Biophysical Journal, 2020

Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes

Laurene Aoun, Alexander Farutin, Nicolas Garcia-Seyda, Paulin Nègre, Mohd Suhail Rizvi, Sham Tlili, Solene Song, Xuan Luo, Martine Biarnes-Pelicot, Rémi Galland, Jean-Baptiste Sibarita, Alphée Michelot, Claire Hivroz, Salima Rafai, Marie-Pierre Valignat, Chaouqi Misbah,Olivier Theodoly
Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fiber traction, whereas the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on two-dimensional and in three-dimensional solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. …
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BioRxiv, 2020

Lattice micropatterning of electron microscopy grids for improved cellular cryo-electron tomography throughput

Leeya Engel, Claudia G. Vasquez, Elizabeth A. Montabana, Belle M. Sow, Marcin P. Walkiewicz, William I. Weis, Alexander R. Dunn
Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environment. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we leverage photo-micropatterning of EM grids to optimally position endothelial cells to enable high-throughput imaging of cell-cell contacts. …
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Journal of Cell Science, 2020

Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins

Xuan Luo, Valentine Seveau de Noray, Laurene Aoun, Martine Biarnes-Pelicot, Pierre-Olivier Strale, Vincent Studer, Marie-Pierre Valignat, Olivier Theodoly
Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules.  …
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Advanced Healthcare Matherials, 2020

Tailoring Common Hydrogels into 3D Cell Culture Templates

Aurélien Pasturel, Pierre‐Olivier Strale, Vincent Studer
Physiologically relevant cell‐based models require engineered microenvironments which recapitulate the topographical, biochemical, and mechanical properties encountered in vivo. In this context, hydrogels are the materials of choice. Here a light‐based toolbox is able to craft such microniches out of common place materials. Extensive use of benzophenone photoinitiators and their interaction with oxygen achieves this. …
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Science Advances, 2020

Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells

S E Stasiak, R R Jamieson, J Bouffard, E J Cram and H Parameswaran
In this study, we report the existence of a communication system among human smooth muscle cells that uses mechanical forces to frequency modulate long-range calcium waves. An important consequence of this mechanical signaling is that changes in stiffness of the underlying extracellular matrix can interfere with the frequency modulation of Ca2+ waves, causing smooth muscle cells from healthy human donors to falsely perceive a much higher agonist dose than they actually received. …
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BioRxiv, 2020

Integer topological defects organize stresses driving tissue morphogenesis

Pau Guillamat, Carles Blanch-Mercader, Karsten Kruse, Aurélien Roux
Tissues acquire their function and shape via differentiation and morphogenesis. Both processes are driven by coordinating cellular forces and shapes at the tissue scale, but general principles governing this interplay remain to be discovered. Here, we report that self-organization of myoblasts around integer topological defects, namely spirals and asters, triggers localized differentiation and, when differentiation is inhibited, drives the growth of cylindrical multicellular protrusions. …
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Biology of the Cell, 2020

Mechanobiology of antigen‐induced T cell arrest

Mélanie Chabaud, Noémie Paillon, Katharina Gaus, Claire Hivroz
To mount an immune response, T cells must first find rare antigens present at the surface of antigen‐presenting cells (APCs). They achieve this by migrating rapidly through the crowded space of tissues and constantly sampling the surface of APCs. Upon antigen recognition, T cells decelerate and polarise towards the APC, ultimately forming a specialised interface known as the immunological synapse. These conjugates form as the result of the interaction between pairs of receptors/ligands that are under mechanical stress due to the continuously reorganising cell cytoskeleton. In this review, …
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ACS Nano, 2020

Coupling Polar Adhesion with Traction, Spring and Torque Forces Allows High Speed Helical Migration of the Protozoan Parasite Toxoplasma

Georgios Pavlou , Bastien Touquet, Luis Vigetti, Patricia Renesto, Alexandre Bougdour, Delphine Debarre, Martial Balland, and Isabelle Tardieux
Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the Plasmodium sporozoite and the Toxoplasma tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two stages and functionally …
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Lab On A Chip, 2020

Microfluidic dialysis using photo-patterned hydrogel membranes in PDMS chips

Hoang-Thanh Nguyen, Morgan Massino, Camille Keita and Jean-Baptiste Salmon
We report the fabrication of permeable membranes for microfluidic dialysis applications in poly(dimethylsiloxane) (PDMS) channels. A maskless UV projection device was used to photo-pattern long hydrogel membranes (mm–cm) with a spatial resolution of a few microns in PDMS chips integrating also micro-valves. We show in particular that multi-layer soft lithography allows one to deplete oxygen from the PDMS walls using a nitrogen gas flow and therefore makes possible in situ UV-induced polymerization of hydrogels. …
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BioRxiv, 2020

Pattern- based Contractility Screening (PaCS), a reference-free traction force microscopy methodology, reveals contractile differences in breast cancer cells

Ajinkya Ghagre, Ali Amini, Luv Kishore Srivastava, Pouria Tirgar, Adele Khavari, Newsha Koushki, Allen Ehrlicher
The sensing and generation of cellular forces are essential aspects of life. Traction Force Microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various constraints, such as time-consuming substrate fabrication techniques, the need to detach cells to measure null force images, followed by complex imaging and analysis, and the unavailability of cells for post-processing. …
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BioRxiv, 2020

Hematopoietic progenitors polarize in contact with bone marrow stromal cells by engaging CXCR4 receptors

Thomas Bessy, Benoit Souquet, Benoit Vianay, Alexandre Schaeffer, Thierry Jaffredo, Jerome Larghero, Laurent Blanchoin, Stephane Brunet, Lionel Faivre, Manuel Théry
Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow, where they regulate the permanent production and renewal of all blood-cell types. HSPC proliferation and differentiation is locally regulated by their interaction with cells forming specific microenvironments close to the bone matrix or close to blood vessels. However, the cellular mechanisms underlying HSPC’s interaction with these cells and their potential impact on HSPC polarity is still poorly understood. Here we modelled the bone-marrow niche using microfluidic technologies in a bone-marrow on a chip device, …
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BioRxiv, 2020

Mechanochemical control of epidermal stem cell divisions by B-plexins

Chen Jiang, Ahsan Javed, Laura Kaiser, Michele M. Nava, Dandan Zhao, Dominique T. Brandt, Javier Fernández-Baldovinos, Luping Zhou, Carsten Höß, Kovilen Sawmynaden, Arkadiusz Oleksy, David Matthews, Lee S. Weinstein, Hermann-Josef Gröne, Carien M. Niessen, Stefan Offermanns, Sara A. Wickström, Thomas Worzfeld
The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. …
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Nature Materials, 2020

Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes

Yue Zhang, Richard De Mets, Cornelia Monzel, Vidhyalakshmi Acharya, Pearlyn Toh, Jasmine Fei Li Chin, Noémi Van Hul, Inn Chuan Ng, Hanry Yu, Soon Seng Ng, S. Tamir Rashid & Virgile Viasnoff
The symmetry breaking of protein distribution and cytoskeleton organization is an essential aspect for the development of apicobasal polarity. In embryonic cells this process is largely cell autonomous, while differentiated epithelial cells collectively polarize during epithelium formation. Here, we demonstrate that the de novo polarization of mature hepatocytes does not require the synchronized development of apical poles on neighbouring cells. De novo polarization at the single-cell level by mere contact with the extracellular matrix and immobilized cadherin defining a polarizing axis. …
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CELL, 2020

Defining the Design Principles of Skin Epidermis Postnatal Growth

Sophie Dekoninck, Edouard Hannezo, Alejandro Sifrim, Yekaterina A. Miroshnikova, Mariaceleste Aragona, Milan Malfait, Souhir Gargouri, Charlotte de Neunheuser, Christine Dubois, Thierry Voet, Sara A. Wickström, Benjamin D. Simons and Cédric Blanpain

During embryonic and postnatal development, or- gans and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single popula- tion of developmental progenitors presenting…

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Programmed transport and release of nanoscale cargo by immune cells

Daniel Meyer, Saba Telele, Anna Zelená, Elsa Neubert, Robert Nißler, Florian Mann, Luise Erpenbeck, Sarah Köster, Sebastian Kruss
Transport and delivery of (nanoscale) materials are crucial for many applications in biomedicine. However, controlled uptake, transport and triggered release of such cargo remains challenging. In this study, we use human immune cells (neutrophilic granulocytes, neutrophils) and program them to perform these tasks in vitro. For this purpose, we let neutrophils phagocytose a nanoscale cargo. As an example, we used DNA-functionalized single-walled carbon nanotubes (SWCNT) that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients …
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Jove, 2019

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cristina Melero*, Aljona Kolmogorova*, Paul Atherton, Brian Derby, Adam Reid, Karin Jansen, Christoph Ballestrem
Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. …
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Extracellular matrix stiffness regulates human airway smooth muscle contraction by altering the cell-cell coupling

Samuel R. Polio, Suzanne E. Stasiak, Ryan R. Jamieson, Jenna L. Balestrini, Ramaswamy Krishnan & Harikrishnan Parameswaran
For an airway or a blood vessel to narrow, there must be a connected path that links the smooth muscle (SM) cells with each other, and transmits forces around the organ, causing it to constrict. Currently, we know very little about the mechanisms that regulate force transmission pathways in a multicellular SM ensemble. Here, we used extracellular matrix (ECM) micropatterning to study force transmission in a two-cell ensemble of SM cells. …
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Nature Methods, 2019

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

Mauricio Toro-Nahuelpan, Levgeniia Zagoriy, Fabrice Senger, Laurent Blanchoin, Manuel Thery & Julia Mahamid
Spatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. …
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NAT PHYS, 2019

Substrate area confinement is a key determinant of cell velocity in collective migration

Danahe Mohammed, Guillaume Charras, Eléonore Vercruysse, Marie Versaevel, Joséphine Lantoine, Laura Alaimo, Céline Bruyère, Marine Luciano, Karine Glinel, Geoffrey Delhaye, Olivier Théodoly & Sylvain Gabriele
Collective cell migration is fundamental throughout development, during wound healing and in many diseases. Although much effort has focused on cell–cell junctions, a role for physical confinement in collective cell migration remains unclear. Here, we used adhesive microstripes of varying widths to mimic the spatial confinement experienced by follower cells within epithelial tissues. Our results reveal that the substrate area confinement is sufficient to modulate the three-dimensional cellular morphology without the need for intercellular adhesive cues. …
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NAT MAT, 2019

Traction forces at the cytokinetic ring regulate cell division and polyploidy in the migrating zebrafish epicardium

Marina Uroz, Anna Garcia-Puig, Isil Tekeli, Alberto Elosegui-Artola, Juan F. Abenza, Ariadna Marín-Llauradó, Silvia Pujals, Vito Conte, Lorenzo Albertazzi, Pere Roca-Cusachs, Ángel Raya & Xavier Trepat
Epithelial repair and regeneration are driven by collective cell migration and division. Both cellular functions involve tightly controlled mechanical events, but how physical forces regulate cell division in migrating epithelia is largely unknown. Here we show that cells dividing in the migrating zebrafish epicardium exert large cell–extracellular matrix (ECM) forces during cytokinesis. These forces point towards the division axis and are exerted through focal adhesions that connect the cytokinetic ring to the underlying ECM. …
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J. Micromech. Microeng., 2019

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

Leeya Engel, Guido Gaietta, Liam P Dow, Mark F Swift, Gaspard Pardon, Niels Volkmann, William I Weis, Dorit Hanein, Beth L Pruitt
Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. …
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A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions

Nisha Bte Mohd Rafiq, Yukako Nishimura, Sergey V. Plotnikov, Visalatchi Thiagarajan, Zhen Zhang, Shidong Shi, Meenubharathi Natarajan, Virgile Viasnoff, Pakorn Kanchanawong, Gareth E. Jones & Alexander D. Bershadsky
The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. …
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Phil. Trans. R. Soc. B, 2019

Forces and constraints controlling podosome assembly and disassembly

Nisha Bte Mohd Rafiq, Gianluca Grenci, Cheng Kai Lim, Michael M Kozlov, Gareth E Jones, Virgile Viasnoff and Alexander D Bershadsky
Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation, and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin- IIA filament assembly. …
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Autonomous induction of hepatic polarity to construct single cell liver

Yue Zhang, Richard de Mets, Cornelia Monzel, Pearlyn Toh, Noemi Van Hul, Soon Seng Ng, S. Tamir Rashid, Virgile Viasnoff
Symmetry breaking of protein distribution and cytoskeleton organization is an essential aspect for development of apico-basal polarity. In embryonic cells this process is largely cell autonomous, while differentiated epithelial cells collectively polarize during epithelium formation. We report here that the de novo polarization of mature hepatocytes is a cell autonomous process. Single hepatocytes developed bona fide secretory hemi-apical lumens upon adhesion to finely tuned substrates bio-functionalized with cadherin and extra cellular matrix. …
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Photoactivatable Hsp47: A Tool to Regulate Collagen Secretion and Assembly

Essak S. Khan, Shrikrishnan Sankaran, Julieta I. Paez, Christina Muth, Mitchell K. L. Han, Aránzazu del Campo
Collagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen‐specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. …
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PLOS One, 2018

Live nanoscopic to mesoscopic topography reconstruction with an optical microscope for chemical and biological samples

Olivier Theodoly, Nicolas Garcia-Seyda, Fréderic Bedu, Xuan Luo, Sylvain Gabriele, Tâm Mignot, Joanna Giermanska, Jean-Paul Chapel, Mélinda Métivier, Marie-Pierre Valignat
Macroscopic properties of physical and biological processes like friction, wetting, and adhesion or cell migration are controlled by interfacial properties at the nanoscopic scale. In an attempt to bridge simultaneously investigations at different scales, we demonstrate here how optical microscopy in Wet-Surface Ellipsometric Enhanced Contrast (Wet-SEEC) mode offers imaging and measurement of thin films at solid/liquid interfaces in the range 1–500 nm with lateral optical resolution. …
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Nano Lett, 2018

Optical magnetometry of single biocompatible micromagnets for quantitative magnetogenetic and magnetomechanical assays

Loïc Toraille, Koceila Aïzel, Elie Balloul, Chiara Vicario, Cornelia Monzel, Mathieu Coppey, Emilie Secret, Jean-Michel Siaugue, Joao Sampaio, Stanislas Rohart, Nicolas Vernier, Louise Bonnemay, Thierry Debuisschert, Loïc Rondin, Jean-Francois ROCH, and Maxime Dahan
The mechanical manipulation of magnetic nanoparticles is a powerful approach to probe and actuate biological processes in living systems. Implementing this technique in high-throughput assays can be achieved using biocompatible micomagnet arrays. Yet, the magnetic properties of these arrays are usually indirectly inferred from simulations or Stokes drag measurements, leaving unresolved questions about the actual profile of the magnetic fields at the micrometer scale and the exact magnetic forces that are applied. …
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Adv Biosys, 2018

A new approach to design artificial 3D micro-niches with combined chemical, topographical and rheological cues

Celine Stoecklin, Zhang Yue, Wilhelm W. Chen, Richard de Mets, Eileen Fong, Vincent Studer, Virgile Viasnoff
The in vitro methods to recapitulate environmental cues around cells are usually optimized to test a specific property of the environment (biochemical nature or the stiffness of the extra cellular matrix (ECM), or nanotopography) for its capability to induce defined cell behaviors (lineage commitment, migration). Approaches that combine different environmental cues in 3D to assess the biological response of cells to the spatial organization of different biophysical and biochemical cues are growingly being developed. …
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Lab Chip, 2018

In situ photopatterning of pressure-resistant hydrogel membranes with controlled permeabilities in PEGDA microfluidic channels.

Jérémy Decock, Mathias Schlenk and Jean-Baptiste Salmon
We report the fabrication of highly permeable membranes in poly(ethylene glycol) diacrylate (PEGDA) channels, for investigating ultra- or micro-filtration, at the microfluidic scale. More precisely, we used a maskless UV projection setup to photopattern PEG-based hydrogel membranes on a large scale (mm–cm), and with a spatial resolution of a few microns. We show that these membranes can withstand trans-membrane pressure drops of up to 7 bar without any leakage, thanks to the strong anchoring of the hydrogel to the channel walls. …
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BioRxiv, 2018

Collagen assembly and turnover imaged with a CRISPR-Cas9 engineered Dendra2 tag

Adam Pickard, Antony Adamson, Yinhui Lu, Joan Chang, Richa Garva, Nigel Hodson, Karl Kadler
Electron microscopy has been the gold standard for studying collagen networks but dynamic information on how cells synthesise the networks has been lacking. Live imaging methods have been unable to distinguish newly-synthesised fibrils from pre-existing fibrils and intracellular collagen. Here, we tagged endogenous collagen-I using CRISPR-Cas9 with photoswitchable Dendra2 and demonstrate live cells synthesising, migrating on, and interacting with, collagen fibrils. …
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Hum Mol Genet. 2016

Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes.

Delépine C, Meziane H, Nectoux J, Opitz M, Smith AB, Ballatore C, Saillour Y, Bennaceur-Griscelli A, Chang Q, Williams EC, Dahan M, Duboin A, Billuart P, Herault Y, Bienvenu T.
Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder, characterized by normal post-natal development followed by a sudden deceleration in brain growth with progressive loss of acquired motor and language skills, stereotypic hand movements and severe cognitive impairment. Mutations in the methyl-CpG-binding protein 2 (MECP2) cause more than 95% of classic cases. …
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Adv Mater. 2016

Multiprotein Printing by Light-Induced Molecular Adsorption.

Strale PO, Azioune A, Bugnicourt G, Lecomte Y, Chahid M, Studer V.
Light-induced molecular adsorption of proteins (LIMAP) allows for quantitative sub-micrometer-resolution printing of multiple biomolecules. Surface-bound gradients are patterned within minutes over an entire glass cover-slip. LIMAP is used to perform selective immuno-assays, to dynamically control the adhesion of individual cells, and to achieve hierarchical co-cultures instrumental for tissue engineering.
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