Contrôle Optogénétique de la Polarité Cellulaire

In this thesis we focus on the mechanisms that establish cell polarization, particularly during cell migration. Despite latest developments that enable visualization of RhoGTPases activity, the underlying principles dictating the cell’s ability to coordinates multiple signaling modules is still unclear. Optogenetic methods have been recognized as promising tools to dissect these intracellular signaling networks by allowing perturbations to be spatially and temporally controlled. We established the quantitative relationship between illumination patterns and the corresponding gradients of induced signaling activity through the characterization of the biophysical properties of CRY2/CIBN. We determined that it is possible to create subcellular gradients of recruited proteins of different shapes of choice up to spatial resolutions of 5μm and temporal ones of ca. 3 minutes.We applied the aforementioned optogenetic approach as a means to perturb the activity of cdc42, Rac1 and RhoA. We characterized the effects of subcellular activation of those RhoGTPases using membrane activity, cell shape changes and cell displacement as reporters of cell polarization and migration. We show that localized activation of RhoGTPases can trigger cellular organization and drive the cell into a migrating state.We also characterized the effects of local activation of RhoA on different cellular effectors as focal adhesion complexes, actin filaments and myosin molecular motors. We measured the dynamics of the newly formed focal adhesion complexes and the acto-myosin complex during retraction events.Altogether, our optogenetic methodology enables simultaneous measurement of the imposed perturbation and the cell response in a straightforward and reproducible way. It provides a quantitative way to control cell polarity and a step forward in the dissection of subcellular signaling networks.Dans cette thèse, nous avons concentré notre étude sur les mécanismes qui génèrent la polarité cellulaire, en particulier dans le cas de la migration cellulaire. Malgré les derniers développements concernant l’observation de l’activité des RhoGTPases, les principes qui dictent la capacité des cellules à coordonner plusieurs modules de signalisation en parallèle ne sont toujours pas compris. L’optogénétique est un outil d’intérêt pour disséquer ces réseaux de signalisation à partir de la création d’une perturbation dont les caractéristiques spatiotemporelles sont contrôlées. Tout d’abord, à partir de la caractérisation des différents processus biophysiques en jeu, nous avons établi les relations quantitatives entre l’illumination et les gradients moléculaires que l’on induit. Nous avons déterminé qu’il est possible de créer des gradients subcellulaires avec une résolution spatiale de l’ordre de 5 μm et temporelle d’environ 3 minutes Ensuite, nous avons utilisé cette approche optogénétique pour contrôler l’activité de Cdc42, Rac1 et RhoA. Nous avons caractérisé les effets subcellulaires de l’activation de ces RhoGTPases en utilisant l’activité de membrane, les changements de forme cellulaire et leurs déplacements comme rapporteurs de la polarisation et de la migration. Nous avons ainsi montré qu’une activation locale de RhoGTPase permet la réorganisation interne des cellules jusqu’à générer un phénotype de migration.Enfin, nous avons caractérisé les effets d’une activation locale de RhoA sur différents acteurs moléculaires comme les points focaux d’adhésion, l’actine et les moteurs moléculaires myosines. Nous avons mesuré alors la dynamique de l’intégration des points focaux dans le cytosquelette et analysé la réponse du réseau d’acto-myosine au cours d’évènements de rétraction.Notre approche optogénétique couple le contrôle d’une perturbation à la mesure de la réponse cellulaire simultanément de manière directe et reproductible. Elle apporte une méthode pour contrôler la polarité cellulaire et une manière de disséquer des réseaux de signalisation à l’échelle subcellulaire

Optogenetic control of cellular forces and mechanotransduction

Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space

Collective cell durotaxis emerges from long-range intercellular force transmission

The ability of cells to follow gradients of extracellular matrix stiffness—durotaxis—has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion.Peer ReviewedPostprint (author's final draft

The cytoplasm of living cells behaves as a poroelastic material

The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at physiologically relevant timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology

Collective cell durotaxis emerges from long-range intercellular force transmission

The ability of cells to follow gradients of extracellular matrix stiffness-durotaxis-has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion

Optogenetic Control of Cell Polarity

Dans cette thèse, nous avons concentré notre étude sur les mécanismes qui génèrent la polarité cellulaire, en particulier dans le cas de la migration cellulaire. Malgré les derniers développements concernant l’observation de l’activité des RhoGTPases, les principes qui dictent la capacité des cellules à coordonner plusieurs modules de signalisation en parallèle ne sont toujours pas compris. L’optogénétique est un outil d’intérêt pour disséquer ces réseaux de signalisation à partir de la création d’une perturbation dont les caractéristiques spatiotemporelles sont contrôlées. Tout d’abord, à partir de la caractérisation des différents processus biophysiques en jeu, nous avons établi les relations quantitatives entre l’illumination et les gradients moléculaires que l’on induit. Nous avons déterminé qu’il est possible de créer des gradients subcellulaires avec une résolution spatiale de l’ordre de 5 μm et temporelle d’environ 3 minutes Ensuite, nous avons utilisé cette approche optogénétique pour contrôler l’activité de Cdc42, Rac1 et RhoA. Nous avons caractérisé les effets subcellulaires de l’activation de ces RhoGTPases en utilisant l’activité de membrane, les changements de forme cellulaire et leurs déplacements comme rapporteurs de la polarisation et de la migration. Nous avons ainsi montré qu’une activation locale de RhoGTPase permet la réorganisation interne des cellules jusqu’à générer un phénotype de migration.Enfin, nous avons caractérisé les effets d’une activation locale de RhoA sur différents acteurs moléculaires comme les points focaux d’adhésion, l’actine et les moteurs moléculaires myosines. Nous avons mesuré alors la dynamique de l’intégration des points focaux dans le cytosquelette et analysé la réponse du réseau d’acto-myosine au cours d’évènements de rétraction.Notre approche optogénétique couple le contrôle d’une perturbation à la mesure de la réponse cellulaire simultanément de manière directe et reproductible. Elle apporte une méthode pour contrôler la polarité cellulaire et une manière de disséquer des réseaux de signalisation à l’échelle subcellulaire.In this thesis we focus on the mechanisms that establish cell polarization, particularly during cell migration. Despite latest developments that enable visualization of RhoGTPases activity, the underlying principles dictating the cell’s ability to coordinates multiple signaling modules is still unclear. Optogenetic methods have been recognized as promising tools to dissect these intracellular signaling networks by allowing perturbations to be spatially and temporally controlled. We established the quantitative relationship between illumination patterns and the corresponding gradients of induced signaling activity through the characterization of the biophysical properties of CRY2/CIBN. We determined that it is possible to create subcellular gradients of recruited proteins of different shapes of choice up to spatial resolutions of 5μm and temporal ones of ca. 3 minutes.We applied the aforementioned optogenetic approach as a means to perturb the activity of cdc42, Rac1 and RhoA. We characterized the effects of subcellular activation of those RhoGTPases using membrane activity, cell shape changes and cell displacement as reporters of cell polarization and migration. We show that localized activation of RhoGTPases can trigger cellular organization and drive the cell into a migrating state.We also characterized the effects of local activation of RhoA on different cellular effectors as focal adhesion complexes, actin filaments and myosin molecular motors. We measured the dynamics of the newly formed focal adhesion complexes and the acto-myosin complex during retraction events.Altogether, our optogenetic methodology enables simultaneous measurement of the imposed perturbation and the cell response in a straightforward and reproducible way. It provides a quantitative way to control cell polarity and a step forward in the dissection of subcellular signaling networks

Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction

International audienceCells and tissues are exposed to multiple mechanical stresses during development, tissue homeostasis and diseases. While we start to have an extensive understanding of the influence of mechanics on cell differentiation and proliferation, how excessive mechanical stresses can also lead to cell death and may be associated with pathologies has been much less explored so far. Recently, the development of new perturbative approaches allowing modulation of pressure and deformation of tissues has demonstrated that compaction (the reduction of tissue size or volume) can lead to cell elimination. Here we discuss the relevant type of stress and the parameters that could be causal to cell death from single cell to multicellular systems. We then compare the pathways and mechanisms that have been proposed to influence cell survival upon compaction. We eventually describe the relevance of compaction-induced death in vivo, and its functions in morphogenesis, size regulation, tissue homeostasis, and cancer progression

Competition for Space Induces Cell Elimination through Compaction-Driven ERK Downregulation

International audienceThe plasticity of developing tissues relies on the adjustment of cell survival and growth rate to environmental cues. This includes the effect of mechanical cues on cell survival. Accordingly, compaction of an epithelium can lead to cell extrusion and cell death. This process was proposed to contribute to tissue homeostasis but also to facilitate the expansion of pretumoral cells through the compaction and elimination of the neighbouring healthy cells. However we know very little about the pathways than can trigger apoptosis upon tissue deformation and the contribution of compaction driven death to clone expansion has never been assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, we show first that tissue compaction induces cell elimination through the downregulation of EGFR/ERK pathway and the upregulation of the pro-apoptotic protein Hid. Those results suggest that the sensitivity of EGFR/ERK pathway to mechanics could play a more general role in the fine tuning of cell elimination during morphogenesis and tissue homeostasis. Secondly, we assessed in vivo the contribution of compaction driven death to pretumoral cell expansion. We found that the activation of the oncogene Ras in clones can downregulate ERK and activate apoptosis in the neighbouring cells through their compaction, which eventually contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumour progression

DeXtrusion: automatic recognition of epithelial cell extrusion through machine learning in vivo

International audienceAccurately counting and localising cellular events from movies is an important bottleneck of high-content tissue/embryo live imaging. Here, we propose a new methodology based on deep learning that allows automatic detection of cellular events and their precise xyt localisation on live fluorescent imaging movies without segmentation. We focused on the detection of cell extrusion, the expulsion of dying cells from the epithelial layer, and devised DeXtrusion: a pipeline based on recurrent neural networks for automatic detection of cell extrusion/cell death events in large movies of epithelia marked with cell contour. The pipeline, initially trained on movies of the Drosophila pupal notum marked with fluorescent E-cadherin, is easily trainable, provides fast and accurate extrusion predictions in a large range of imaging conditions, and can also detect other cellular events, such as cell division or cell differentiation. It also performs well on other epithelial tissues with reasonable re-training. Our methodology could easily be applied for other cellular events detected by live fluorescent microscopy and could help to democratise the use of deep learning for automatic event detections in developing tissues

An RNAi Screen in a Novel Model of Oriented Divisions Identifies the Actin-Capping Protein Z β as an Essential Regulator of Spindle Orientation

International audienc