Université PSL

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RECHERCHER

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Large scale control and programming of gene expression using CRISPR.
Laboratoire Biochimie - Deyell M, Ameta S, Nghe P
Semin Cell Dev Biol. - S1084-9521(18 30110-1 - doi: 10.1016/j.semcdb.2019.05.013 - 2019
The control of gene expression in cells and organisms allows to unveil gene to function relationships and to reprogram biological responses. Several systems, such as Zinc fingers, TALE (Transcription activator-like effectors), and siRNAs (small-interfering RNAs), have been exploited to achieve this. However, recent advances in Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 (CRISPR-Cas9) have overshadowed them due to high specificity, compatibility with many different organisms, and design flexibility. In this review we summarize state-of-the art for CRISPR-Cas9 technology for large scale gene perturbation studies, including single gene and multiple genes knock-out, knock-down, knock-up libraries, and their associated screening assays. We feature in particular the combination of these methods with single-cell transcriptomics approaches. Finally, we highlight the application of CRISPR-Cas9 systems in building synthetic circuits that can be interfaced with gene networks to control cellular states.
Polarization of Myosin II Refines Tissue Material Properties to Buffer Mechanical Stress
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Maria Duda, Natalie J Kirkland, Nargess Khalilgharibi, Melda Tozluoglu, Alice C Yuen, Nicolas Carpi, Anna Bove, Matthieu Piel, Guillaume Charras, Buzz Baum, Yanlan Mao
Dev Cell - 245-260.e7 - DOI : S1534-5807(18)31088-8 - 2019
As tissues develop, they are subjected to a variety of mechanical forces. Some of these forces are instrumental in the development of tissues, while others can result in tissue damage. Despite our extensive understanding of force-guided morphogenesis, we have only a limited understanding of how tissues prevent further morphogenesis once the shape is determined after development. Here, through the development of a tissue-stretching device, we uncover a mechanosensitive pathway that regulates tissue responses to mechanical stress through the polarization of actomyosin across the tissue. We show that stretch induces the formation of linear multicellular actomyosin cables, which depend on Diaphanous for their nucleation. These stiffen the epithelium, limiting further changes in shape, and prevent fractures from propagating across the tissue. Overall, this mechanism of force-induced changes in tissue mechanical properties provides a general model of force buffering that serves to preserve the shape of tissues under conditions of mechanical stress
Reconstitution of cell migration at a glance
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Juan Manuel Garcia-Arcos, Renaud Chabrier, Mathieu Deygas, Guilherme Nader, Lucie Barbier, Pablo José Sáez, Aastha Mathur, Pablo Vargas, Matthieu Piel
Frontiers in immunology - 188.e5 171 - DOI : jcs225565 - 2019
Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.
Macropinocytosis Overcomes Directional Bias in Dendritic Cells Due to Hydraulic Resistance and Facilitates Space Exploration
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Hélène D Moreau, Carles Blanch-Mercader, Rafaele Attia, Mathieu Maurin, Zahraa Alraies, Doriane Sanséau, Odile Malbec, Maria-Graciela Delgado, Philippe Bousso, Jean-François Joanny, Raphaël Voituriez, Matthieu Piel, Ana-Maria Lennon-Duménil
Frontiers in immunology - 188.e5 171 - S1534-5807(19)30235-7 - 2019
The migration of immune cells can be guided by physical cues imposed by the environment, such as geometry, rigidity, or hydraulic resistance (HR). Neutrophils preferentially follow paths of least HR in vitro, a phenomenon known as barotaxis. The mechanisms and physiological relevance of barotaxis remain unclear. We show that barotaxis results from the amplification of a small force imbalance by the actomyosin cytoskeleton, resulting in biased directional choices. In immature dendritic cells (DCs), actomyosin is recruited to the cell front to build macropinosomes. These cells are therefore insensitive to HR, as macropinocytosis allows fluid transport across these cells. This may enhance their space exploration capacity in vivo. Conversely, mature DCs down-regulate macropinocytosis and are thus barotactic. Modeling suggests that HR may help guide these cells to lymph nodes where they initiate immune responses. Hence, DCs can either overcome or capitalize on the physical obstacles they encounter, helping their immune-surveillance function
Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Vargas P, Maiuri P, Bretou M, Sáez PJ, Pierobon P, Maurin M, Chabaud M, Lankar D, Obino D, Terriac E, Raab M, Thiam H-R, Brocker T, Kitchen-Goosen SM, Alberts AS, Sunareni P, Xia S, Li R, Voituriez R, Piel M, Lennon-Duménil A-M
Nat. Cell Biol. - 18(1): 43-53 - DOI: 10.1016/j.jim.2015.12.005 - 2019
Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.
Optical volume and mass measurements show that mammalian cells swell during mitosis.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Zlotek-Zlotkiewicz E, Monnier S, Cappello G, Le Berre M, Piel M
J. Cell Biol. - 211( 4): 765-74 - DOI: 10.1016/j.jim.2015.12.005 - 2019
The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells.
Actin Flows Mediate a Universal Coupling between Cell Speed and Cell Persistence.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Maiuri P, Rupprecht J-F, Wieser S, Ruprecht V, Bénichou O, Carpi N, Coppey M, De Beco S, Gov N, Heisenberg C-P, Lage Crespo C, Lautenschlaeger F, Le Berre M, Lennon-Dumenil A-M, Raab M, Thiam H-R, Piel M, Sixt M, Voituriez R
Cell - 161(2) 374-86 - DOI: 10.1016/j.cell.2015.01.056 - 2019
Cell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.
Deterministic patterns in cell motility
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Ido Lavi, Matthieu Piel, Ana-Maria Lennon-Duménil , Raphaël Voituriez and Nir S. Gov
Nature Physics - 12 1146–1152 - DOI: : 10.1038/NPHYS3836 - 2019
Cell migration paths are generally described as random walks, associated with both intrinsic and extrinsic noise. However, complex cell locomotion is not merely related to such fluctuations, but is often determined by the underlying machinery. Cell motility is driven mechanically by actin and myosin, two molecular components that generate contractile forces. Other cell functions make use of the same components and, therefore, will compete with the migratory apparatus. Here, we propose a physical model of such a competitive system, namely dendritic cells whose antigen capture function and migratory ability are coupled by myosin II. The model predicts that this coupling gives rise to a dynamic instability, whereby cells switch from persistent migration to unidirectional self-oscillation, through a Hopf bifurcation. Cells can then switch to periodic polarity reversals through a homoclinic bifurcation. These predicted dynamic regimes are characterized by robust features that we identify through in vitro trajectories of dendritic cells over long timescales and distances. We expect that competition for limited resources in other migrating cell types can lead to similar deterministic migration modes.
Polarization of Myosin II Refines Tissue Material Properties to Buffer Mechanical Stress.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Duda M, Kirkland NJ, Khalilgharibi N, Tozluoglu M, Yuen AC, Carpi N, Bove A, Piel M, Charras G, Baum B, Mao Y.
Dev Cell - 48(2) 245-260. - doi: 10.1016/j.devcel.2018.12.020. - 2019
As tissues develop, they are subjected to a variety of mechanical forces. Some of these forces are instrumental in the development of tissues, while others can result in tissue damage. Despite our extensive understanding of force-guided morphogenesis, we have only a limited understanding of how tissues prevent further morphogenesis once the shape is determined after development. Here, through the development of a tissue-stretching device, we uncover a mechanosensitive pathway that regulates tissue responses to mechanical stress through the polarization of actomyosin across the tissue. We show that stretch induces the formation of linear multicellular actomyosin cables, which depend on Diaphanous for their nucleation. These stiffen the epithelium, limiting further changes in shape, and prevent fractures from propagating across the tissue. Overall, this mechanism of force-induced changes in tissue mechanical properties provides a general model of force buffering that serves to preserve the shape of tissues under conditions of mechanical stress.
Myosin II Activity Is Selectively Needed for Migration in Highly Confined Microenvironments in Mature Dendritic Cells.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Lucie Barbier, Pablo J Sáez, Rafaele Attia, Ana-Maria Lennon-Duménil, Ido Lavi, Matthieu Piel, Pablo Vargas
Frontiers in immunology - 747 - DOI : 10.3389/fimmu.2019.00747 - 2019
Upon infection, mature dendritic cells (mDCs) migrate from peripheral tissue to lymph nodes (LNs) to activate T lymphocytes and initiate the adaptive immune response. This fast and tightly regulated process is tuned by different microenvironmental factors, such as the physical properties of the tissue. Mechanistically, mDCs migration mostly relies on acto-myosin flow and contractility that depend on non-muscular Myosin IIA (MyoII) activity. However, the specific contribution of this molecular motor for mDCs navigation in complex microenvironments has yet to be fully established. Here, we identified a specific role of MyoII activity in the regulation of mDCs migration in highly confined microenvironments. Using microfluidic systems, we observed that during mDCs chemotaxis in 3D collagen gels under defined CCL21 gradients, MyoII activity was required to sustain their fast speed but not to orientate them toward the chemokine. Indeed, despite the fact that mDCs speed declined, these cells still migrated through the 3D gels, indicating that this molecular motor has a discrete function during their motility in this irregular microenvironment. Consistently, using microchannels of different sizes, we found that MyoII activity was essential to maintain fast cell speed specifically under strong confinement. Analysis of cell motility through micrometric holes further demonstrated that cell contractility facilitated mDCs passage only over very small gaps. Altogether, this work highlights that high contractility acts as an adaptation mechanism exhibited by mDCs to optimize their motility in restricted landscapes. Hence, MyoII activity ultimately facilitates their navigation in highly confined areas of structurally irregular tissues, contributing to the fine-tuning of their homing to LNs to initiate adaptive immune responses.

391 publications.