Scientific papers
Read our users’ scientific papers and see how PRIMO photopatterning helped them in various fields: control of cell polarity, cell adhesion, cryo-ET, microfabrication, etc.
BioRxiv, 2024
Engineered microvasculature using maskless photolithography and on-chip hydrogel patterning: a facile approach
In vitro models of human microvasculature are increasingly used to understand blood vessel diseases and to support drug development. Most engineered models, however, are slow and labor-intensive to produce. Here, we used a single commercial digital micromirror device (DMD)-based setup for maskless photolithography to both fabricate microfluidic chips and pattern the inside of these chips with gelatin methacrylate (GelMA) hydrogels. ...
Voir l'articleBioRxiv, 2024
Actin dynamics sustains spatial gradients of membrane tension in adherent cells
Tension propagates in lipid bilayers over hundreds of microns within milliseconds, precluding the formation of tension gradients. Nevertheless, plasma membrane tension gradients have been evidenced in migrating cells and along axons. Here, using a fluorescent membrane tension probe, we show that membrane tension gradients exist in all adherent cells, whether they migrate or not. ...
Voir l'articleACS Nano, 2024
Engineering the Cellular Microenvironment: Integrating Three-Dimensional Nontopographical and Two-Dimensional Biochemical Cues for Precise Control of Cellular Behavior
The development of biomaterials capable of regulating cellular processes and guiding cell fate decisions has broad implications in tissue engineering, regenerative medicine, and cell-based assays for drug development and disease modeling. Recent studies have shown that three-dimensional (3D) nanoscale physical cues such as nanotopography can modulate various cellular processes like adhesion and endocytosis by inducing nanoscale curvature on the plasma and nuclear membranes. Two-dimensional (2D) biochemical cues such as protein micropatterns can also regulate cell function and fate by controlling cellular geometries. Development of biomaterials with precise control over nanoscale physical and biochemical cues can significantly influence programming cell function and fate. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. ...
Voir l'articleBioRxiv, 2024
Vimentin promotes collective cell migration through collagen networks via increased matrix remodeling and spheroid fluidity
The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin’s role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. ...
Voir l'articleNature, 2024
Endoplasmic reticulum–plasma membrane contact gradients direct cell migration
Directed cell migration is driven by the front–back polarization of intracellular signalling. Receptor tyrosine kinases and other inputs activate local signals that trigger membrane protrusions at the front. Equally important is a long-range inhibitory mechanism that suppresses signalling at the back to prevent the formation of multiple fronts. However, the identity of this mechanism is unknown. Here we report that endoplasmic reticulum–plasma membrane (ER–PM) contact sites are polarized in single and collectively migrating cells. ...
Voir l'articleBioRxiv, 2024
Light-induced Extracellular Vesicle Adsorption
The role of extracellular vesicles (EVs) in human health and disease has garnered considerable attention over the past two decades. However, while several types of EVs are known to interact dynamically with the extracellular matrix and there is great potential value in producing high-fidelity EV micropatterns, there are currently no label-free, high-resolution, and tunable platform technologies with this capability. We introduce Light-induced Extracellular Vesicle Adsorption (LEVA) as a powerful solution to rapidly advance the study of matrix- and surface-bound EVs and other particles. ...
Voir l'articleBiofabrication, 2024
Steering cell orientation through light-based spatiotemporal modulation of the mechanical environment
The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved, in vitro techniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3D in vitro model systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. ...
Voir l'articleThesis, 2024
Micropatterning subcellulaire pour étudier la connectivité neuronale
Micropatterning was initially employed to replicate and understand the influence of the extracellular matrix on cells and some of their components. Over the past decade, subcellular printing has emerged, enabling the study of protein interactions and their role in signaling pathways as well as in the formation of synaptic, immunological, or neuronal pathways.The synaptic connection is mediated by synaptic adhesion proteins present on each side of the synapse. Due to the complexity of the synaptic environment and the lack of in vitro models to study synaptic connection in a biomimetic and controlled environment, the exact roles of these proteins in synaptogenesis remain uncertain. Subcellular protein printing presents a potential solution to address this gap. ...
Voir l'articleBiorxiv, 2024
Shape dynamics and migration of branched cells on complex networks
Migratory and tissue resident cells exhibit highly branched morphologies to perform their function and to adapt to the microenvironment. Immune cells, for example, display transient branched shapes while exploring the surrounding tissues. In another example, to properly irrigate the tissues, blood vessels bifurcate thereby forcing the branching of cells moving on top or within the vessels. In both cases microenvironmental constraints force migrating cells to extend several highly dynamic protrusions. Here, we present a theoretical model for the shape dynamics and migration of cells that simultaneously span several junctions, which we validated by using micropatterns with an hexagonal array, and a neuronal network image analysis pipeline to monitor the macrophages and endothelial cell shapes and migration. ...
Voir l'articlePNAS, 2024
Dual topologies of myotomal collagen XV and Tenascin C act in concert to guide and shape developing motor axons
During development, motor axons are guided toward muscle target by various extrinsic cues including extracellular matrix (ECM) proteins whose identities and cellular source remain poorly characterized. Here, using single-cell RNAseq of sorted GFP+ cells from smyhc1:gfp-injected zebrafish embryos, we unravel the slow muscle progenitors (SMP) pseudotemporal trajectory at the single-cell level and show that differentiating SMPs are a major source of ECM proteins. (...) Importantly, bioprinted micropatterns that mimic this in vivo ECM topology were sufficient to drive directional motor axon growth. …
Plos Biology, 2024
IntAct: A nondisruptive internal tagging strategy to study the organization and function of actin isoforms
Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. ...
Voir l'articleBioRxiv, 2024
Dissecting the binding mechanisms of synaptic membrane adhesion complexes using a micropattern based cellular model
The formation of adhesive cell-cell contacts is based on the intrinsic binding properties between specific transmembrane ligand-receptor pairs. In neurons, synaptic adhesion molecules provide a physical linkage between pre- and post-synaptic compartments, but the dynamics of these complexes in their actual membrane environments remain essentially unknown. To access such information, we developed a versatile assay to measure the affinity and binding kinetics of synaptic ligand-receptor interactions, based on the immobilization of Fc-tagged ligands on micropatterned substrates combined with live-cell imaging of fluorescently-tagged counter receptors in heterologous cells. …
BioRxiv, 2024
Doublecortin reinforces microtubules to promote growth cone advance in soft environments
Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, the molecular function of DCX in neuron morphogenesis remains unknown and controversial. We demonstrate that DCX function is intimately linked to its microtubule-binding activity. (…) Together with high resolution traction force microscopy data, we propose a model in which DCX-decorated, rigid growth cone microtubules provide intracellular mechanical resistance to actomyosin generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated forces in the growth cone periphery may be insufficient for productive growth cone advance. …
BioRxiv, 2024
Limiting Brownian Motion to Enhance Immunogold Phenotyping and Superimpose Optical and Non-Optical Single-EP Analyses
Optical and non-optical techniques propelled the field of single extracellular particle (EP) research through phenotypic and morphological analyses, revealing the similarities, differences, and co-isolation of EP subpopulations. Overcoming the challenges of optical and non-optical techniques motivates the use of orthogonal techniques while analyzing extracellular particles (EPs), which require varying concentrations and preparations. Herein, we introduce the nano-positioning matrix (NPMx) technique capable of superimposing optical and non-optical modalities for a single-EP orthogonal analysis. The NPMx technique is realized by ultraviolet-mediated micropatterning to reduce the stochasticity of Brownian motion. …
Mol Syst Biol, 2024
The population context is a driver of the heterogeneous response of epithelial cells to interferons
Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. …
BioRxiv, 2024
Optogenetic generation of leader cells reveals a force-velocity relation for collective cell migration
The front of migratory cellular clusters during development, wound healing and cancer invasion is typically populated with highly protrusive cells that are called leader cells. Leader cells are thought to physically pull and direct their cohort of followers, but how leaders and followers are mechanically organized to migrate collectively remains controversial. (…) Here we show that the effectiveness of leader-follower organization is proportional to the asymmetry of traction and tension within the cellular cluster. By combining hydrogel micropatterning and optogenetic activation of Rac1, we locally generate highly protrusive leaders at the edge of minimal cell groups. …
BioRxiv, 2024
EasyGrid: a versatile platform for automated cryo-EM sample preparation and quality control
Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. …
Sichuan Da Xue Xue Bao Yi Xue Ban, 2024
Spatial Constraints of Rectangular Hydrogel Microgrooves Regulate the Morphology and Arrangement of Human Umbilical Vein Endothelial Cells
The objective was to construct microscale rectangular hydrogel grooves and to investigate the morphology and alignment of human umbilical vein endothelial cells (HUVECs) under spatial constraints. Vascular endothelial cell morphology and alignment are important factors in vascular development and the maintenance of homeostasis. Hydrogel microgrooves can regulate the morphology and orientation of HUVECs and mimic to a certain extent the in vivo microenvironment of vascular endothelial cells, providing an experimental model that bears better resemblance to human physiology for the study of the unique physiological functions of vascular endothelial cells. …
Sensors and Actuators, 2024
Microfluidic integration of the single cell adhesion dot array (SCADA) technology for the real-time quantification of cell affinity
Understanding cell affinity to substrates and biomolecules is of great importance in disease research, drug development and general cell biology studies. Established techniques to measure cell affinity involve either expensive and cumbersome techniques that quantify the binding between cells and proteins in suspension (e.g., flow cytometry), or indirect methods that quantify the amount of cells on a surface (e.g., impedance sensors). Novel approaches exploit microtechnologies to reduce the number of cells needed and reach single cell resolution. However, the examples so far fail to provide a simple device to measure cell affinity with single cell resolution that can be adapted to several purposes and cell biology laboratories. Herein, we describe a tool for the real-time optical monitoring of cell affinity. It is based in the integration of the cell-based biosensing platform Single Cell Adhesion Dot Arrays (SCADA) into a microfluidic device. …
Bio-protocol, 2023
Correlative Light and Electron Cryo-Microscopy Workflow Combining Micropatterning, Ice Shield, and an In-Chamber Fluorescence Light Microscope
In situ cryo-electron tomography (cryo-ET) is the most current, state-of-the-art technique to study cell machinery in its hydrated near-native state. The method provides ultrastructural details at sub-nanometer resolution for many components within the cellular context. Making use of recent advances in sample preparation techniques and combining this method with correlative light and electron microscopy (CLEM) approaches have enabled targeted molecular visualization. Nevertheless, the implementation has also added to the complexity of the workflow and introduced new obstacles in the way of streamlining and achieving high throughput, sample yield, and sample quality. Here, we report a detailed protocol by combining multiple newly available technologies to establish an integrated, high-throughput, optimized, and streamlined cryo-CLEM workflow for improved sample yield. …
BioRxiv, 2023
1D confinement mimicking microvessel geometry controls pericyte shape and motility
Pericytes are mural cells of the microvasculature, characterised by their elongated distinct shape. Pericytes span along the axis of the vessels they adhere to, therefore they experience extreme lateral and longitudinal confinement. Pericyte shape is key for their function during vascular regulation and their spatial distribution is established by cell migration during the embryonic stage and maintained through controlled motility in the adult. However, how pericyte morphology is associated with migration and function remains unknown. We use micropatterns to mimic pericyte adhesion to vessels, and to reproduce in vitro the shapes adopted by pericytes in vivo. …
Journal of Cell Science, 2023
Microtubules under mechanical pressure can breach dense actin networks
The crosstalk between actin network and microtubules is key to the establishment of cell polarity. It ensures that the asymmetry of actin architec ture along cell periphery directs the organization of microtubules in cell interior. In particular, the way the two networks are physically inter-twined regulates the spatial organization and the distribution of forces in the microtubule network. While their biochemical crosstalk is getting uncovered, their mechanical crosstalk is still poorly understood. Here we designed an in vitro reconstitution assay to study the physical interaction between dynamic microtubules with various structures made of actin filaments. …
BioRxiv, 2023
Unravelling the metastasis-preventing effect of miR-200c in vitro and in vivo
Advanced breast cancer as well as insufficient treatment can lead to the dissemination of malignant cells from the primary tumor to distant organs. Recent research has shown that miR-200c can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear, whether sole miR-200c expression is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. …
Journal of Extracellular Vesicles, 2023
Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ
The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVPPRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVPPRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVPPRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. …
BioRxiv, 2023
Nuclear deformation and dynamics of migrating cells in 3D confinement reveal adaptation of pulling and pushing forces
Eukaryotic cells show an astounding ability to migrate through pores and constrictions smaller than their nuclear diameter. However, the forces engaged in nuclear deformation and their effect on confined cell dynamics remain unclear. Here, we study the mechanics and dynamics of nuclei of mesenchymal cancer cells as they spontaneously and repeatedly transition through 3D compliant hydrogel channels. …
BioRxiv, 2023
Comparative profiling of cellular gait on adhesive micropatterns defines statistical patterns of activity that underlie native and cancerous cell dynamics
Cell dynamics are powered by patterns of activity, but it is not straightforward to quantify these patterns or compare them across different environmental conditions or cell-types. Here we digitize the long-term shape fluctuations of metazoan cells grown on micropatterned fibronectin islands to define and extract statistical features of cell dynamics without the need for genetic modification or fluorescence imaging. These shape fluctuations generate single-cell morphological signals that can be decomposed into two major components: a continuous, slow-timescale meandering of morphology about an average steady-state shape; and short-lived “events” of rapid morphology change that sporadically occur throughout the timecourse. …
BioRxiv, 2023
Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context
Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. …
Nature Structural and Molecular Biology, 2023
Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection
Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. (…) Human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. …
Advanced Healthcare Materials, 2023
Mechanoresponse of Curved Epithelial Monolayers Lining Bowl-Shaped 3D Microwells
The optimal functioning of many organs relies on the curved architecture of their epithelial tissues. However, the mechanoresponse of epithelia to changes in curvature remains misunderstood. Here, bowl-shaped microwells in hydrogels are designed via photopolymerization to faithfully replicate the shape and dimensions of lobular structures. Leveraging these hydrogel-based microwells, curved epithelial monolayers are engineered, and how in-plane and Gaussian curvatures at the microwell entrance influence epithelial behavior is investigated. …
Biorxiv, 2023
Spatial Patterning of Laminin and N-Cadherin for Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have key physiological differences from primary human cardiomyocytes (CMs), including lower sarcomere alignment and contractility, smaller area and lower aspect ratio, and lower force production. Protein micropatterning has been demonstrated to make hiPSC-CMs behave more like primary human CMs across these metrics. However, these micropatterned models typically use only extracellular matrix (ECM) proteins and have not investigated whether providing a protein associated with CM-CM interactions, such as N-cadherin, further enhances hiPSC-CM structure and function. Here, we developed a novel dual-protein patterning process to geometrically control single-cell CM placement on deformable hydrogels suitable for traction force microscopy (TFM). The patterns were comprised of rectangular laminin islands for attachment across the majority of the cell area, with N-cadherin “end-caps” imitating cell-cell interactions. …
Nature Methods, 2023
Correlative montage parallel array cryo-tomography for in situ structural cell biology
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 parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis. …
Nature Materials, 2023
The laminin-keratin link shields the nucleus from mechanical deformation and signalling
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. …
Thesis, 2023
Modelling physics with deep learning: An experimental case of cell contractility
Deep Learning (DL) algorithms have been used to model physical systems. However, the success heavily relies on how DL methodologies accommodate the properties of a system and data. (…) Under this context, we study the case of Traction Force Microscopy (TFM), a class of experimental procedures and algorithms for measuring cell traction. (…) We introduce Deep Morphology Traction Microscopy (DeepMorphoTM), a DL approach that infers cell traction from a shape sequence of a cell. By employing a deterministic framework, DeepMorphoTM effectively mitigates the biological variability in cell contractility for a given cell shape. …
BioRxiv, 2023
A platform for dissecting force sensitivity and multivalency in actin networks
The physical structure and dynamics of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them into specific functional architectures. Mechanical force has been shown to modulate the interactions between several ABPs and individual actin filaments, but it is unclear how this phenomenon contributes to the emergent force-responsive functional dynamics of actin networks. Here, we engineer filament linker complexes and combine them with photo-micropatterning of myosin motor proteins to produce an in vitro reconstitution platform for examining how force impacts the behavior of ABPs within multi-filament assemblies. …
iScience, 2023
Naïve T lymphocytes chemotax long distance to CCL21 but not to a source of bioactive S1P
Naïve T lymphocytes traffic through the organism in search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on CCL21 chemokine sensing by CCR7 receptors, while exit into efferent lymphatics relies on sphingolipid S1P sensing by S1PR1 receptors. While both molecules are claimed chemotactic, a quantitative analysis of naïve T lymphocyte migration along defined gradients is missing. Here, we used a reductionist approach to study the real-time single-cell response of naïve T lymphocytes to CCL21 and serum rich in bioactive S1P. Using microfluidic and micropatterning ad hoc tools, we show that CCL21 triggers stable polarization and long-range chemotaxis of cells, whereas S1P-rich serum triggers a transient polarization only and no significant displacement, potentially representing a brief transmigration step through exit portals. …
Biorxiv, 2023
Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. …
Biophysical Journal, 2023
Force generation in human blood platelets by filamentous actomyosin structures
Blood platelets are central elements of the blood clotting response after wounding. Upon vessel damage, they bind to the surrounding matrix and contract the forming thrombus, thus helping to restore normal blood circulation. The hemostatic function of platelets is directly connected to their mechanics and cytoskeletal organization. The reorganization of the platelet cytoskeleton during spreading occurs within minutes and leads to the formation of contractile actomyosin bundles, but it is not known if there is a direct correlation between the emerging actin structures and the force field that is exerted to the environment. In this study, we combine fluorescence imaging of the actin structures with simultaneous traction force measurements in a time-resolved manner. …
biorxiv, 2023
Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster
Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. …
Nature Communications, 2023
Mapping mechanical stress in curved epithelia of designed size and shape
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. …
Journal of Cell Biology, 2023
Mechanical control of the mammalian circadian clock via YAP/TAZ and TEAD
Autonomous circadian clocks exist in nearly every mammalian cell type. These cellular clocks are subjected to a multilayered regulation sensitive to the mechanochemical cell microenvironment. Whereas the biochemical signaling that controls the cellular circadian clock is increasingly well understood, mechanisms underlying regulation by mechanical cues are largely unknown. Here we show that the fibroblast circadian clock is mechanically regulated through YAP/TAZ nuclear levels. We use high-throughput analysis of single-cell circadian rhythms and apply controlled mechanical, biochemical, and genetic perturbations to study the expression of the clock gene Rev-erbα. …
Nature Communications, 2023
PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell Complex
The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. …
Nature Communications, 2023
A convolutional neural network STIFMap reveals associations between stromal stiffness and EMT in breast cancer
Intratumor heterogeneity associates with poor patient outcome. Stromal stiffening also accompanies cancer. Whether cancers demonstrate stiffness heterogeneity, and if this is linked to tumor cell heterogeneity remains unclear. We developed a method to measure the stiffness heterogeneity in human breast tumors that quantifies the stromal stiffness each cell experiences and permits visual registration with biomarkers of tumor progression. We present Spatially Transformed Inferential Force Map (STIFMap) which exploits computer vision to precisely automate atomic force microscopy (AFM) indentation combined with a trained convolutional neural network to predict stromal elasticity with micron-resolution using collagen morphological features and ground truth AFM data. …
Thesis, 2023
Spatiotemporal Analysis of Metazoan Cell Morphological Dynamics on Micropatterned Substrates
Spatial patterns of adhesive substrates can dictate cell motility trajectories.
Mammalian cells demonstrate idiosyncratic trajectories when subjected to barbell-shaped micropatterns with thin constrictions. However, the morphologies of these cells over time remained uncharacterized. We subjected 3T3 mouse fibroblast cells to an array of micropatterns, and compared their shape dynamics to a panel of triple-negative breast cancer cells. When exposed to a gradient of size, we found that the morphological dynamics of 3T3 cells display trends analogous to the potential energy curve of a molecule, where energy is high at small micropattern sizes, drops off at a minimum point, and rises again at large micropattern sizes. We also
found the breast cancer cell panel exhibited a gradient of morphological activity and could be classified using principal component analysis. …
biorxiv, 2023
In vitro modelling of anterior primitive streak patterning with hESC reveals the dynamic of WNT and NODAL signalling required to specify notochord progenitors
Notochord progenitors (NotoPs) are a rare, yet vital embryonic cell population that give rise to the cells that form and maintain intervertebral discs. An unlimited access to NotoPs would open new opportunities for basic biomedical research and regenerative medicine of the discs. However, the mechanisms responsible for the specification and the maintenance of NotoPs are not understood. This gap in understanding stems from the fact that NotoPs emerge during the gastrulation to axial elongation transition; an event that is ethically and technically challenging to investigate. Here, to circumvent this issue, we use micropatterning to guide the development of human ESCs into standardised patterns of anterior primitive streak cell fates. …
biorxiv, 2023
Engineering cell and nuclear morphology on nano topography by contact-free protein micropatterning
Platforms with nanoscale topography have recently become powerful tools in cellular biophysics and bioengineering. Recent studies have shown that nanotopography affects various cellular processes like adhesion and endocytosis, as well as physical properties such as cell shape. To engineer nanopillars more effectively for biomedical applications, it is crucial to gain better control and understanding of how nanopillars affect cell and nuclear physical properties, such as shape and spreading area, and impact cellular processes like endocytosis and adhesion. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. …
Advanced Science, 2023
Downregulation of YAP Activity Restricts P53 Hyperactivation to Promote Cell Survival in Confinement
Cell migration through confining three dimensional (3D) topographies can lead to loss of nuclear envelope integrity, DNA damage, and genomic instability. Despite these detrimental phenomena, cells transiently exposed to confinement do not usually die. Whether this is also true for cells subjected to long-term confinement remains unclear at present. To investigate this, photopatterning and microfluidics are employed to fabricate a high-throughput device that circumvents limitations of previous cell confinement models and enables prolonged culture of single cells in microchannels with physiologically relevant length scales. …
biorxiv, 2023
Frictiotaxis underlies adhesion-independent durotaxis
Cells move directionally along gradients of substrate stiffness, a process called durotaxis. The current consensus is that durotaxis relies on cell-substrate focal adhesions to transmit forces that drive directed motion. Such a mechanism implies that focal adhesion-independent durotaxis is impossible, although this assumption has never been tested. Here, we show that confined cells can perform durotaxis despite lacking strong or specific adhesions. We show that the mechanism of this adhesion-independent durotaxis is that stiffer substrates offer higher friction. We develop a physical model that predicts that non-adherent cells polarize and migrate towards regions of higher friction — a process that we call frictiotaxis. …
Biorxiv, 2023
Population context drives cell-to-cell variability in interferon response in epithelial cells
Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to the neighboring cells) of human intestinal epithelial cells affects single cell response to interferons. …
biorxiv, 2023
Identification of the intracellular protein targets of a bio-active clickable half-sandwich iridium complex by chemical proteomics
Identification of intracellular targets of anticancer drug candidates provides key information on their mechanism of action. Exploiting the ability of the anticancer (C^N)-chelated half-sandwich iridium(III) complexes to covalently bind proteins, click chemistry with a bioorthogonal azido probe was used to localize a phenyloxazoline-chelated iridium complex within cells and profile its interactome at the proteome-wide scale. Proteins involved in protein folding and actin cytoskeleton regulation were identified as high affinity targets. Upon iridium complex treatment, HSP90 folding activity was inhibited in vitro and major cytoskeleton disorganization was observed. We used a wide array of imaging and biochemical methods to validate selected targets and obtain a multiscale overview of the effects of this complex on live human cells. …
ACS Applied Nano Materials, 2023
Micropatterning of Quantum Dots for Biofunctionalization and Nanoimaging
Micron-scale patterning of colloidal quantum dots (QDs) is extremely important for the fabrication of high-performance Quantum dot Light-Emitting Diode (QLED) displays, biosensing, and super-resolution imaging. Thus, several nondestructive methods have been recently proposed, such as spatial self-organization. However, none of them can be useful for biofunctionalization or nanoimaging. To address this limitation, we propose a method to create micropatterns of QDs of any shape and size. UV photolithography assisted by a digital micromirror device (DMD) and silanization allow creating an adhesive layer, on which QDs micropatterns can be assembled with a 2 μm resolution. …
biorxiv, 2023
Vimentin intermediate filaments structure and mechanically support microtubules in cells
The eukaryotic cytoskeleton comprises three types of mechanically distinct biopoly-mers – actin filaments, microtubules and intermediate filaments (IFs)– along with pas-sive crosslinkers and active molecular motors. Among these filament types, IFs are expressed in a cell-type specific manner and vimentin is found in cells of mesenchymal origin. The composite cytoskeletal network determines the mechanical and dynamic properties of the cell and is specifically governed by the interplay of the three different filament systems. We study the influence of vimentin IFs on the mechanics and net-work structure of microtubules by analyzing fluorescence micrographs of fibroblasts on protein micropatterns. We develop and apply quantitative, automated data analysis to a large number of cells, thus mitigating the considerable natural variance in data from biological cells. …
Thesis, 2023
Engineered 3D-Vessels-on-Chip to study effects of dynamic fluid flow on human induced pluripotent stem cell derived endothelial cells
Realistic models of the human vasculature would benefit understanding of normal physiology and disease pathology in the blood circulatory systems. Here we used a photo patterning system to form near vertical hydrogel walls inside a microfluidic device, generating a perfusable network. The hydrogels had realistic (tissue-like) viscoelastic properties and were permeable to large molecules like 70kD dextran. Endothelial cells from human pluripotent stem cells (hiPSC-ECs) cultured in the hydrogels could be guided to form complex networks. …
Biorxiv, 2023
Self-organization of Long-lasting Human Endothelial Capillary Networks guided by DLP Bioprinting
Tissue engineering holds great promise for regenerative medicine, drug discovery and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential and is a major challenge to be met. In this work, we explore a promising Digital Light Processing (DLP) bioprinting approach to encapsulate endothelial progenitor cells (EPCs) in 3D photopolymerized hydrogel scaffolds to guide them towards vascular network formation. We observed that EPCs encapsulated in the appropriate photopolymerized hydrogel can proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of various diameters (in the range of 10 to 100 μm). …
Biophysical Journal, 2023
Engineering cell morphology using maskless 2D protein micropatterning on 3D nanostructures
Mechanical cues such as the 2D and 3D shape of cellular microenvironments affect several cellular processes including adhesion and proliferation. Recent studies provide controlled conditions to recapitulate the 2D and 3D microenvironments to understand the mechanisms of cellular response to these mechanical cues. (…) In this study, we present a micropatterning technique on nanostructured surfaces based on a maskless laser-assisted technique to study the cellular response to 3D nano-topographies in a controlled 2D microenvironment. We used a two-step dry and wet etching technique to fabricate transparent (SiO2) 3D nanostructured surfaces. Next, we micropatterned extracellular matrix proteins directly on the fabricated nanostructures by maskless micropatterning (PRIMO, Alvéole) system mounted on an inverted microscope. …
Nature Communications, 2023
Plasma FIB milling for the determination of structures in situ
Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. 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 that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication. …
Biorxiv, 2023
The distance between the plasma membrane and the actomyosin cortex acts as a nanogate to control cell surface mechanics
Animal cell shape changes are controlled by the actomyosin cortex, a peripheral actin network tethered to the plasma membrane by membrane-to-cortex attachment (MCA) proteins. Previous studies have focused on how myosin motors or actin turnover can generate the local deformations required for morphogenesis. However, how the cell controls local actin nucleation remains poorly understood. By combining molecular engineering with biophysical approaches and in situ cryo-electron tomography characterization of cortical actin network architecture, we show that membrane-to-cortex tethering determines the distance between the plasma membrane and the actomyosin cortex at the nanoscale of single actin nucleators. …
Biorxiv, 2023
Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion
Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here we employ an organotypic microfluidic platform capable of recapitulating and dissecting three-dimensional (3D) morphogenic features of a ductal epithelium, to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. …
ACS Synth. Biol., 2023
Extracellular Cues Govern Shape and Cytoskeletal Organization in Giant Unilamellar Lipid Vesicles
Spontaneous and induced front-rear polarization and a subsequent asymmetric actin cytoskeleton is a crucial event leading to cell migration, a key process involved in a variety of physiological and pathological conditions such as tissue development, wound healing, and cancer. Migration of adherent cells relies on the balance between adhesion to the underlying matrix and cytoskeleton-driven front protrusion and rear retraction. A current challenge is to uncouple the effect of adhesion and shape from the contribution of the cytoskeleton in regulating the onset of front-rear polarization. Here, we present a minimal model system that introduces an asymmetric actin cytoskeleton in synthetic cells, which are resembled by giant unilamellar lipid vesicles (GUVs) adhering onto symmetric and asymmetric micropatterned surfaces. Surface micropatterning of streptavidin-coated regions with varying adhesion shape and area was achieved by maskless UV photopatterning. …
Biomaterials, 2023
Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies
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. …
Biorxiv, 2022
Friction patterns guide actin network contraction
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. …
Nature Communications, 2022
Morphological control enables nanometer-scale dissection of cell-cell signaling complexes
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. …
STAR Protocols, 2022
Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
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. …
Nature Physics, 2022
Mechanical stress driven by rigidity sensing governs epithelial stability
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. …
Cell Reports, 2022
A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1
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. …
Current Opinion in Structural Biology, 2022
Electron microscopy of cellular ultrastructure in three dimensions
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. …
Thesis, 2022
Spatial control of angiogenesis by engineered patterns of Notch ligands
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. …
BioRxiv, 2022
ARHGEF18 participates in Endothelial Cell Mechano-sensitivity in Response to Flow
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. …
Cell Reports, 2022
Pressure and curvature control of the cell cycle in epithelia growing under spherical confinement
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. …
Nature Communications, 2022
Intrinsic cell rheology drives junction maturation
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. …
Biorxiv, 2022
Cryo-electron tomography reveals enrichment and identifies microtubule lumenal particles in neuronal differentiation
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. …
Trends in Cell Biology, 2022
Mechanobiological approaches to synthetic morphogenesis: learning by building
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. …
Current Biology, 2022
MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization
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. …
Lab on a Chip, 2022
Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis
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. …
Methods in Molecular Biology, 2022
Controlling Cell Shape and Microtubule Organization by Extracellular Matrix Micropatterning
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. …
Adv. Funct. Mater., 2022
Printing and Erasing of DNA-Based Photoresists Inside Synthetic Cells
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. …
Biorxiv, 2022
Conserved basal lamina proteins, laminin and nidogen, are repurposed to organize mechanosensory complexes responsible for touch sensation
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. …
Nature Materials, 2022
Integer topological defects organize stresses driving tissue morphogenesis
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. …
Jove, 2022
Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy
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). …
PNAS, 2022
ATP allosterically stabilizes integrin-linked kinase for efficient force generation
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. …
ACS Appl. Mater. Interfaces, 2022
Facile and Versatile Method for Micropatterning Poly(acrylamide) Hydrogels Using Photocleavable Comonomers
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. …
Micromachines, 2021
Rapid Prototyping of Organ-on-a-Chip Devices Using Maskless Photolithography
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. …
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. …
Biomaterials, 2021
Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography
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. …
Journal of Cell Biology, 2021
Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1
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. …
Journal of structural biology, 2021
Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts
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. …
Jove, 2021
Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows
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). …
nano letters, 2021
Nanoscale Surface Topography Reduces Focal Adhesions and Cell Stiffness by Enhancing Integrin Endocytosis
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. …
ACS Appl. Mater. Interfaces, 2021
Composite Elastomer-Enabled Rapid Photofabrication of Microfluidic Devices
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. …
Biology of the Cell, 2021
Direct measurement of near-nano-Newton forces developed by self-organizing actomyosin fibers bound α-catenin
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. …
biorxiv, 2021
Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids
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. …
Methods in Molecular Biology, 2021
Manufacturing a Bone Marrow-On-A-Chip Using Maskless Photolithography
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. …
PNAS, 2021
Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates
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. …
ACS Applied Materials & Interfaces, 2021
Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments
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). …
ACS Applied Materials & Interfaces, 2021
Pattern-Based Contractility Screening, a Reference-Free Alternative to Traction Force Microscopy Methodology
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. …
Scientific Reports, 2021
Collective migration during a gap closure in a two-dimensional haptotactic model
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. …
Jove, 2021
Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions
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. …
Nature Communications, 2021
Mechanochemical control of epidermal stem cell divisions by B-plexins
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. …
Cell Press, 2021
Acto-myosin network geometry defines centrosome position
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. …
Methods in Microbiology, 2021
Micropatterning of cells on EM grids for efficient cryo-correlative light electron microscopy
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. …
Phys. Rev. Lett., 2021
Quantifying Material Properties of Cell Monolayers by Analyzing Integer Topological Defects
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. …
Scientific Reports, 2020
A minimalist model to measure interactions between proteins and synaptic vesicles
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. …
Nature Materials, 2020
Stress fibres are embedded in a contractile cortical network
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. …
Biology of the Cell, 2020
Human neutrophils swim and phagocytise bacteria
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. …
Nature Communications, 2020
T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration
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. …
Biophysical Journal, 2020
Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes
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. …
Journal of Cell Science, 2020
Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins
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. …
Advanced Healthcare Matherials, 2020
Tailoring Common Hydrogels into 3D Cell Culture Templates
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. …
Science Advances, 2020
Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells
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. …
Biology of the Cell, 2020
Mechanobiology of antigen‐induced T cell arrest
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, …
ACS Nano, 2020
Coupling Polar Adhesion with Traction, Spring and Torque Forces Allows High Speed Helical Migration of the Protozoan Parasite Toxoplasma
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 …
Lab On A Chip, 2020
Microfluidic dialysis using photo-patterned hydrogel membranes in PDMS chips
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. …
Nature Materials, 2020
Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes
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. …
CELL, 2020
Defining the Design Principles of Skin Epidermis Postnatal Growth
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…
Nanoscale, 2020
Transport and programmed release of nanoscale cargo from cells by using NETosis
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 …
Jove, 2019
Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins
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. …
SCIENTIFIC REPORTS, 2019
Extracellular matrix stiffness regulates human airway smooth muscle contraction by altering the cell-cell coupling
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. …
Nature Methods, 2019
Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies
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. …
NAT PHYS, 2019
Substrate area confinement is a key determinant of cell velocity in collective migration
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. …
NAT MAT, 2019
Traction forces at the cytokinetic ring regulate cell division and polyploidy in the migrating zebrafish epicardium
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. …
J. Micromech. Microeng., 2019
Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies
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. …
NATURE MATERIALS, 2019
A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions
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. …
Phil. Trans. R. Soc. B, 2019
Forces and constraints controlling podosome assembly and disassembly
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. …
BIORXIV, 2019
Autonomous induction of hepatic polarity to construct single cell liver
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. …
ADVANCED SCIENCE, 2019
Photoactivatable Hsp47: A Tool to Regulate Collagen Secretion and Assembly
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. …
PLOS One, 2018
Live nanoscopic to mesoscopic topography reconstruction with an optical microscope for chemical and biological samples
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. …
Nano Lett, 2018
Optical magnetometry of single biocompatible micromagnets for quantitative magnetogenetic and magnetomechanical assays
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. …
Adv Biosys, 2018
A new approach to design artificial 3D micro-niches with combined chemical, topographical and rheological cues
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. …
Lab Chip, 2018
In situ photopatterning of pressure-resistant hydrogel membranes with controlled permeabilities in PEGDA microfluidic channels.
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. …
BioRxiv, 2018
Collagen assembly and turnover imaged with a CRISPR-Cas9 engineered Dendra2 tag
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. …
Hum Mol Genet. 2016
Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes.
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. …
Adv Mater. 2016
Multiprotein Printing by Light-Induced Molecular Adsorption.
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.