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Two-step local functionalization of fluoropolymer Dyneon THV microfluidic materials by scanning electrochemical microscopy combined to click reaction
Cyrine Slim, Eva Ratajovà, Sophie Griveau, Frédéric Kanoufi, David Ferraro, Camille Perréard, Fanny d’Orlyé, Anne Varenne and Fethi Bedioui
Electrochemistry Communications - 60 (5–8) - doi:10.1016/j.elecom.2015.07.019 - 2015
We propose an original two-step strategy combining the use of scanning electrochemical microscopy (SECM) and molecular chemistry via a “click” reaction (copper (I)-catalyzed azide alkyne cycloaddition (CuAAC)) to locally functionalize Dyneon THV surfaces, an attractive fluoropolymer for microfluidic applications. The first step consists in the local reduction of THV using a SECM tip to activate the surface by the creation of a locally carbonized zone and notably the formation of surface alkyne functions. This is then followed by a direct CuAAC reaction with an azide-bearing ligand for its local immobilization. The proof of concept is demonstrated by efficient local functionalization of the substrate with a fluorescent dye stable up to 6 months. Surface modifications were characterized by IR-ATR, XPS, and fluorescence microscopy.
Dynamics of CRISPR-Cas9 genome interrogation in living cells
Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, Zhang ET, El Beheiry M, Masson J-B, Dahan M, Liu Z, Doudna JA, Tjian R
Science - 350(6262) 823-6 - DOI: 10.1126/science.aac6572 - 2015
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.
Droplets in Microchannels: Dynamical Properties of the Lubrication Film
Axel Huerre, Olivier Theodoly, Alexander M. Leshansky, Marie-Pierre Valignat, Isabelle Cantat and Marie-Caroline Jullien
Phys. Rev. Lett. - 115 (064501) 064501 - http://dx.doi.org/10.1103/PhysRevLett.115.064501 - 2015
We study the motion of droplets in a confined, micrometric geometry, by focusing on the lubrication film between a droplet and a wall. When capillary forces dominate, the lubrication film thickness evolves nonlinearly with the capillary number due to the viscous dissipation between the meniscus and the wall. However, this film may become thin enough (tens of nanometers) that intermolecular forces come into play and affect classical scalings. Our experiments yield highly resolved topographies of the shape of the interface and allow us to bring new insights into droplet dynamics in microfluidics. We report the novel characterization of two dynamical regimes as the capillary number increases: (i) at low capillary numbers, the film thickness is constant and set by the disjoining pressure, while (ii) above a critical capillary number, the interface behavior is well described by a viscous scenario. At a high surfactant concentration, structural effects lead to the formation of patterns on the interface, which can be used to trace the interface velocity, that yield direct confirmation of the boundary condition in the viscous regime.
Optical volume and mass measurements show that mammalian cells swell during mitosis.
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 - 2015
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.
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 - 2015
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.
A computational mechanics approach to assess the link between cell morphology and forces during confined migration.
Aubry D, Thiam H, Piel M, Allena R
Biomech Model Mechanobiol - 14(1): 143-57 - DOI: 10.1016/bs.mcb.2014.11.007 - 2015
Confined migration plays a fundamental role during several biological phenomena such as embryogenesis, immunity and tumorogenesis. Here, we propose a two-dimensional mechanical model to simulate the migration of a HeLa cell through a micro-channel. As in our previous works, the cell is modelled as a continuum and a standard Maxwell model is used to describe the mechanical behaviour of both the cytoplasm (including active strains) and the nucleus. The cell cyclically protrudes and contracts and develops viscous forces to adhere to the substrate. The micro-channel is represented by two rigid walls, and it exerts an additional viscous force on the cell boundaries. We test four channels whose dimensions in terms of width are i) larger than the cell diameter, ii) sub-cellular, ii) sub-nuclear and iv) much smaller than the nucleus diameter. The main objective of the work is to assess the necessary conditions for the cell to enter into the channel and migrate through it. Therefore, we evaluate both the evolution of the cell morphology and the cell-channel and cell-substrate surface forces, and we show that there exists a link between the two, which is the essential parameter determining whether the cell is permeative, invasive or penetrating.
Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells.
Liu Y-J, Le Berre M, Lautenschlaeger F, Maiuri P, Callan-Jones A, Heuzé M, Takaki T, Voituriez R, Piel M
Cell - 160( 4): 659-72 - DOI: 10.1016/j.cell.2015.01.007 - 2015
The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration-one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.
Laser induced wounding of the plasma membrane and methods to study the repair process.
Jimenez AJ, Maiuri P, Lafaurie-Janvore J, Perez F, Piel M
Methods Cell Biol. - 5.2083333333 391-408 - DOI: 10.1016/bs.mcb.2014.11.007 - 2015
Cells are constantly exposed to agents that can trigger the perforation of their plasma membrane. This damage occurs naturally, and the frequency and intensity depends on how much cells are exposed to damaging threats. The following protocol is a simple and powerful method to damage the plasma membrane using laser ablation. It allows the induction of a single and localized wound at the plasma membrane of cultured cells, which can be followed with fast time-lapse imaging. The first part of the protocol describes simple cell culture techniques and the material ideal to make the experiments. A second part of the protocol gives advice about the procedures to make effective wounds in cells while ensuring a good survival rate. We also propose different ways to follow the opening and closure of the plasma membrane. Finally, we describe the procedure to efficiently analyze the data acquired after single cell photodamage to characterize the wounding process.
Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells.
Chabaud M, Heuzé ML, Bretou M, Vargas P, Maiuri P, Solanes P, Maurin M, Terriac E, Le Berre M, Lankar D, Piolot T, Adelstein RS, Zhang Y, Sixt M, Jacobelli J, Bénichou O, Voituriez R, Piel M, Lennon-Duménil A-M
Nat Commun - 0.25 7526 - DOI: 110.1016/j.jim.2015.12.005 - 2015
The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.
How to control proteins with light in living systems
Gautier A., Gauron C., Volovitch M., Bensimon D., Jullien L. and Vriz S.
Nature Chemical Biology - 10 (2014) 33–541 - DOI::10.1038/nchembio.1534 - 2014
The possibility offered by photocontrolling the activity of biomolecules in vivo while recording physiological parameters is opening up new opportunities for the study of physiological processes at the single-cell level in a living organism. For the last decade, such tools have been mainly used in neuroscience, and their application in freely moving animals has revolutionized this field. New photochemical approaches enable the control of various cellular processes by manipulating a wide range of protein functions in a noninvasive way and with unprecedented spatiotemporal resolution. We are at a pivotal moment where biologists can adapt these cutting-edge technologies to their system of study. This user-oriented review presents the state of the art and highlights technical issues to be resolved in the near future for wide and easy use of these powerful approaches.

326 publications.