Biomaterials, 2023

Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies

Nicolas Rose, Berenice Estrada Chavez, Surabhi Sonam, Thao Nguyen, Gianluca Grenci, Anne Bigot, Antoine Muchir, Benoît Ladoux, Bruno Cadot, Fabien Le Grand, Léa Trichet
Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three-dimensional “Organ-on-chip” models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. …
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Biorxiv, 2022

Friction patterns guide actin network contraction

Alexandra Colin, Magali Orhant-Prioux, Christophe Guérin, Mariya Savinov, Ilaria Scarfone, Aurelien Roux, Enrique M. De La Cruz, Alex Mogilner, Manuel Théry and Laurent Blanchoin
The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile acto-myosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we micropatterned actin nucleation promoting factors on glass or on a lipid bilayer. …
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Nature Communications, 2022

Morphological control enables nanometer-scale dissection of cell-cell signaling complexes

Liam P. Dow, Guido Gaietta, Yair Kaufman, Mark F. Swift, Moara Lemos, Kerry Lane, Matthew Hopcroft, Armel Bezault, Cécile Sauvanet, Niels Volkmann, Beth L. Pruitt & Dorit Hanein
Protein micropatterning enables robust control of cell positioning on electron-microscopy substrates for cryogenic electron tomography (cryo-ET). However, the combination of regulated cell boundaries and the underlying electron-microscopy substrate (EM-grids) provides a poorly understood microenvironment for cell biology. Because substrate stiffness and morphology affect cellular behavior, we devised protocols to characterize the nanometer-scale details of the protein micropatterns on EM-grids by combining cryo-ET, atomic force microscopy, and scanning electron microscopy. …
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STAR Protocols, 2022

Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells

Linda E. Franken, Rene Rosch, Ulrike Laugks, Kay Grünewald
Here, we present a protocol for assessing virus-infected cells using electron cryo-tomography (cryoET). It includes the basic workflows of seeding cells, plunge-freezing, clipping, cryo-focused ion beam milling (cryoFIB-milling), and cryoET, as well as two optional modules: micropatterning and live-cell fluorescence microscopy. We use an A549 human cell line and the virus HAdV5-pIX-mcherry in this protocol, but the comprehensive workflow can be easily transferred to other cell types and different types of virus infection or treatment. …
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Nature Physics, 2022

Mechanical stress driven by rigidity sensing governs epithelial stability

Surabhi Sonam, Lakshmi Balasubramaniam, Shao-Zhen Lin, Ying Ming Yow Ivan, Irina Pi-Jaumà, Cecile Jebane, Marc Karnat, Yusuke Toyama, Philippe Marcq, Jacques Prost, René-Marc Mège, Jean-François Rupprecht & Benoît Ladoux
Epithelia act as barriers against environmental stresses. They are continuously exposed to various mechanical stress and abrasion, which impact epithelial integrity. The impact of the environment on epithelial integrity remains elusive. By culturing epithelial cells on two-dimensional hydrogels, we observe a loss of epithelial monolayer integrity on soft substrates through spontaneous hole formation. These monolayer ruptures are associated with local cellular stretching and cell-division events. (…) Our results thus show that substrate stiffness provides feedback on the mechanical state of epithelial monolayers with potential application towards a mechanistic understanding of compromised epithelial integrity during normal and pathological human ontogenesis. …
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Research Square, 2022

STIFMap employs a convolutional neural network to reveal spatial mechanical heterogeneity and tension-dependent activation of an epithelial to mesenchymal transition within human breast cancers

Valerie Weaver, Connor Stashko, Mary-Kate Hayward, Jason Northey, Neil Pearson, Alastair Ironside, Johnathon Lakins, Marie-Anne Goyette, Lakyn Mayo, Hege Russnes, E Hwang, Matthew Kutys, Kornelia Polyak
Intratumor heterogeneity in breast cancer associates with poor patient outcome. Tissue fibrosis and stromal stiffening accompany breast cancer development and associate with the aggressiveness of human breast cancer subtypes. Whether human breast cancers demonstrate stiffness heterogeneity, and if this is linked to breast tumor aggression remains unclear. To answer these questions, we developed a spatial method to measure the stiffness heterogeneity in human breast tumor tissues that also quantifies the local stromal stiffness each cell experiences and permits correlation with biomarkers of tumor aggression. Here, we present Spatially Transformed Inferential Force Maps (STIFMaps) to predict the elasticity across whole tissue sections with micron-resolution. …
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Cell Reports, 2022

A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1

Lisa Donker, Ronja Houtekamer, Marjolein J. Vliem, François Sipieter, Helena Canever, Manuel Gómez-González, Miquel Bosch-Padrós, Willem-Jan Pannekoek, Xavier Trepat, Nicolas Borghi, Martijn Gloerich
Epithelial cell divisions are coordinated with cell loss to preserve epithelial integrity. However, how epithelia adapt their rate of cell division to changes in cell number, for instance during homeostatic turnover or wounding, is not well understood. Here, we show that epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell-cycle progression. Micropatterning is used for monolayer stress microscopy, to map those intercellular forces. …
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Current Opinion in Structural Biology, 2022

Electron microscopy of cellular ultrastructure in three dimensions

Neta Varsano, Sharon Grayer Wolf
Electron microscopy in three dimensions (3D) of cells and tissues can be essential for understanding the ultrastructural aspects of biological processes. The quest for 3D information reveals challenges at many stages of the workflow, from sample preparation, to imaging, data analysis and segmentation. Here, we outline several available methods, including volume SEM imaging, cryo-TEM and cryo-STEM tomography, each one occupying a different domain in the basic tradeoff between field-of-view and resolution. We discuss the considerations for choosing a suitable method depending on research needs and highlight recent developments that are essential for making 3D volume imaging of cells and tissues a standard tool for cellular and structural biologists. …
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Thesis, 2022

Spatial control of angiogenesis by engineered patterns of Notch ligands

Laura Tiemeijer
In tissue engineering and regenerative medicine, proper vascularization of engineered tissues is imperative, as diffusion of nutrients, oxygen and waste is limited in constructs larger than a few cells thick. Therefore, tissue vascularization has been extensively researched. However, most approaches rely on preset structural support for the cells provided by scaffolds and microfluidic chips to instruct vascular organization, which limit the integration into engineered and native tissues. (…) In this thesis, we have developed an in vitro method where we used spatial patterns of parallel lines of Notch signaling ligands to locally modulate endothelial tip/stalk cell selection and thereby gained spatial control over endothelial sprouting. …
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BioRxiv, 2022

ARHGEF18 participates in Endothelial Cell Mechano-sensitivity in Response to Flow

Surya Prakash Rao Batta, Marc Rio, Corentin Lebot, Céline Baron-Menguy, Robin Le Ruz, Gervaise Loirand, Anne-Clémence Vion
Hemodynamic forces play an important role in vascular network development and homeostasis. In physiological conditions, shear stress generated by laminar flow promotes endothelial cells (EC) health and induces their alignment in the direction of flow. In contrast, altered hemodynamic forces induce endothelial dysfunction and lead to the development of vascular disorders such as atherosclerosis and aneurysms. Following mechano-sensor activation, Rho protein-mediated cytoskeletal rearrangement is one of the first steps in transforming flow-induced forces into intracellular signals in EC via guanine nucleotide exchange factors (RhoGEFs) that mediate the spatio-temporal activation of these Rho proteins. Here we identified ARHGEF18 as a flow-sensitive RhoGEF specifically activating RhoA. (…) Our study therefore characterized ARHGEF18 as the first flow-sensitive RhoA GEF in ECs, whose activity is essential for the maintenance of intercellular junctions and a properly organized endothelial monolayer under physiological flow conditions. …
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Cell Reports, 2022

Pressure and curvature control of the cell cycle in epithelia growing under spherical confinement

Ilaria Di Meglio, Anastasiya Trushko, Pau Guillamat, Carles Blanch-Mercader, Shada Abuhattum, Aurélien Roux
Morphogenesis requires spatiotemporal regulation of proliferation, both by biochemical and mechanical cues. In epithelia, this regulation is called contact inhibition of proliferation, but disentangling biochemical from mechanical cues remains challenging. Here, we show that epithelia growing under confinement accumulate pressure that inhibits proliferation above a threshold value. (…) Altogether, our results suggest that different mechanical cues resulting from pressure inhibition of proliferation are at play through different mechano-sensing pathways: the β-catenin pathway sustains cell division under high pressure, and the YAP pathway senses local curvature. …
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Nature Communications, 2022

Intrinsic cell rheology drives junction maturation

K. Sri-Ranjan, J. L. Sanchez-Alonso, P. Swiatlowska, S. Rothery, P. Novak , S. Gerlach, D. Koeninger, B. Hoffmann, R. Merkel, M. Stevens, S. X. Sun, J. Gorelik, Vania Braga
A fundamental property of higher eukaryotes that underpins their evolutionary success is stable cell-cell cohesion. Yet, how intrinsic cell rheology and stiffness contributes to junction stabilization and maturation is poorly understood. We demonstrate that localized modulation of cell rheology governs the transition of a slack, undulated cell-cell contact (weak adhesion) to a mature, straight junction (optimal adhesion). Cell pairs confined on different geometries have heterogeneous elasticity maps and control their own intrinsic rheology co-ordinately. (…) Our data inform on the minimal intrinsic rheology to generate a mature junction and provide a springboard towards understanding elements governing tissue-level mechanics. …
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Biorxiv, 2022

Engineering a Single Extracellular Vesicle Protein and RNA Assay (siEVPRA) via In Situ Fluorescence Microscopy in a UV Micropatterned Array

Jingjing Zhang, Xinyu Wang, Xilal Y. Rima, Luong T. H. Nguyen, Kristin Huntoon, Yifan Ma, Nicole Walters, Kwang Joo Kwak, Min Jin Yoon, Daeyong Lee, Yifan Wang, Jonghoon Ha, Kelsey Scherler, Shannon Fallen, Inyoul Lee, Andre F. Palmer, Wen Jiang, Kai Wang, Betty Y.S. Kim, L. James Lee, Eduardo Reátegui
The physical and molecular heterogeneity of extracellular vesicles (EVs) confounds bulk biomarker characterization, thus encouraging the development of novel assays capable of profiling EVs at a single-vesicle resolution. Here, we present a single EV (siEV) protein and RNA assay (siEVPRA) to simultaneously detect proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs) in siEVs. The siEVPRA consists of an array of microdomains embedded on a polyethylene glycol (PEG)-coated glass surface produced via UV photopatterning, functionalized with antibodies to target siEV subpopulations. Fluorescently labeled antibodies and RNA-targeting molecular beacons (MBs) were used to generate signals for proteins, mRNAs, and miRNAs on siEVs detected by total internal reflection fluorescence microscopy (TIRFM). (…) Together, these results indicate that the siEVPRA provides an effective platform to investigate the heterogeneity of proteins and RNAs in subpopulations of EVs. …
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Biorxiv, 2022

Plasma FIB milling for the determination of structures in situ

Casper Berger, Maud Dumoux, Thomas Glen, Neville B.-y. Yee, John M. Mitchels, Zuzana Patáková, James H Naismith, Michael Grange
Structural biology inside cells and tissues requires methods able to thin vitrified specimens to electron transparent thicknesses. Until now, focused ions beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit Gallium as an ion beam source. Here, we show that plasma-coupled ion sources can produce cryogenic lamella of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. In addition, these lamellae were produced in a prototype microscope equipped for long cryogenic run times (>1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate for the first time that plasma ion sources can be used for structural biology within cells, determining a structure in-situ to 4.9 Å and describing a workflow upon which different plasmas can be examined to streamline lamella fabrication further. …
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Biorxiv, 2022

Cryo-electron tomography reveals enrichment and identifies microtubule lumenal particles in neuronal differentiation

Saikat Chakraborty, Antonio Martinez-Sanchez, Florian Beck, Mauricio Toro-Nahuelpan, In-Young Hwang, Kyung-Min Noh, Wolfgang Baumeister, Julia Mahamid
Functional architecture of the neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which bind to the MT outer surface. Yet, electron microscopy (EM) has long revealed hitherto unknown electron-dense particles inside the lumens of neuronal MTs. Here, we use cryogenic electron tomography (cryo-ET) to analyze the native three-dimensional (3D) structures and organization of MT lumenal particles inside vitrified rodent primary neurons, pluripotent P19 cells and human induced pluripotent stem cell-derived neurons. We obtain 3D maps of several lumenal particles at molecular resolution that periodically decorate neuronal MTs. We show that increased lumenal particle localization is concomitant with neuronal differentiation and correlates with higher MT curvatures. Lumenal particles binding topology in MTs, their structural resemblance to tubulin binding cofactors (TBCs), enrichment around MT lattice defects and at plus-ends indicated their potential role in tubulin proteostasis for the maintenance of neuronal MTs. …
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Trends in Cell Biology, 2022

Mechanobiological approaches to synthetic morphogenesis: learning by building

Marija Matejčić, Xavier Trepat
Tissue morphogenesis occurs in a complex physicochemical microenvironment with limited experimental accessibility. This often prevents a clear identification of the processes that govern the formation of a given functional shape. By applying state-of-the-art methods to minimal tissue systems, synthetic morphogenesis aims to engineer the discrete events that are necessary and sufficient to build specific tissue shapes. Here, we review recent advances in synthetic morphogenesis, highlighting how a combination of microfabrication and mechanobiology is fostering our understanding of how tissues are built. …
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Current Biology, 2022

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization

Ana M. Pasapera, Sarah M. Heissler, Masumi Eto, Yukako Nishimura, Robert S. Fischer, Hawa R. Thiam, Clare M. Waterman
Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. (…) Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration. …
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Lab on a Chip, 2022

Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis

Xilal Y. Rima, Jingjing Zhang, Luong TH Nguyen, Aaron Rajasuriyar, Min Jin Yoon, Chi-Ling Chiang, Nicole Walters, Kwang Joo Kwak, L.James Lee, Eduardo Reátegui
Investigating cellular and vesicular heterogeneity in breast cancer remains a challenge, which encourages the development of controllable in vitro systems that mimic the tumor microenvironment. Although three-dimensional cell culture better recapitulates the heterogeneity observed in tumor growth and extracellular vesicle (EV) biogenesis, the physiological relevance is often contrasted with the control offered by two-dimensional cell culture. Therefore, to challenge this misconception we developed a novel microfluidic system harboring highly tunable three-dimensional EV microbioreactors (EVµBRs) to model micrometastatic EV release in breast cancer while capitalizing on the convenient, low-volume, and sterile interface provided by microfluidics. (…) To immobilize the EVµBRs within a microchannel and facilitate EV extraction, oxygen inhibition in free-radical polymerization was repurposed to rapidly generate two-layer hydrodynamic traps in situ using a digital-micromirror device (DMD)-based ultraviolet (UV) projection system. …
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Biorxiv, 2022

Mapping mechanical stress in curved epithelia of designed size and shape

Ariadna Marín-Llauradó, Sohan Kale, Adam Ouzeri, Raimon Sunyer, Alejandro Torres-Sánchez, Ernest Latorre, Manuel Gómez-González, Pere Roca-Cusachs, Marino Arroyo, Xavier Trepat
The function of organs such as lungs, kidneys and mammary glands relies on the three-dimensional geometry of their epithelium. To adopt shapes such as spheres, tubes and ellipsoids, epithelia generate mechanical stresses that are generally unknown. Here we engineered curved epithelial monolayers of controlled size and shape and mapped their state of stress. We designed pressurized epithelia with circular, rectangular and ellipsoidal footprints. (…) Besides interrogating the fundamental mechanics of epithelia over a broad range of sizes and shapes, our approach will enable a systematic study of how geometry and stress influence epithelial fate and function in three-dimensions. …
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Methods in Molecular Biology, 2022

Controlling Cell Shape and Microtubule Organization by Extracellular Matrix Micropatterning

Alessandro Dema, Shima Rahgozar, Laurent Siquier, Jeffrey van Haren, Torsten Wittmann
Micropatterning of extracellular matrix proteins enables defining cell position and shape in experiments investigating intracellular dynamics and organization. While such standardization is advantageous in automated and quantitative analysis of many cells, the original methods generating such patterns are cumbersome and inflexible. However, recent development of contact-less methods that allow photochemical generation of protein patterns robustly and rapidly is boosting the broader availability of micropatterning approaches. Here, we describe an optimized protocol to achieve large micropatterned areas with high fidelity using a commercially available microscope-mounted UV projection system. …
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Adv. Funct. Mater., 2022

Printing and Erasing of DNA-Based Photoresists Inside Synthetic Cells

Tobias Walther, Kevin Jahnke, Tobias Abele, Kerstin Göpfrich
In the pursuit of producing functioning synthetic cells from the bottom-up, DNA nanotechnology has proven to be a powerful tool. However, the crowded yet highly organized arrangement in living cells, bridging from the nano- to the micron-scale, remains challenging to recreate with DNA-based architectures. Here, laser microprinting is established to print and erase shape-controlled DNA hydrogels inside the confinement of water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). (…) Overall, DNA-based photoresists for laser printing in confinement allow to build up architectures on the interior of synthetic cells with light, which diversifies the toolbox of bottom-up synthetic biology. …
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Biorxiv, 2022

The laminin-keratin link shields the nucleus from mechanical deformation and signalling

Zanetta Kechagia, Pablo Sáez, Manuel Gómez-González, Martín Zamarbide, Ion Andreu, Thijs Koorman, Amy E.M. Beedle, Patrick W.B. Derksen, Xavier Trepat, Marino Arroyo, Pere Roca-Cusachs
The mechanical properties of the extracellular matrix (ECM) dictate tissue behaviour. In epithelial tissues, laminin is both a very abundant ECM component, and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111, unlike fibronectin or collagen I, impairs cell response to substrate rigidity, and YAP nuclear localization. …
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Biorxiv, 2022

Conserved basal lamina proteins, laminin and nidogen, are repurposed to organize mechanosensory complexes responsible for touch sensation

Alakananda Das, Joy Franco, Lingxin Wang, Dail Chapman, Lucy Wang, Chandni Jaisinghani, Beth Pruitt, Miriam Goodman
The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in all metazoans. Using Caenorhabditis elegans nematodes, we show that mechanosensory complexes essential for touch sensation reside at this interface and contain laminin, nidogen, and the MEC-4 mechano-electrical transduction channel proteins. …
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Nature Materials, 2022

Integer topological defects organize stresses driving tissue morphogenesis

Pau Guillamat, Carles Blanch-Mercader, Guillaume Pernollet, Karsten Kruse, Aurélien Roux
Tissues acquire 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, suffices to establish complex multicellular architectures. …
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biorxiv, 2022

Mechanical control of the mammalian circadian clock via YAP/TAZ and TEAD

Juan F. Abenza, Leone Rossetti, Malèke Mouelhi, Javier Burgués, Ion Andreu, Keith Kennedy, Pere Roca-Cusachs, Santiago Marco, Jordi García-Ojalvo, Xavier Trepat
Circadian rhythms are a key survival mechanism that dictates biological activity according to the day-night cycle. Although these peripheral clocks are remotely controlled by the master clock in the brain, they are also sensitive to their immediate physical microenvironment through mechanisms that are still unknown. Here we show that the circadian clock in fibroblasts is regulated mechanically through YAP/TAZ and TEAD. We use high-throughput analysis of single-cell circadian rhythms and apply controlled mechanical, biochemical, and genetic perturbations to study the expression of the core clock gene Rev-erbα. …
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Jove, 2022

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Joel Christian, Johannes W. Blumberg, Dimitri Probst, Cristina Lo Giudice, Sandra Sindt, Christine Selhuber-Unkel, Ulrich S. Schwarz, Elisabetta Ada Cavalcanti-Adam
For 2D-TFM (Traction Force Microscopy) on polyacrylamide, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography on PA hydrogels of different elasticity, and their displacement is tracked by embedded fluorescent beads. To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). …
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PNAS, 2022

ATP allosterically stabilizes integrin-linked kinase for efficient force generation

Isabel M. Martin, Michele M. Nava, Sara A. Wickström, Frauke Gräter
Focal adhesions link the actomyosin cytoskeleton to the extracellular matrix regulating cell adhesion, shape, and migration. Adhesions are dynamically assembled and disassembled in response to extrinsic and intrinsic forces, but how the essential adhesion component integrin-linked kinase (ILK) dynamically responds to mechanical force and what role adenosine triphosphate (ATP) bound to this pseudokinase plays remain elusive. Here, we apply force–probe molecular-dynamics simulations of human ILK:α-parvin coupled to traction force microscopy to explore ILK mechanotransducing functions. Our study proposes a role of ATP as an obligatory binding partner for structural and mechanical integrity of the pseudokinase ILK, ensuring efficient cellular force generation and migration. …
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ACS Appl. Mater. Interfaces, 2022

Facile and Versatile Method for Micropatterning Poly(acrylamide) Hydrogels Using Photocleavable Comonomers

Dimitris Missirlis, Miguel Baños, Felix Lussier, Joachim Spatz
We here present a micropatterning strategy to introduce small molecules and ligands on patterns of arbitrary shapes on the surface of poly(acrylamide)-based hydrogels. To achieve the above, a monomer containing a caged amine was co-polymerized in the hydrogel network; upon UV light illumination using a commercially available setup, primary amines were locally deprotected and served as reactive groups for further functionalization. Cell patterning on various cell adhesive ligands was demonstrated, with cells responding to a combination of pattern shape and substrate elasticity. The approach is compatible with standard traction force microscopy (TFM) experimentation and can further be extended to reference-free TFM. …
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biorxiv, 2022

Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

Jie E. Yang, Matthew R. Larson, Bryan S. Sibert, Joseph Y. Kim, Daniel Parrell, Juan C. Sanchez, Victoria Pappas, Anil Kumar, Kai Cai, Keith Thompson, Elizabeth Wright
Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis. …
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Micromachines, 2021

Rapid Prototyping of Organ-on-a-Chip Devices Using Maskless Photolithography

Dhanesh Kasi, Mees de Graaf, Paul Motreuil-Ragot, Jean-Phillipe Frimat, Michel Ferrari, Pasqualina Sarro, Massimo Mastrangeli, Arn M. J. M. van den Maagdenberg, Christine Mummery, Valeria Orlova
Organ-on-a-chip (OoC) and microfluidic devices are conventionally produced using microfabrication procedures that require cleanrooms, silicon wafers, photomasks, and multiple iterations of design steps. Here, we describe a rapid and cleanroom-free microfabrication method using a commercial digital micromirror device-based setup. Using this approach: digital photomasks can be designed, projected, and quickly
adjusted if needed; and SU-8 molds can be fabricated without cleanroom availability, which in turn reduces microfabrication time and costs and expedites prototyping of new OoC devices. …
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Biomaterials, 2021

Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography

Elsa Mazari-Arrighi, Dmitry Ayollo, Wissam Farhat, Auriane Marret, Emilie Gontran, Pascale Dupuis-Williams, Jerome Larghero, Francois Chatelain, Alexandra Fuchs
Cholangiocytes, biliary epithelial cells, are known to spontaneously self-organize into spherical cysts with a central lumen. In this work, we explore a promising biocompatible stereolithographic approach to encapsulate cholangiocytes into geometrically-controlled 3D hydrogel structures to guide them towards the formation of branched tubular networks. We demonstrate that within the appropriate mix of hydrogels, normal rat cholangiocytes can proliferate, migrate and organize into branched tubular structures, form walls consisting of a cell monolayer, transport fluorescent dyes into the luminal space and show markers of epithelial maturation such as primary cilia. …
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Journal of Cell Biology, 2021

Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1

Thomas Bessy, Adrian Candelas, Benoit Souquet, Khansa Saadallah, Alexandre Schaeffer, Benoit Vianay, Damien Cuvelier, Samy Gobaa, Cecilia Nakid-Cordero, Julien Lion, Jean-Christophe Bories, Nuala Mooney, Thierry Jaffredo, Jerome Larghero, Laurent Blanchoin, Lionel Faivre, Stephane Brunet, 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 modeled the bone-marrow niche using microfluidic technologies in a bone-marrow on a chip device. …
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Journal of structural biology, 2021

Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts

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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Jove, 2021

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Bryan S. Sibert, Joseph Y. Kim, Jie E. Yang, Elizabeth R. Wright
There are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB). …
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nano letters, 2021

Nanoscale Surface Topography Reduces Focal Adhesions and Cell Stiffness by Enhancing Integrin Endocytosis

Xiao Li, Lasse H. Klausen, Wei Zhang, Zeinab Jahed, Ching-Ting Tsai, Thomas L. Li, and Bianxiao Cui
Both substrate stiffness and surface topography regulate cell behavior through mechanotransduction signaling pathways. However, the mechanisms by which cells recognize topographical features are not fully understood. Here we demonstrate that the presence of nanotopography drastically alters cell behavior such that neurons and stem cells cultured on rigid glass substrates behave as if they were on soft hydrogels. We further show that rigid nanotopography resembles the effect of soft hydrogels in reducing cell stiffness and membrane tension as measured by atomic force microscopy. Finally, we demonstrate that nanotopography reduces focal adhesions and cell stiffness by enhancing the endocytosis and the subsequent removal of integrin receptors. …
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ACS Appl. Mater. Interfaces, 2021

Composite Elastomer-Enabled Rapid Photofabrication of Microfluidic Devices

Futianchun Zhu , Yu He, Zefan Lu, Hongliang Fan, Tao Zhang
Recently, photocurable resins, as a huge class of materials, have attracted extensive interest. However, very few of them can now be used in device fabrication due to the challenge in machining these materials. In response, we herein propose a novel concept of composite elastomers, which can covalently link with and consequently offer a flexible support to photocured thin films. This effect would allow most photocurable resins to be used in microfluidic device fabrication, greatly enriching the material choices for diverse applications. Moreover, the whole fabrication process becomes very simple and rapid, with an impressive throughput of at least hundreds of replicas per day. …
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Biology of the Cell, 2021

Direct measurement of near-nano-Newton forces developed by self-organizing actomyosin fibers bound α-catenin

Surabhi Sonam, Clémence Vigouroux, Antoine Jégou, Guillaume Romet-Lemonne, Christophe Le Clainche, Benoit Ladoux, René Marc Mège
Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell–cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. (…) A key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with micro force sensor arrays used so far to measure cell-generated forces. …
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biorxiv, 2021

Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

Bryan S Sibert, Joseph Y Kim, Jie E Yang, Elizabeth R Wright
Culture or adhere cells on TEM grids in a manner that is suitable for tomography while preserving the physiological state of the cells remains a challenge in whole-cell cryo-electron tomography (cryo-ET). Here, we demonstrate the versatility of micropatterning to direct and promote growth of both cultured and primary eukaryotic cells on TEM grids, by studying host-pathogen interactions using respiratory syncytial virus infected BEAS-2B cells as an example. We demonstrate the ability to use whole-cell tomography of primary Drosophila neuronal cells to identify organelles and cytoskeletal stuctures in cellular axons and the potential for micropatterning to dramatically increase throughput for these studies. …
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Methods in Molecular Biology, 2021

Manufacturing a Bone Marrow-On-A-Chip Using Maskless Photolithography

Benoit Souquet, Matthieu Opitz, Benoit Vianay, Stéphane Brunet, Manuel Théry
The bone marrow (BM) is a complex microenvironment in which hematopoietic stem and progenitor cells (HSPCs) interact with multiple cell types that regulate their quiescence, growth, and differentiation. These cells constitute local niches where HSPCs are confined and subjected to specific set of physical and biochemical cues. (…) Here, we present a method to manufacture a pseudo BM-on-a-chip with separated compartments mimicking the vascular and the endosteal niches. Such a configuration with connected but distant compartments allowed the investigation of the specific contribution of each niche to the regulation of HSPC behavior in vitro. …
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PNAS, 2021

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates

Robert Fischer, Xiaoyu Sun, Michelle Baird, Matt Hourwitz, Bo Ri Seo, Ana Pasapera, Shalin Mehta, Wolfgang Losert, Claudia Fischbach, John Fourkas, and Clare Waterman
Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. …
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ACS Applied Materials & Interfaces, 2021

Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments

Cas van der Putten, Antonetta Buskermolen, Maike Werner, Hannah Brouwer, Paul Bartels, Patricia Dankers, Carlijn Bouten, and Nicholas Kurniawan
Here, we present a new approach to investigate cellular responses in multi-cue environments, by combining optics-based protein patterning and lithography-based substrate microfabrication. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). …
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ACS Applied Materials & Interfaces, 2021

Pattern-Based Contractility Screening, a Reference-Free Alternative to Traction Force Microscopy Methodology

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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Scientific Reports, 2021

Collective migration during a gap closure in a two-dimensional haptotactic model

Marie Versaevel, Laura Alaimo, Valentine Seveau, Marine Luciano, Danahe Mohammed, Céline Bruyère, Eléonore Vercruysse, Olivier Théodoly, Sylvain Gabriele
The ability of cells to respond to substrate-bound protein gradients is crucial for many physiological processes, such as immune response, neurogenesis and cancer cell migration. Here we use a photopatterning technique to create well-controlled circular, square and linear fibronectin (FN) gradients on two-dimensional (2D) culture substrates, to understand collective cell migration in response to haptotaxis. Our findings provide a better understanding of the wound healing process over protein gradients, which are reminiscent of haptotaxis. …
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Jove, 2021

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Joris Guyon, Pierre-Olivier Strale, Irati Romero-Garmendia, Andreas Bikfalvi, Vincent Studer, Thomas Daubon
Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity. …
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Nature Communications, 2021

Mechanochemical control of epidermal stem cell divisions by B-plexins

Chen Jiang, Ahsan Javed, Laura Kaiser, Michele M. Nava, Rui Xu, Dominique T. Brandt, Dandan Zhao, Benjamin Mayer, Javier Fernández-Baldovinos, Luping Zhou, Carsten Höß, Kovilen Sawmynaden, Arkadiusz Oleksy, David Matthews, Lee S. Weinstein, Heidi Hahn, Hermann-Josef Gröne, Peter L. Graumann, Carien M. Niessen, Stefan Offermanns, Sara A. Wickström, Thomas Worzfeld

Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease. We identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. We show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness; and provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. …

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Cell Press, 2021

Acto-myosin network geometry defines centrosome position

Ana Joaquina Jimenez, Alexandre Schaeffer, Chiara De Pascalis, ..., Matthieu Piel, Laurent Blanchoin, Manuel Théry

Jimenez et al. show that the centrosome, which has long been thought to sit at the geometric center of the cell, is actually positioned at the center of a subcellular zone defined by the absence of contractile acto-myosin bundles. Centrosome position is defined by dyneins exerting pulling forces on microtubules specifically in this zone. …

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Methods in Microbiology, 2021

Micropatterning of cells on EM grids for efficient cryo-correlative light electron microscopy

Lea Swistak, Anna Sartori-Ruppc, Matthijn Vos, Jost Enninga
Cryo-transmission electron microscopy (cryo-TEM) provides access to high resolution information of adherent cell ultrastructures in a close to native environment but only volumes of less than 500 nm can be imaged. Cryo-focused ion beam (FIB) milling overcomes this obstacle through the generation of thin lamella of less than 200 nm. These lamellas can be imaged by cryo-electron tomography (cryo-ET) giving access to ultrastructural data within the volume of the imaged cells. Nevertheless, a lack of control on the positioning of the samples on the electron microscopy (EM) grids drastically constrains its throughput. The use of custom-designed micropatterned EM grids bypasses these issues by accurately positioning cells in areas that allow FIB milling followed by cryo-ET. Combined with fluorescent light microscopy in correlative light electron microscopy (CLEM) pipelines to pinpoint specific events and automated FIB milling, micropatterning of cells on EM grids has the potential of dramatically accelerating the workflow of cryo-ET. Here, a detailed description is provided of the key steps necessary to implement photomicropatterning of EM grids for improved CLEM pipelines. …
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Phys. Rev. Lett., 2021

Quantifying Material Properties of Cell Monolayers by Analyzing Integer Topological Defects

Carles Blanch-Mercader, Pau Guillamat, Aurélien Roux, and Karsten Kruse
In developing organisms, internal cellular processes generate mechanical stresses at the tissue scale. The resulting deformations depend on the material properties of the tissue, which can exhibit long-ranged orientational order and topological defects. It remains a challenge to determine these properties on the time scales relevant for developmental processes. (…) We illustrate our approach by analyzing monolayers of C2C12 cells in small circular confinements, where they form a single topological defect with integer charge. We find that such monolayers exert compressive stresses at the defect centers, where localized cell differentiation and formation of three-dimensional shapes is observed. …
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Scientific Reports, 2020

A minimalist model to measure interactions between proteins and synaptic vesicles

Eleonora Perego, Sofiia Reshetniak, Charlotta Lorenz, Christian Hoffmann, Dragomir Milovanović, Silvio O. Rizzoli & Sarah Köster
Protein dynamics in the synaptic bouton are still not well understood, despite many quantitative studies of synaptic structure and function. The complexity of the synaptic environment makes investigations of presynaptic protein mobility challenging. Here, we present an in vitro approach to create a minimalist model of the synaptic environment by patterning synaptic vesicles (SVs) on glass coverslips. …
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Nature Materials, 2020

Stress fibres are embedded in a contractile cortical network

Timothée Vignaud, Calina Copos, Christophe Leterrier, Mauricio Toro-Nahuelpan, Qingzong Tseng, Julia Mahamid, Laurent Blanchoin, Alex Mogilner, Manuel Théry & Laetitia Kurzawa
Contractile actomyosin networks are responsible for the production of intracellular forces. There is increasing evidence that bundles of actin filaments form interconnected and interconvertible structures with the rest of the network. In this study, we explored the mechanical impact of these interconnections on the production and distribution of traction forces throughout the cell. By using a combination of hydrogel micropatterning, traction force microscopy and laser photoablation, we measured the relaxation of traction forces in response to local photoablations. …
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Biology of the Cell, 2020

Human neutrophils swim and phagocytise bacteria

Nicolas Garcia‐Seyda, Valentine Seveau, Fabio Manca, Martine Biarnes‐Pelicot, Marie‐Pierre Valignat, Marc Bajénoff, Olivier Theodoly
Leukocytes migrate in an amoeboid fashion while patrolling our organism in the search for infection or tissue damage. Their capacity to migrate has been proven integrin independent, however, non‐specific adhesion or confinement remain a requisite in current models of cell migration. This idea has been challenged twice within the last decade with human neutrophils and effector T lymphocytes, which were shown to migrate in free suspension, a phenomenon termed swimming. …
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Nature Communications, 2020

T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration

Damien Garbett, Anjali Bisaria, Changsong Yang, Dannielle G. McCarthy, Arnold Hayer, W. E. Moerner, Tatyana M. Svitkina & Tobias Meyer
Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. …
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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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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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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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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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Nanoscale, 2020

Transport and programmed release of nanoscale cargo from cells by using NETosis

Daniel Meyer, Saba Telele, Anna Zelená, Alice J. Gillen, Alessandra Antonucci, Elsa Neubert, Robert Nißler, Florian A. Mann, Luise Erpenbeck, Ardemis A. Boghossian, 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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