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

Suzanne E Stasiak, Ryan R Jamieson, Jeff Bouffard, Erin J Cram, Harikrishnan Parameswaran
Agonist-induced calcium oscillations constitute a critical part of the machinery by which smooth muscle cells (SMCs) regulate constriction in airways and the vasculature. The frequency of these Ca2+ oscillations encodes the concentration of contractile agonist detected by the surface receptors on the SMC, with higher frequencies corresponding to higher agonist concentrations. Here, we report a collective phenomenon in SMCs where cells in multicellular ensembles exhibit  …
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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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Different integrins mediate haptotaxis of T lymphocytes towards either lower or higher adhesion zones

Xuan Luo, Laurene Aoun, Martine Biarnes-Pelicot, Pierre-Olivier Strale, Vincent Studer, Marie-Pierre Valignat, Olivier Theodoly
Guidance of cells by molecules anchored on a substrate, known as haptotaxis, is arguably crucial in development, immunology and cancer, however the exact cues and mechanisms driving cell orientation in vivo are hardly identified. Adhesive haptotaxis has been described in the case of mesenchymatous cells that develop strong pulling forces with their substrates and orient via a tug of war mechanism, a competition between cells pulling edges. In the case of amoeboid cells that migrate with minimal interaction with their substrate, existence of adhesive haptotaxis remains unclear. …
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Mammalian Amoeboid Swimming is propelled by molecular and not protrusion-based paddling in Lymphocytes

Laurene Aoun, Paulin Negre, Alexander Farutin, Nicolas Garcia-Seyda, Mohd Suhail Rivzi, Remi Galland, Alphee Michelot, Xuan Luo, Martine Biarnes-Pelicot, Claire Hivroz, Salima Rafai, Jean-Baptiste Sibareta, Marie-Pierre Valignat, Chaouqi Misbah, Olivier Theodoly
Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like fibroblasts crawling, relies on maturation of adhesion complexes and actin fiber traction, while the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on 2D and in 3D 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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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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Tailoring 3D cell culture templates with common hydrogels

Aurelien Pasturel, Pierre-Olivier Strale, Vincent Studer
3D cell culture aims at reconciliating the simplicity of in vitro models with the human like properties encountered in vivo. Soft permeable hydrogels have emerged as user-friendly materials to grow cells in more physiological conditions. With the intent on turning these homogeneous substrates into biomimetic templates, we introduce a generic solution compatible with the most biologically relevant and often frail materials. Here we take control of the chemical environment driving generic radical reactions to craft common gels with patterned light. …
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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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