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International audience; The barrier function of epithelia is one of the cornerstones of the body plan organisation of metazoans. It relies on the polarity of epithelial cells which organises along the apico-basal axis the mechanical properties, signalling as well as transport. This barrier function is however constantly challenged by the fast turnover of epithelia occurring during morphogenesis or adult tissue homeostasis. Yet, the sealing property of thetissue can be maintained thanks to cell extrusion: a series of remodelling steps involving the dying cell and its neighbours leading to seamless cell expulsion. Alternatively, the tissue architecture can also be challenged by local damages or the emergence of mutant cells that may alter its organisation. This includes mutants of the polarity complexes which can generate neoplastic overgrowths or be eliminated by cell competition when surrounded by wild type cells. In this review, we will provide an overview of the regulation of cell extrusion in various tissues focusing on the relationship between cell polarity, cell organisation and the direction of cell expulsion. We will then describe how local perturbations of polarity can also trigger cell elimination either by apoptosis or by cell exclusion, focusing specifically on how polarity defects can be directly causal to cell elimination. Overall, we propose a general framework connecting the influence of polarity on cell extrusion and its contribution to aberrant cell elimination.

Epithelial cell death is essential for tissue homeostasis, robustness and morphogenesis. The expulsion of epithelial cells following caspase activation requires well-orchestrated remodeling steps leading to cell elimination without impairing tissue sealing. While numerous studies have provided insight about the process of cell extrusion, we still know very little about the relationship between caspase activation and the remodeling steps of cell extrusion. Moreover, most studies of cell extrusion focused on the regulation of actomyosin and steps leading to the formation of a supracellular contractile ring. However, the contribution of other cellular factors to cell extrusion has been poorly explored. Using the Drosophila pupal notum, a single layer epithelium where most extrusion events are caspase-dependent, we first showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration/dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. We confirmed that local and rapid increase/decrease of microtubules is sufficient to respectively expand/constrict cell apical area. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting steps of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation., This a pre-release prior to the publication of the article. It will be later updated

Epithelia are tissues made up of cells that are highly adherent to each other. Thanks to this property, epithelia act as a barrier and protect the organs they encase from external dangers such as infections. However, epithelia are far from static and exhibit high rates of cell turnover through cell division and death. This is a challenge for the tissue because the fragmentation of dying cells can result in a loss of tissue sealing and thus potentially endanger the organs they protect. To address this problem, apoptotic cells are expelled from the epithelia by a sequence of remodelling events that contract the cell while bringing its neighbours together. This process is called cell extrusion and it helps maintain tissue sealing during cell death. However, how the cell makes the decision to extrude and what are the mechanisms that lead to its contraction are not well known. To study this phenomenon, we use the monolayer epithelium of the Drosophila pupa notum. In this tissue, caspase activation precedes and is necessary for cell extrusion. Cell extrusion has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. We showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion. Additionally, microtubule stabilisation strongly impairs cell extrusion. This demonstrates for the first time that microtubules disassembly by caspases is an important rate-limiting step of extrusion. Moreover, it outlines a more general function of microtubules in epithelial cell shape stabilisation. Additionally, because caspase cleave a large amount of substrate, caspase activation was long seen as a point of no-return irreversibly leading to apoptosis. However, recent studies showed non-apoptotic functions of caspases. These non-apoptotic functions are broad and involved for instance in cell differentiation, proliferation, maintenance of pluripotency and many others. Similarly, our work and other’s showed events of transient caspase activation. Therefore, what sets the balance between non-lethal caspase levels and the irreversible commitment in apoptosis remains unclear. It has been proposed that cells can survive up to a certain threshold of caspase activation but was never tested. If such threshold indeed exists, how its value is set and modulated is unclear. We used a live caspase reporter and systematic segmentation of cells in the notum to quantify the engagement of cell in extrusion. Using logistic regression, we showed that there is no common threshold of effector caspase activity leading to extrusion. Rather, caspases levels correlate linearly with the probability to engage in extrusion. Furthermore, using Bayesian statistical analysis we were able to identify parameters predictive of cell sensitivity to caspase. We showed for instance that past caspase activation seems to be associated with the engagement in extrusion at a lower caspase level. This is the first quantitative analysis of the engagement process in extrusion or apoptotis in vivo and at the single cell level.; Les épithéliums sont des tissus constitués de cellules très adhérentes les unes aux autres. Grâce à cela, les épithéliums agissent comme une barrière et protègent les organes qu'ils enveloppent des dangers extérieurs tels que les infections. Cependant, les épithéliums sont loin d'être statiques et présentent des taux élevés de renouvellement cellulaire par division et mort cellulaire. C'est un défi pour le tissu car la fragmentation des cellules mourantes peut entraîner une perte d'étanchéité du tissu et donc mettre en danger les organes qu'ils protègent. Pour pallier cela, les cellules apoptotiques sont expulsées des épithéliums par une séquence d'événements de remodelage qui contractent la cellule tout en rapprochant ses voisines. Ce processus, appelé extrusion cellulaire, contribue à maintenir l'étanchéité des tissus pendant la mort cellulaire. Cependant, la façon dont la cellule prend la décision d'extruder, et les mécanismes qui conduisent à sa contraction sont mal connus. Pour étudier ce phénomène, nous utilisons l'épithélium monocouche du notum de la pupe de drosophile. Dans ce tissu, l'activation des caspases précède et est nécessaire à l'extrusion cellulaire. L'extrusion cellulaire a été principalement analysée à travers l'étude de la régulation de l'actomyosine. Cependant, la relation entre l'activation des caspases et l'extrusion cellulaire est encore mal comprise. Nous avons montré que, contrairement à ce qui était anticipé, l'initiation de l'extrusion cellulaire et la constriction apicale ne sont pas associées à la modulation de la concentration ou de la dynamique de l'actomyosine. Au contraire, la constriction apicale des cellules est initiée par le désassemblage d'un réseau médio-apical de microtubules par l’action des caspases effectrices. Il est important de noter que la déplétion des microtubules est suffisante pour passer outre le besoin de caspases pour l'extrusion cellulaire. De plus, la stabilisation des microtubules nuit fortement à l'extrusion cellulaire. Ces résultats démontrent pour la première fois que le désassemblage des microtubules par les caspases est une importante étape limitante de l’extrusion. En outre, elle met en évidence une fonction plus générale des microtubules dans la stabilisation de la forme des cellules épithéliales. En outre, comme les caspases clivent une grande quantité de substrat, l'activation des caspases a longtemps été considérée comme un point de non-retour conduisant irréversiblement à l'apoptose. Cependant, des études récentes ont montré des fonctions non apoptotiques des caspases. Ces fonctions sont vastes et impliquées dans la différenciation cellulaire, la prolifération, le maintien de la pluripotence et bien d'autres. De même, plusieurs travaux montrent des événements d'activation transitoire des caspases. Par conséquent, ce qui établit la balance entre les niveaux de caspase non létaux et l'engagement dans l'apoptose reste peu clair. Il a été proposé que les cellules survivent jusqu'à un seuil d'activation des caspases, mais cela n'a jamais été testé. Si un tel seuil existe, la manière dont sa valeur est fixée et est modulée n'est pas claire. Pour quantifier l’engagement des cellules dans l’extrusion, nous avons utilisé un rapporteur live de caspase et systématiquement segmenté et suivi les cellules du notum. Par régression logistique, nous avons montré qu'il n'y a pas de seuil commun d'activité des caspases effectrices menant à l'extrusion. Au contraire, les niveaux de caspases sont corrélés linéairement avec la probabilité d’engagement dans l'extrusion. De plus, par statistique bayésienne, nous avons pu identifier des paramètres prédictifs de la sensibilité des cellules aux caspases. Par exemple l'activation antérieure des caspases semble être associée à l'engagement dans l'extrusion à un niveau de caspase inférieur. Ceci est la première analyse quantitative du processus d'engagement dans l'extrusion ou l'apoptose in vivo et au niveau de la cellule unique.

During this PhD, I studied different phenomena pertaining to the mechanics of cells and tissues. The first project describes lamellipodial initiation during cell spreading. The model introduces a coupling between membrane curvature and cortical actin orientation, that generates substrate tractions by friction. A full wetting transition exists when edge curvature, actin orientation and tractions enter into a positive feed-back loop. A bi-stable transition allows a polar nucleation. The second project is an experimental collaboration that studies the way Cancer-Associated Fibroblasts (CAFs) cells could reshape tumours. CAFs surrounding a cluster of Cancer Cells (CCs) assemble a contractile ring to end up on top of CCs, and sometimes build a 3D bud through a shear stress. We use an elasto-plastic model for CAFs, compared with CAF closure dynamics and tractions. We combine vertex and continuous mechanical models to describe the CC monolayer shape, until the appearance of rearrangements. Finally, we describe the mechanical stability of buds by combining elasticity with plastic rearrangements. The last project reports preliminary results on the mechanics of cell extrusion in an epithelial monolayer under compression. Using the same vertex model, we look at the mechanical properties able to drive the basal-to-lateral and lateral-to-apical transitions. Then, we search for a mechanical instability on a minimal 3-cell system, and identify conditions that favour extrusion., Durant ce doctorat, j’ai étudié différents phénomènes liés à la mécanique de cellules et tissus. Le premier projet décrit l’initiation lamellipodiale durant l’étalement cellulaire. Le modèle introduit un couplage entre la courbure membranaire et l’orientation d’actine corticale, qui génère des tractions sur le substrat par friction. Une transition de mouillage total existe quand la courbure du bord, l’orientation d’actine et les tractions entrent dans une boucle rétro-active positive. Une transition bi-stable autorise une nucléation polarisée. Le second projet est une collaboration expérimentale qui étudie comment des Fibroblastes-Associés au Cancer (FACs) pourraient remodeler des tumeurs. Des FACs entourant un amas de Cellules Cancéreuses (CCs) assemblent un anneau contractile pour finir au-dessus des CCs, et génèrent parfois un bourgeon 3D par contrainte de cisaillement. On utilise un modèle élasto-plastique pour les FACs, comparé à la dynamique de fermeture des FACs et les tractions. On combine des modèles mécaniques à «vertex» et continu pour décrire la forme de la monocouche de CCs, jusqu’à l’apparition des réarrangements. Finalement, on décrit la stabilité mécanique des bourgeons en combinant élasticité et réarrangements plastiques. Le dernier projet rapporte des résultats préliminaires sur la mécanique de l’extrusion cellulaire dans une monocouche épithéliale sous compression. Utilisant le même modèle de vertex, on regarde les propriétés mécaniques capables de générer les transitions basal-latéral et latéral-apical. Ensuite, on cherche une instabilité mécanique pour un système minimal à trois cellules, identifiant les conditions qui favorisent l’extrusion.

Significance This study shows that an oncogenic mammary epithelial cell surrounded by normal cells can extrude basally in vivo and invade surrounding tissues without formation of a primary tumor. Here, we show that overexpression of the key polarity protein atypical protein kinase C ι (aPKCi) is sufficient for triggering basally oriented epithelial cell extrusion and early cell invasion into the mammary gland stroma. Moreover, we highlight the importance of the difference between the mechanical properties of aPKCi-overexpressing cells and those of the normal surrounding cells associated with the decrease of vinculin at the cell junction, which triggers cell segregation, the first step toward promoting and controlling the direction of cell extrusion., Metastasis is the main cause of cancer-related deaths. How a single oncogenic cell evolves within highly organized epithelium is still unknown. Here, we found that the overexpression of the protein kinase atypical protein kinase C ι (aPKCi), an oncogene, triggers basally oriented epithelial cell extrusion in vivo as a potential mechanism for early breast tumor cell invasion. We found that cell segregation is the first step required for basal extrusion of luminal cells and identify aPKCi and vinculin as regulators of cell segregation. We propose that asymmetric vinculin levels at the junction between normal and aPKCi+ cells trigger an increase in tension at these cell junctions. Moreover, we show that aPKCi+ cells acquire promigratory features, including increased vinculin levels and vinculin dynamics at the cell–substratum contacts. Overall, this study shows that a balance between cell contractility and cell–cell adhesion is crucial for promoting basally oriented cell extrusion, a mechanism for early breast cancer cell invasion.