BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation.

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Coupled mechanical mapping and interference contrast microscopy reveal viscoelastic and adhesion hallmarks of monocyte differentiation into macrophages.

Monocytes activated by pro-inflammatory signals adhere to the vascular endothelium and migrate from the bloodstream to the tissue ultimately differentiating into macrophages. Cell mechanics and adhesion play a crucial role in macrophage functions during this inflammatory process. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we used various tools to quantify the morphology, adhesion, and viscoelasticity of monocytes and differentiatted macrophages. Combination of atomic force microscopy (AFM) high resolution viscoelastic mapping with interference contrast microscopy (ICM) at the single-cell level revealed viscoelasticity and adhesion hallmarks during monocyte differentiation into macrophages. Quantitative holographic tomography imaging revealed a dramatic increase in cell volume and surface area during monocyte differentiation and the emergence of round and spread macrophage subpopulations. AFM viscoelastic mapping showed important stiffening (increase of the apparent Young's modulus, E0) and solidification (decrease of cell fluidity, ß) on differentiated cells that correlated with increased adhesion area. These changes were enhanced in macrophages with a spread phenotype. Remarkably, when adhesion was perturbed, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the stiffer and more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activities. Thus, our results revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

Multi-Step Extracellular Matrix Remodelling and Stiffening in the Development of Idiopathic Pulmonary Fibrosis.

The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young's Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.

αvß3 integrin expression increases elasticity in human melanoma cells.

Living cells interact with the extracellular matrix (ECM) transducing biochemical signals into mechanical cues and vice versa. Thanks to this mechano-transduction process, cells modify their internal organization and upregulate their physiological functions differently. In this complex mechanism integrins play a fundamental role, connecting the extracellular matrix with the cytoskeleton. Cytoskeletal rearrangements, such as the increase of the overall contractility, impact cell mechanical properties, the entire cell stiffness, and cell deformability. How cell mechanics is influenced via different integrins and their interaction with ECM in health and disease is still unclear. Here, we investigated the influence of αvß3 integrin expression on the mechanics of human melanoma M21 cells using atomic force microscopy and micro-constriction. Evidence is provided that (i) αvß3 integrin expression in human melanoma cells increases cell stiffness in both adherent and non-adherent conditions; (ii) replacing αvß3 with αIIbß3 integrin in melanoma cells, cell stiffness is increased under adherent, while decreased under non-adherent conditions; (iii) αvß3 integrin cell stiffening is also maintained when cells adhere to fibronectin, but this phenomenon does not strongly depend on the fibronectin concentration. In all, this study sheds light on the role of αvß3 in regulating cellular mechanics.

Genome editing retraces the evolution of toxin resistance in the monarch butterfly.

Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness1. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits2-4, and new genome editing technology is facilitating functional validation of these mutations in whole organisms1,5. We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins6-11. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na+/K+-ATPase)7-11, the physiological target of cardiac glycosides12. Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization13,14. We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage11,15. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides16, culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators17-19. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis13,14,20-22. Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities2,6-11,15,17-19.

High-frequency microrheology reveals cytoskeleton dynamics in living cells.

Living cells are viscoelastic materials, with the elastic response dominating at long timescales (≳1 ms)1. At shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce2. Here, we develop high-frequency microrheology (HF-MR) to probe the viscoelastic response of living cells from 1Hz to 100 kHz. We report the viscoelasticity of different cell types and upon cytoskeletal drug treatments. At previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequency, providing a univocal mechanical fingerprint. Microrheology over a wide dynamic range up to the frequency of action of the molecular components provides a mechanistic understanding of cell mechanics.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro.

Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

Elongated membrane tethers, individually anchored by high affinity α4ß1/VCAM-1 complexes, are the quantal units of monocyte arrests.

The α4ß1 integrin facilitates both monocyte rolling and adhesion to the vascular endothelium and is physiologically activated by monocyte chemoattractant protein (MCP-1). The current study investigated the initial events in the adhesion of THP-1 cells to immobilized Vascular Cell Adhesion Molecule 1 (VCAM-1). Using AFM force measurements, cell adhesion was shown to be mediated by two populations of α4ß1/VCAM-1 complexes. A low affinity form of α4ß1 was anchored to the elastic elements of the cytoskeleton, while a higher affinity conformer was coupled to the viscous elements of the cell membrane. Within 100 ms of contact, THP-1 cells, stimulated by co-immobilized MCP-1, exhibited a tremendous increase in adhesion to VCAM-1. Enhanced cell adhesion was accompanied by a local decoupling of the cell membrane from the cytoskeleton and the formation of long membrane tethers. The tethers were individually anchored by multiple α4ß1/VCAM-1 complexes that prolonged the extension of the viscous tethers. In vivo, the formation of these membrane tethers may provide the quantal structural units for the arrest of rolling monocytes within the blood vessels.

Structural and mechanical heterogeneity of the erythrocyte membrane reveals hallmarks of membrane stability.

The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosphorylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

Thrombin and histamine induce stiffening of alveolar epithelial cells.

The mechanical properties of alveolar epithelial cells play a central role in maintaining the physical integrity of the alveolar epithelium. We studied the viscoelastic properties of alveolar epithelial cells (A549) in response to thrombin and histamine with optical magnetic twisting cytometry. Ferrimagnetic beads coated with Arg-Gly-Asp (RGD)-peptide or acetylated low-density lipoprotein were bound to cell surface receptors and subsequently twisted in an oscillatory magnetic field (0.1-100 Hz). The cell storage (G') and loss (G'') moduli were computed from twisting torque and bead displacement. In measurements with RGD-coated beads, thrombin (0.5 U/ml) induced a rapid and sustained threefold increase in G' and G'' at approximately 100 s after challenge. Histamine (100 microM) induced a rapid but transient twofold increase in G' and G'' with maximum values 60 s after challenge. Posttreatment with cytochalasin D abolished thrombin-induced cell stiffening. G' increased with frequency following a power law with exponent 0.214. G'' increased proportionally to G' up to 10 Hz but showed a steeper rise at higher frequencies. Thrombin caused a fall in the power-law exponent (0.164). In measurements with acetylated low-density lipoprotein-coated beads, minor changes (<20%) were observed in G' and G'' after the addition of thrombin and histamine. F-actin staining revealed that thrombin and histamine induced a profound reorganization of the actin cytoskeleton at the cell periphery and formation of actin bundles. In the mechanically dynamic environment of the lung, cell stiffening induced by thrombin and histamine increases centripetal tension, which could contribute to alveolar barrier dysfunction.