulation du volume en réponse à une compression mécanique rapide (échelle de temps : millisecondes) indépendante de l'adhérence dépend également de l'état du cortex d'actomyosine. Les cellules perdent jusqu'à 30% de leur volume lorsqu'elles sont confinées, car la membrane plasmique est attachée au cortex et ne peux pas être dépliée en réponse à l'augmentation de la tension. La perturbation du cortex d'actine induit le détachement de la membrane et limite la perte de volume. Enfin, nous avons montré que la réponse du volume à un choc osmotique (échelle de temps : secondes) est plus que complexe que décrite dans la littérature. Nos données indiquent qu'au niveau de la cellule unique, la réponse initiale du volume au changement de l'osmolarité extérieure n'est pas un processus passif uniforme. En utilisant la technique du choc osmotique, nous avons également confirmé que les cellules ont un large excès de membrane repliée dans des réservoirs. Nos résultats montrent que le volume et l'aire cellulaires sont couplés par l'homéostasie de la tension de surface, et, étant donné que les déformations induisent une augmentation de la tension de surface, elles conduisent à des modifications du volume et de l'aire de la cellule. / The field of biomechanics significantly progressed in the last two decades. The importance of the feedback between biochemical signaling and physical properties was revealed in many studies. Cells within tissues constantly generate and experience mechanical forces. Biochemical perturbations inside the cells as well as alterations in the mechanical environment can shift the tiny balance of normal physiological state and lead to pathologies, e.g. cancer. Although the mechanical properties of individual cells can alter when they are within the tissues, the understanding of single cell mechanics is still important. Differentiation, immune cell migration, and cancer invasion strongly depend on the mechanical properties of individual cells. Mechanical deformations can lead to a change in cell surface area and volume. We are particularly interested in single mammalian cell volume regulation in the context of deformations of different timescales. For the moment, volume regulation in this context was out from the research interest, probably due to the difficulties of accurate measurements, and cell volume often considered as a constant parameter. We developed a method for cell volume measurements based on a fluorescent exclusion that allowed us to perform precise volume measurements of individual live cells. In the present study, we mainly focused on cell volume regulation while dynamic spreading on a substrate (timescale – minutes). We demonstrated that there are different regimes for volume regulation while spreading: cells decrease, increase or do not change volume, and a type of the regime depends on the state of the actomyosin cortex and spreading speed. We obtained that faster-spreading cells tend to lose more volume. Our hypothesis is that during fast Arp2/3-driven lamellipodia extension actin pull on the membrane that generates tension and activation of ion transport and regulatory volume loss. Inhibition of actin polymerization or Arp2/3-dependent actin branching decreases spreading speed and volume loss. Next, we showed that inhibition of contractility increases spreading speed and volume loss. However, inhibition of Arp2/3 complex in cells with low contractility leads to fast spreading without volume loss. Our explanation is that inhibition of Arp2/3 induces cell blebbing and even fast deformation does not lead to volume loss as a cell can relax tension by membrane unfolding. We also showed that volume regulation in response to fast mechanical compression (timescale – milliseconds) independent of adhesion also depends on the actomyosin cortex state. Control cells lose up to 30% of volume under confinement, as the cell membrane is attached to the cortex and cannot be unfolded in response to the tension increase. Disruption of actin cortex leads to membrane detachment and prevents volume loss under confinement. Additionally, we showed that cell volume response to the osmotic shock (timescale – seconds) is more complex than it used to be known in the literature. For instance, our data indicate that at the level of individual cells initial volume response to the change of external osmolarity is not a uniform passive process. Using osmotic shock technique, we also confirmed that cells have a large excess of membrane folded in reservoirs. Taken together, our data show that cell volume and surface area are coupled through surface tension homeostasis and as deformations induce surface tension increase, they lead to change volume and surface area.

Development of a single-cell embryo into an adult multi-cellular organism features the establishment of upto three anatomical body axes - anteroposterior, dorsoventral and left-right. It has been observed in many organisms that these body axes can consistently orient relative with respect to the geometric features of the embryo in many organisms. One such example is observed in the model organism Caenorhabditis elegans (C. elegans), where the Anteroposterior (AP) axis coincides with the geometric long axis of the ellipsoidal embryo -- the shape being imposed by the surrounding eggshell. In C. elegans, the Anteroposterior axis is established at the one-cell stage via its polarization by PAR polarity proteins. This cell polarization proceeds via a self-organized mechanochemical feedback between the PAR proteins and mechanical flows in the actomyosin cortex, resulting in the formation of two mutually exclusive domains of Anterior PAR and Posterior PAR proteins on the cortex denoting the future anterior and posterior end of the embryo -- and thus establishing the Anteroposterior axis. The initial orientation of the Anteroposterior axis is determined by the site of sperm entry at fertilization. However, the nascent Anteroposterior axis that forms after fertilization is observed to actively re-orient -- indicated by the movement of the PAR domains and concurrent migration (here termed posteriorisation) of the sperm-donated male pronucleus -- such that it aligns with the long axis of the ellipsoidal embryo, if it is not already aligned. In effect, the site of sperm entry only determines which half of the embryo becomes the posterior half of the embryo. This phenomenon of active re-orientation of the Anteroposterior axis, that ensures that the Anteroposterior axis aligns with the long axis of the embryo, is termed Anteroposterior axis alignment. The work described in this thesis investigates the mechanism of this Anteroposterior axis alignment in the C. elegans embryo. Anterior-directed flows in the actomyosin cortex observed during Anteroposterior axis establishment have also been found to be essential for Anteroposterior axis alignment. In this thesis, two possible mechanisms of Anteroposterior axis alignment are considered, both of which are consequences of these cortical flows. Cortical flows at the embryo surface can drive flows in the bulk cytoplasm in the embryo, generating cytoplasmic flows which point towards the sperm-donated male pronucleus as it posteriorises. Previous studies have proposed that these cytoplasmic flows could push onto the male pronucleus, and due to the ellipsoidal geometry of the embryo, drive it towards the closest tip of the embryo. This proposed mechanism is referred to as the cytoplasmic flow-dependent mechanism in this thesis. Another mechanism proposed in this thesis postulates that the reorientation of the Anteroposterior axis occurs via the repositioning of the pseudocleavage furrow. The pseudocleavage furrow is a contractile ring-like structure that forms at the boundary of the two PAR domains during Anteroposterior axis establishment. The pseudocleavage furrow forms as a result of compressive alignment of actin filaments in the actomyosin cortex due to cortical flows. In cases where the Anteroposterior axis is not aligned with the long axis of the embryo, the pseudocleavage furrow is not perpendicular to the long axis of the embryo. In such cases, active anisotropic stresses generated in the actomyosin cortex could force the rotation of the pseudocleavage furrow akin to an elastic rubber-band on an ellipsoid, and cause the Anteroposterior axis to re-orient towards the long axis of the embryo. This proposed mechanism is referred to as the pseudocleavage furrow-dependent mechanism in this thesis. This thesis investigates the role played by the two mechanisms in Anteroposterior axis alignment. This is accomplished in the following way: a theoretical model of the Anteroposterior axis alignment is introduced, consisting of a description of the actomyosin cortex as an active nematic fluid present on the 2D surface of a fixed ellipsoid representing the embryo. This description of the cortex incorporates both the cytoplasmic flow-dependent mechanism and the pseudocleavage furrow-dependent mechanism. RNAi experiments in the C. elegans embryo that remove the pseudocleavage furrow, in conjuction with numerical simulations using the theoretical model, show that the pseudocleavage furrow-dependent mechanism is the predominant mechanism that drives Anteroposterior axis alignment, while cytoplasmic flow-dependent mechanism plays only a minor role. RNAi experiments that modify the geometry of the C. elegans embryo -- specifically, generate rounder embyros -- show that embryo geometry can influence the rate of re-orientation of the Anteroposterior axis during Anteroposterior axis alignment -- with slower Anteroposterior axis alignment in rounder embryos. Such an relation between embryo geometry and Anteroposterior axis alignment is found to be consistent with pseudocleavage furrow-dependent mechanism, both via predictions made using the theoretical model and using a simplified effective model of a contractile ring (or elastic rubber-band) on a fixed ellipsoid. Altogether, the work presented in this thesis shows Anteroposterior axis alignment observed in the C. elegans embryo is driven primarily by the anisotropic stresses in the actomyosin cortex that generate the pseudocleavage furrow. The work here also shows that the Anteroposterior axis alignment process is sensitive to the geometry of the embryo. In effect, active mechanical flows in the actomyosin cortex translate the ellipsoidal geometry of the embryo into a robust orientation of the Anteroposterior axis of the C. elegans embryo. Mechanical flows such as these are not exclusive to C. elegans, nor are specific orientations of the body axes with respect to the embryo geometry. The results in this thesis thus point towards a possibly general role of the interactions between mechanical flows and embryo geometry to properly orient the body axes of the developing embryos of many multi-cellular organisms.:Contents Abbreviations iii Abstract iv 1 Introduction 1 1.1 Cytoskeleton 3 1.1.1 Main constituents of the cytoskeleton 3 1.1.2 Actomyosin cortex 7 1.2 Hydrodynamic theory of active fluids 8 1.2.1 Conservation Laws 9 1.2.2 Continuously broken symmetries 11 1.2.3 Irreversible thermodynamics of active fluids 13 1.2.4 Constitutive equations of active nematic fluids 19 1.3 C. elegans as a model organism 21 1.3.1 Early embryogenesis in C. elegans 22 1.4 AP axis establishment in C. elegans 24 1.4.1 PAR polarity system . 24 1.4.2 Mechanism of AP axis establishment 26 1.4.3 AP axis alignment 27 1.5 Overview 29 2 A theoretical model for AP axis alignment 30 2.1 A model of AP axis establishment in C. elegans 30 2.1.1 Turing-like system for PAR polarity system 31 2.1.2 Active isotropic description of actomyosin cortex 33 2.1.3 Guiding cues for AP axis establishment 34 2.1.4 Full model of AP axis establishment in [1] 35 2.2 A model of pseudocleavage furrow formation in C. elegans 36 2.2.1 Dynamics of Actin alignment 37 2.2.2 Active stress generated by alignment of actin filaments 38 2.3 A model of AP axis alignment in C. elegans 39 2.3.1 A thin film active nematic description of the cortex 40 2.3.2 Description of the Cytoplasm and Male pronucleus 46 2.3.3 Numerical simulations of the theoretical model 48 3 Materials and Methods 52 3.1 Culture conditions, strains and worm handling 52 3.2 Genetic perturbations by RNAi 53 3.3 Time-lapse microscopy 53 3.4 Image analysis 54 3.4.1 Pre-processing 54 3.4.2 Tracking posteriorisation of the male pronucleus 56 3.4.3 Measuring cortical flows 66 3.4.4 Measuring cytoplasmic flows 67 3.5 Data analysis 67 4 Experimental investigation of AP axis alignment 71 4.1 Characterising AP axis alignment in unperturbed embryos 71 4.2 Cortical flows are required for AP axis alignment 76 4.3 Role of Pseudocleavage furrow in AP axis alignment 83 4.3.1 Removing Pseudocleavage furrow via RNAi 83 4.3.2 Comparing numerical simulations to experimental results 88 4.4 Role of embryo geometry in AP axis alignment 99 4.4.1 Rounder embryos show slower AP axis alignment 99 4.4.2 Relation between embryo geometry and AP axis alignment 108 4.5 Additional experiments 118 4.5.1 Exploring relation between embryo geometry and AP axis alignment in ima-3 RNAi embryos 118 4.5.2 Are pseudocleavage furrow-dependent and cytoplasmic flow-dependent mechanisms sufficient to explain AP axis alignment? 121 4.5.3 Role of microtubules in AP axis alignment 127 5 Conclusions and Outlook 134 Appendix 139 Bibliography 142 List of publications 156 Acknowledgements 157

The cytoskeleton is an intrinsic aspect of all cells, and is essential for many cellular events including cell motility, endocytosis, cell division and wound healing. Remodeling of the cytoskeleton in response to these cellular activities leads to significant alterations in the morphology of the cell. One such alteration is the formation of an actomyosin contractile array required for cytokinesis, wound healing and embryonic development. / Cellular structure and shape depends upon tensional prestress brought about by the organization of cytoskeletal components. Using the Xenopus laevis oocyte wound healing model, it is first described how diminished cellular tension affects the balance of the Rho family of GTPases, and subsequently prevents the formation of actomyosin contractile arrays. This suggests that cellular tension in the cell is not created at the level of the cytoskeletal elements but rather via the upstream signaling molecules: RhoA and Cdc42. / The role of N-WASP (Neural-Wiscott Aldrich Syndrome Protein), a mediator of Arp2/3 based actin polymerization, is next examined for its putative role in cellular wound healing. Xenopus laevis oocytes injected with mutant N-WASP constructs reveals in vivo evidence that functional N-WASP is required for appropriate contractile array formation and wound closure. / Lastly, it is revealed that the cellular structures involved with single cell wound healing in other model systems are also important for the initial repair of severed muscle cells. Actin, non-muscle myosin-II, microtubules, sarcomeric myosin and Cdc42 are all recruited and reorganized at the edge of damaged C2C12 myotubes. This data promotes the possibility that an actomyosin array may be established in injured muscle cells as well.

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

Les cellules neuroendocrines possèdent d’une part la voie de sécrétion constitutive, existant dans tous les types cellulaires, qui permet le renouvellement continu de la membrane plasmique et de la matrice extracellulaire, et d’autre part la voie de sécrétion régulée, spécifique aux cellules sécrétrices, qui permet la sécrétion d’hormones suite à la stimulation de la cellule. Les organites impliqués dans cette dernière voie sont des granules de sécrétion à cœur dense (GS), sui stockent les hormones ainsi que les glycoprotéines solubles, les granines. Parmi ces dernières, la chromogranine A (CgA) joue un rôle majeur dans la biogénèse des GS mais les mécanismes moléculaires ne sont pas clairement définis. Dans une lignée de cellules non-endocrines COS7 (dépourvues de granines et donc de voie de sécrétion régulée), mon équipe d’accueil a démontré que l’expression de la CgA induit la formation de vésicules présentant une structure et des fonctions caractéristiques des GS. L’analyse du protéome des GS purifiés à partir d’une lignée de cellules COS7 exprimant de manière stable la CgA (COS7-CgA) a révélé la présence de protéines liant les éléments du cytosquelette et le calcium. Durant ma thèse, nous avons focalisé notre attention sur la myosine 1b (myo1b), l’actine et le complexe de nucléation de l’actine Arp2/3 du fait de leur capacité à induire le bourgeonnement de compartiments post-golgiens dans des cellules non-endocrines. Nous avons montré (i) que la myo1b contrôle la formation des GS ainsi que la sécrétion régulée au sein des cellules COS7-CgA et des cellules neuroendocriniennes PC12, et (ii) que la myo1b et le complexe Arp2/3 permettent le recrutement d’actine fibrillaire dans la région golgienne et la formation des GS. Ces travaux montrent pour la première fois l’implication du complexe actomyosine dans la formation des GS. Afin d’identifier le lien moléculaire entre la CgA luminale et la myo1b cytosolique, nous avons recherché les interactions potentielles de la CgA avec les lipides de la membrane du réseau trans-golgien (TGN). Nous avons montré (i) que la CgA interagit avec l’acide phosphatidique (PA), (ii) que les espèces de PA prédominantes sont communes dans les membranes golgienne et granulaire, (iii) que la CgA est capable d’interagir spécifiquement avec des espèces de PA intégrées avec des membranes artificielles et (iv) que l’inhibition de la production du PA au niveau golgien altère significativement la formation des GS et la sécrétion régulée dans les cellules neuroendocrines. L’ensemble des résultats obtenus dans le cadre de ma thèse suggère que l’interaction entre la CgA et le PA est cruciale pour la biogenèse de GS à partir de la membrane du TGN. Nous émettons l’hypothèse que cette interaction est à l’origine de la formation de microdomaines enrichis en PA qui contrôleraient la courbure de la membrane du TGN et le recrutement du complexe actomyosine. / Neuroendocrine cells exhibit the constitutive secretory pathway which is common all cell types and allows the continuous renewal of the plasma membrane and the extracellular matrix, and the regulated secretory pathway, which is specific to secretory cells and allows hormone secretion following cell stimulation. The organelles supporting the latter pathway are dense-core secretory granules (SG), which store hormones and soluble glycoproteins, called granins. Among these, chromogranin A (CgA) plays a major role in the biogenesis of SG but the molecular mechanisms underlying this process are not clearly understood. Using non-endocrine COS7 cell line (which are devoid of granins and regulated secretory pathway), my host team has demonstrated that the CgA expression induces the formation of vesicles with structural and functional characteristic of SG. The proteome analysis of purified SG from a COS7 cell line stably expressing CgA (COS7-CgA) revealed the presence of cytoskeleton- and calcium-binding proteins. During my thesis, we focused our attention on myosin 1b (myo1b), actin and actin nucleation complex Arp2/3 due to their ability to induce the budding of post-Golgi compartments in non-endocrine cells. We have shown (i) that myo1b controls SG formation as welle as the regulated secretion in COS7-CgA and PC12 neuroendocrine cells, (ii) that myo1b and Arp2/3 complex are required to recruit fibrillar actin (F-actin) to the Golgi region and to SG formation. These results highlight for the first time the involvement of the actomyosin complex in SG formation. In order to identify the molecular link between luminal CgA and Cytosolic myo1b, we investigated the potential interactions of CgA with lipids of the trans-Golgi network (TGN) membrane. We showed (i) that CgA interacts with phosphatidic acid (PA), (ii) that the predominant PA species are common in Golgi and granular membranes, (iii) that Cg Ais able to interact specifically with these PA species included in artificial membranes, and (iv) that inhibition of PA production at the Golgi level significantly alters SG formation and regulated secretion in neuroendocrine cells. All these results obtained during my thesis suggest that the interaction between CgA and PA is crucial for SG biogenesis from the TGN membrane. We suggest that this interaction is at the origin of the formation of PA-enriched microdomains that could control the curvature of the TGN membrane and the recruitment of the actomyosin complex.

No description available.

Macrophages are innate immune cells responsible for engulfing microbes and cell debris through phagocytosis and orchestrating immune responses to maintain homeostasis. While conducting immune surveillance over all types of organs and tissues, macrophages face inherently heterogeneous microenvironments with unique biophysical features. For instance, microglia residing in the brain, Kupffer cells living in the skin and bone osteoclasts are exposed to very distinct tissue stiffnesses. Despite the research done in the last decade clearly indicates that macrophages are sensitive to physical factors, how mechanical cues modulate their inflammatory response remains poorly understood. The present study aims at investigating how microenvironment stiffness influences the pro-inflammatory behaviour of macrophages. Besides characterising the regulatory effect on pro-inflammatory gene expression and cytokine production, this work examines the impact of stiffness on the inflammasome, one of the main macrophage signalling platforms. For this, an in vitro system based in 2D polyacrylamide hydrogels whose stiffness can be independently tuned was established. Using substrates with an elastic moduli between 0.2 and 33.1 kPa, bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff polyacrylamide. Upon priming with lipopolysaccharide, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels, yet there were no significant differences in the expression of other major pro-inflammatory genes. Additionally stimulating macrophages with the ionophore nigericin revealed higher secreted protein levels of IL-1β and IL-6 on compliant substrates. Interestingly, macrophages challenged on compliant polyacrylamide also displayed an enhanced formation of the NLRP3 inflammasome as well as increased levels of pyroptotic cell death. The upregulation of inflammasome assembly on compliant hydrogels was not primarily attributed to the reduced cell spreading, since spatially confining cells on micropatterns led to a decrease of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, these results show that substrate stiffness affects the pro-inflammatory response of macrophages and for the first time describe that the NLRP3 inflammasome is one of the signalling components affected by stiffness mechanosensing. The work presented here expands our understanding of how microenvironment stiffness affects macrophage behaviour and which elements of their machinery might contribute to integrate mechanical cues into the regulation of their inflammatory functions. The onset of pathological processes or the implant of foreign bodies represent immune challenges in which macrophages can face a mechanically changing environment. Therefore, a better insight on how macrophages detect and process biophysical signals could potentially provide a basis for new strategies to modulate inflammatory responses.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS / Als Teil des angeborenen Immunsystems sind Makrophagen dafür verantwortlich Pathogene und Zellrückstände durch Phagozytose zu beseitigen. Sie orchestrieren Immunantworten um homöostatische Bedingungen von Organen und Geweben aufrechtzuerhalten. Dabei sind sie extrem heterogenen Mikroumgebungen ausgesetzt, welche sich jeweils durch eine einzigartige Kombination von (bio)chemischen und mechanischen Eigenschaften, vor allem Gewebesteifigkeiten, auszeichnen. Dies veranschaulichen beispielsweise im Gehirn residierende Mikroglia, Kupffer-Zellen in der Haut und Osteoklasten in Knochen. Obwohl diverse Studien aus dem letzten Jahrzehnt eindeutig zeigen, dass Makrophagen auf mechanische Signale reagieren, ist der zugrunde liegende Mechanismus, wie diese Signale eine Entzündungsreaktion modulieren, noch immer unzureichend verstanden. Die vorliegende Studie beinhaltet die systematische Untersuchung, wie die Steifigkeit der Mikroumgebung das proinflammatorische Verhalten von Makrophagen beeinflusst. Neben der Charakterisierung der regulatorischen Wirkung auf die proinflammatorische Genexpression und Zytokinproduktion untersucht diese Arbeit auch den Einfluss der Steifigkeit auf das Inflammasom; eine der wichtigsten Signalplattformen für Makrophagen. Zu diesem Zweck wurde zunächst ein Zellkultursystem mit 2D-Polyacrylamid-Hydrogelen als Zellsubstrat entwickelt, bei dem das Elastizitätsmodul der Gelsubstrate gezielt eingestellt werden kann. Unter Verwendung von Substraten mit einem Elastizitätsmodul zwischen 0,2 kPa und 33,1 kPa zeigt die mikroskopische Analyse, dass aus Knochenmark stammende Makrophagen im Vergleich zu steifem Polyacrylamid eine weniger ausgebreitete und rundere Morphologie annehmen. Nach dem Primen mit Lipopolysaccharid waren die Expressionsniveaus des Gens, das für TNF-α kodiert, auf deformierbareren Hydrogelen höher, jedoch gab es keine signifikanten Unterschiede in der Expression anderer wichtiger pro-inflammatorischer Gene. Eine zusätzliche Stimulierung von Makrophagen mit dem Ionophor Nigericin bewirkte höhere sekretierte Proteinspiegel von IL-1β und IL-6 auf deformierbaren Substraten. Makrophagen, die deformierbarem Polyacrylamid ausgesetzt waren, zeigten auch eine verstärkte Bildung des NLRP3-Inflammasoms sowie ein erhöhtes Ausmaß an pyroptotischem Zelltod. Die Hochregulierung der Inflammasom-Assemblierung auf deformierbaren Hydrogelen wurde nicht primär auf die reduzierte Zellausbreitung zurückgeführt, da räumlich begrenzte Zellen auf Mikromustern zu einer Abnahme von Inflammasom-positiven Zellen im Vergleich zu stark ausgebreiteten Zellen führten. Schließlich verringerte eine Störung der Aktomyosin-Kontraktilität die Unterschiede in der Inflammasombildung zwischen deformierbaren und steifen Substraten. Zusammenfassend zeigen diese Ergebnisse, dass die Substratsteifigkeit die proinflammatorische Reaktion von Makrophagen beeinflusst und beschreiben erstmalig, dass das NLRP3-Inflammasom eine der Signalkomponenten ist, die von der zellulären Steifheitswahrnehmung beeinflusst werden. Die hier vorgestellte Arbeit erweitert unser Verständnis davon, wie die Steifigkeit der Mikroumgebung das Verhalten von Makrophagen beeinflusst und welche Elemente ihrer Maschinerie dazu beitragen könnten mechanische Signale in die Regulierung ihrer Entzündungsfunktionen zu integrieren. Das Einsetzen pathologischer Prozesse oder die Implantation von Fremdkörpern stellen Immunherausforderungen dar, bei denen Makrophagen einer sich mechanisch verändernden Umgebung ausgesetzt sein können. Daher könnte ein besserer Einblick in die Art und Weise, wie Makrophagen biophysikalische Signale erkennen und verarbeiten, möglicherweise eine Grundlage für neue Strategien zur Modulation von Entzündungsreaktionen bieten.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

Three novel mutations (G159D, L29Q and E59D/D75Y) in cardiac troponin C (CTnC) associate their clinical outcomes with a given cardiomyopathy. Current paradigms propose that sarcomeric mutations associated with dilated cardiomyopathy (DCM) decrease the myofilament calcium sensitivity while those associated with hypertrophic (HCM) cardiomyopathy increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the calcium regulation of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. This required the development of new calculator programs to solve complex ionic equilibria to more accurately buffer and expand the free calcium range of our test solutions. In accordance with the DCM paradigms, our result show that G159D and E59D/D75Y CTnC decrease the myofilament calcium sensitivity and force generating capabilities which would likely increase the rate of muscle relaxation and weaken the contractile force of the myocardium. Alternatively, the lack of myofilament change from L29Q CTnC (associated with HCM) may explain why the only proband is seemingly unaffected. Notably, the changes in the calcium sensitivity of tension (in fibers) do not necessarily occur in the isolated CTnC and vice versa. These counter-intuitive findings are justified through a transition in calcium affinity occurring at the level of cardiac troponin (CTn) and higher, implying that the true effects of these mutations become apparent as the hierarchal level of the myofilament increases. Despite these limitations, the regulated thin filament (RTF) retains its role as the calcium regulatory unit and best indicates a mutation's ability to sensitize (+) or desensitize (-) the muscle to calcium. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse (+ or -) these aberrant changes in myofilament sensitivity. Therefore, we have developed an RTF mediated High Throughput Screening assay to identify compounds in libraries of molecules that can specifically modulate the calcium sensitivity of cardiac contraction. The knowledge gained from these studies will help us and others to uncover new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases.