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

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, 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 using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC). 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). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Tuning Epithelial Cell-Cell Adhesion and Collective Dynamics with Functional DNA-E-Cadherin Hybrid Linkers.

The binding strength between epithelial cells is crucial for tissue integrity, signal transduction and collective cell dynamics. However, there is no experimental approach to precisely modulate cell-cell adhesion strength at the cellular and molecular level. Here, we establish DNA nanotechnology as a tool to control cell-cell adhesion of epithelial cells. We designed a DNA-E-cadherin hybrid system consisting of complementary DNA strands covalently bound to a truncated E-cadherin with a modified extracellular domain. DNA sequence design allows to tune the DNA-E-cadherin hybrid molecular binding strength, while retaining its cytosolic interactions and downstream signaling capabilities. The DNA-E-cadherin hybrid facilitates strong and reversible cell-cell adhesion in E-cadherin deficient cells by forming mechanotransducive adherens junctions. We assess the direct influence of cell-cell adhesion strength on intracellular signaling and collective cell dynamics. This highlights the scope of DNA nanotechnology as a precision technology to study and engineer cell collectives.

Nanophysical Mapping of Inflammasome Activation by Nanoparticles via Specific Cell Surface Recognition Events.

Silica nanoparticles (SiNP) trigger a range of innate immune responses in relevant essential organs, such as the liver and the lungs. Inflammatory reactions, including NLRP3 inflammasome activation, have been linked to particulate materials; however, the molecular mechanisms and key actors remain elusive. Although many receptors, including several scavenger receptors, were suggested to participate in SiNP cellular uptake, mechanistic evidence of their role on innate immunity is lacking. Here we present an atomic force microscopy-based approach to physico-mechanically map the specific interaction occurring between nanoparticles and scavenger receptor A1 (SRA1) in vitro on living lung epithelial cells. We find that SiNP recognition by SRA1 on human macrophages plays a key role in mediating NLRP3 inflammasome activation, and we identify cellular mechanical changes as clear indicators of inflammasome activation in human macrophages, greatly advancing our knowledge on the interplay among nanomaterials and innate immunity.

Reovirus directly engages integrin to recruit clathrin for entry into host cells.

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, ß1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct ß1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and ß1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and ß1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for ß1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and ß1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems.

During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cell biological systems under physiological conditions. Here, we review key technological improvements that have established AFM as an analytical tool to observe and quantify native biological systems from the micro- to the nanoscale. Native biological systems include living tissues, cells, and cellular components such as single or complexed proteins, nucleic acids, lipids, or sugars. We showcase the procedures to customize nanoscopic chemical laboratories by functionalizing AFM tips and outline the advantages and limitations in applying different AFM modes to chemically image, sense, and manipulate biosystems at (sub)nanometer spatial and millisecond temporal resolution. We further discuss theoretical approaches to extract the kinetic and thermodynamic parameters of specific biomolecular interactions detected by AFM for single bonds and extend the discussion to multiple bonds. Finally, we highlight the potential of combining AFM with optical microscopy and spectroscopy to address the full complexity of biological systems and to tackle fundamental challenges in life sciences.

Mechanochemical Activation of Class-B G-Protein-Coupled Receptor upon Peptide-Ligand Binding.

Glucagon binding to the class-B G-protein-coupled glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting. Recently, GCGR crystal structures have highlighted the conformation and molecular details of inactive and active receptor states. However, the dynamics of the conformational changes accompanying GCGR activation remains unclear. Here, we use multiplex force-distance curve-based atomic force microscopy (FD-based AFM) to probe in situ glucagon binding to individual GCGRs and monitor dynamically the transition to the active conformer. After a "dock" step, in which glucagon is partially bound to the GCGR extracellular domain, further interactions of the N-terminus with the transmembrane domain trigger an increase in the stiffness of the complex, adopting a highly stable and rigid "lock" conformer. This mechanotransduction is key for G-protein recruitment.

Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide variety of molecules, the strategy presented here provides new opportunities to study specific interactions and deliver single molecules with high spatiotemporal resolution in a wide range of applications, including AFM-based cell biology.

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.

Probing ligand-receptor bonds in physiologically relevant conditions using AFM.

Cell surface receptors, often called transmembrane receptors, are key cellular components as they control and mediate cell communication and signalling, converting extracellular signals into intracellular signals. Elucidating the molecular details of ligand binding (cytokine, growth factors, hormones, pathogens,...) to cell surface receptors and how this binding triggers conformational changes that initiate intracellular signalling is needed to improve our understanding of cellular processes and for rational drug design. Unfortunately, the molecular complexity and high hydrophobicity of membrane proteins significantly hamper their structural and functional characterization in conditions mimicking their native environment. With its piconewton force sensitivity and (sub)nanometer spatial resolution, together with the capability of operating in liquid environment and at physiological temperature, atomic force microscopy (AFM) has proven to be one of the most powerful tools to image and quantify receptor-ligand bonds in situ under physiologically relevant conditions. In this article, a brief overview of the rapid evolution of AFM towards quantitative biological mapping will be given, followed by selected examples highlighting the main advances that AFM-based ligand-receptor studies have brought to the fields of cell biology, immunology, microbiology, and virology, along with future prospects and challenges. Graphical abstract.

The puzzling issue of silica toxicity: are silanols bridging the gaps between surface states and pathogenicity?

BACKGROUND: Silica continues to represent an intriguing topic of fundamental and applied research across various scientific fields, from geology to physics, chemistry, cell biology, and particle toxicology. The pathogenic activity of silica is variable, depending on the physico-chemical features of the particles. In the last 50 years, crystallinity and capacity to generate free radicals have been recognized as relevant features for silica toxicity. The 'surface' also plays an important role in silica toxicity, but this term has often been used in a very general way, without defining which properties of the surface are actually driving toxicity. How the chemical features (e.g., silanols and siloxanes) and configuration of the silica surface can trigger toxic responses remains incompletely understood. MAIN BODY: Recent developments in surface chemistry, cell biology and toxicology provide new avenues to improve our understanding of the molecular mechanisms of the adverse responses to silica particles. New physico-chemical methods can finely characterize and quantify silanols at the surface of silica particles. Advanced computational modelling and atomic force microscopy offer unique opportunities to explore the intimate interactions between silica surface and membrane models or cells. In recent years, interdisciplinary research, using these tools, has built increasing evidence that surface silanols are critical determinants of the interaction between silica particles and biomolecules, membranes, cell systems, or animal models. It also has become clear that silanol configuration, and eventually biological responses, can be affected by impurities within the crystal structure, or coatings covering the particle surface. The discovery of new molecular targets of crystalline as well as amorphous silica particles in the immune system and in epithelial lung cells represents new possible toxicity pathways. Cellular recognition systems that detect specific features of the surface of silica particles have been identified. CONCLUSIONS: Interdisciplinary research bridging surface chemistry to toxicology is progressively solving the puzzling issue of the variable toxicity of silica. Further interdisciplinary research is ongoing to elucidate the intimate mechanisms of silica pathogenicity, to possibly mitigate or reduce surface reactivity.

High-resolution mapping and recognition of lipid domains using AFM with toxin-derivatized probes.

Cellular membrane lateral organization, in particular the assembly of lipids in domains, is difficult to evaluate at high resolution. Here, we used atomic force microscopy (AFM) to investigate at high-resolution lipid membranes containing variable amounts of sphingomyelin (SM) and cholesterol (Chol), two abundant membrane lipids. To this end, we developed new AFM tip functionalization strategies to specifically probe SM and Chol. Multiparametric AFM imaging allowed us to highlight the lateral submicrometric organization of these two lipids within lipid bilayers through the simultaneous topographic evidence of different phase regimes together with the extraction of their nanomechanical properties and the specific detection of lipid moieties by functionalized AFM probes. The combination of AFM topography and nanomechanical mapping with specific probes for molecular recognition of lipids represents a novel approach to identify lipid-enriched domains in supported bilayers and offers a unique perspective to directly observe lipid assemblies in living cells.