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.

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds.

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

Advances in mechanical biomarkers.

Mechanical biomarkers distinguish health conditions through quantitative mechanical measurements. The emergence and establishment of nanotechnology in the last decades have provided new tools to obtain mechanical biomarkers at the nanoscale. Mechanical measurements are reproducible, label-free, start to be applied in vivo can be high throughput, and require small samples. Mechanical protocols in clinical practice at the macro scale like palpation or blood pressure measurement are routinely used by medical doctors. Nanotechnology brought mechanical sensing to the next scale, where cells, tissues, and proteins can be probed and linked to medical conditions. Mechanical changes in cells and tissues may be detected before other markers, such as protein expression, providing an important advantage as biomarkers. In the present review, we explore the biomarker's historical evolution, describe mechanical biomarkers on various diseases and novel discoveries in the nanomechanical field for their characterization. We conclude that mechanical biomarkers are establishing novel hallmarks in diseases, in several cases for early diagnostics of diseases and discovery of drug targets in the proteins involved in the mechanical changes, while advances in instrumentation are bringing commercial products into the clinical practice. Mechanical biomarkers along with clinical testing are establishing an important niche in the market, whose demand is increasing due to the expansion of personalized medicine and unmet needs in the clinics.

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.

Heterogeneous and rate-dependent streptavidin-biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations.

Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds.

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.

Biological physics by high-speed atomic force microscopy.

While many fields have contributed to biological physics, nanotechnology offers a new scale of observation. High-speed atomic force microscopy (HS-AFM) provides nanometre structural information and dynamics with subsecond resolution of biological systems. Moreover, HS-AFM allows us to measure piconewton forces within microseconds giving access to unexplored, fast biophysical processes. Thus, HS-AFM provides a tool to nourish biological physics through the observation of emergent physical phenomena in biological systems. In this review, we present an overview of the contribution of HS-AFM, both in imaging and force spectroscopy modes, to the field of biological physics. We focus on examples in which HS-AFM observations on membrane remodelling, molecular motors or the unfolding of proteins have stimulated the development of novel theories or the emergence of new concepts. We finally provide expected applications and developments of HS-AFM that we believe will continue contributing to our understanding of nature, by serving to the dialogue between biology and physics. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.

Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed.

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.

Monitoring Unfolding of Titin I27 Single and Bi Domain with High-Pressure NMR Spectroscopy.

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular dynamics (MD) has often been combined with force spectroscopy experiments to decipher the unfolding mechanism of titin immunoglobulin-like single or multidomain, the giant multimodular protein from sarcomeres, yielding information on the sequential events during titin unfolding under stretching. Here, we used high-pressure NMR to monitor the unfolding of titin I27 Ig-like single domain and tandem. Because this method brings residue-specific information on the folding process, it can provide quasiatomic details on this process without the help of MD simulations. Globally, the results of our high-pressure analysis are in agreement with previous results obtained by the combination of experimental measurements and MD simulation and/or protein engineering, although the intermediate folding state caused by the early detachment of the AB ß-sheet, often reported in previous works based on MD or force spectroscopy, cannot be detected. On the other hand, the A'G parallel ß-sheet of the ß-sandwich has been confirmed as the Achilles heel of the three-dimensional scaffold: its disruption yields complete unfolding with very similar characteristics (free energy, unfolding volume, kinetics rate constants) for the two constructs.

History, rare, and multiple events of mechanical unfolding of repeat proteins.

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

Mutation in the Core Structure of Desmin Intermediate Filaments Affects Myoblast Elasticity.

Elastic properties of cells are mainly derived from the actin cytoskeleton. However, intermediate filaments are emerging as major contributors to the mechanical properties of cells. Using atomic force microscopy, we studied the elasticity of mouse myoblasts expressing a mutant form of the gene encoding for desmin intermediate filaments, p.D399Y. This variant produces desmin aggregates, the main pathological symptom of myofibrillar myopathies. Here we show that desmin-mutated cells display a 39% increased median elastic modulus compared to wild-type cells. Desmin-mutated cells required higher forces than wild-type cells to reach high indentation depths, where desmin intermediate filaments are typically located. In addition, heat-shock treatment increased the proportion of cells with aggregates and induced a secondary peak in the distribution of Young's moduli. By performing atomic force microscopy mechanical mapping combined with fluorescence microscopy, we show that higher Young's moduli were measured where desmin aggregates were located, indicating that desmin aggregates are rigid. Therefore, we provide evidence that p.D399Y stiffens mouse myoblasts. Based on these results, we suggest that p.D399Y-related myofibrillar myopathy is at least partly due to altered mechanical properties at the single-cell scale, which are propagated to the tissue scale.

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.

MechanoProDB: a web-based database for exploring the mechanical properties of proteins.

The mechanical stability of proteins is crucial for biological processes. To understand the mechanical functions of proteins, it is important to know the protein structure and mechanical properties. Protein mechanics is usually investigated through force spectroscopy experiments and simulations that probe the forces required to unfold the protein of interest. While there is a wealth of data in the literature on force spectroscopy experiments and steered molecular dynamics simulations of forced protein unfolding, this information is spread and difficult to access by non-experts. Here, we introduce MechanoProDB, a novel web-based database resource for collecting and mining data obtained from experimental and computational works. MechanoProDB provides a curated repository for a wide range of proteins, including muscle proteins, adhesion molecules and membrane proteins. The database incorporates relevant parameters that provide insights into the mechanical stability of proteins and their conformational stability such as the unfolding forces, energy landscape parameters and contour lengths of unfolding steps. Additionally, it provides intuitive annotations of the unfolding pathways of each protein, allowing users to explore the individual steps during mechanical unfolding. The user-friendly interface of MechanoProDB allows researchers to efficiently navigate, search and download data pertaining to specific protein folds or experimental conditions. Users can visualize protein structures using interactive tools integrated within the database, such as Mol*, and plot available data through integrated plotting tools. To ensure data quality and reliability, we have carefully manually verified and curated the data currently available on MechanoProDB. Furthermore, the database also features an interface that enables users to contribute new data and annotations, promoting community-driven comprehensiveness. The freely available MechanoProDB aims to streamline and accelerate research in the field of mechanobiology and biophysics by offering a unique platform for data sharing and analysis. MechanoProDB is freely available at https://mechanoprodb.ibdm.univ-amu.fr.

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.

Mechanics of proteins with a focus on atomic force microscopy.

The capacity of proteins to function relies on a balance between molecular stability to maintain their folded state and structural flexibility allowing conformational changes related to biological function. Among many others, four different examples can be chosen. The giant protein titin is stretched and can unfold during muscle contraction providing passive elasticity to muscle tissue; myoglobin adsorbs and releases oxygen molecules thank to conformational changes in its structure; the outer membrane protein G (OmpG) is a bacterial porin with a long and flexible loop that modulates gating; and the proton pump bacteriorhodopsin adapts its cytosolic half to allow proton pumping. All these conformational changes triggered either by chemical or by physical cues, require mechanical flexibility or elasticity of certain protein domains. While the methods to determine protein structure, X-ray crystallography above all, have been dramatically improved over the last decades, the number of tools that directly measure the mechanical flexibility of proteins and protein domains is still limited. In this tutorial, after a brief introduction to protein structure, we present some of the available techniques to estimate protein flexibility, then focusing on atomic force microscopy (AFM). We describe the principles of the technique and its various imaging and force spectroscopy modes of operation that allow probing the elasticity of proteins, protein domains and their surrounding environment.

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.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

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.

Direct measurement of the mechanical properties of lipid phases in supported bilayers.

Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties. Here, we acquired high-resolution topographic and quantitative mechanics data of phase-separated SLBs using a recent atomic force microscopy (AFM) imaging mode based on force measurements. This technique allows us to quantitatively map at high resolution the mechanical differences of lipid phases at different loading forces. We have applied this approach to evaluate the contribution of the underlying hard support in the determination of the elastic properties of SLBs and to determine the adequate indentation range for obtaining reliable elastic moduli values. At ~200 pN, elastic forces dominated the force-indentation response and the sample deformation was <20% of the bilayer thickness, at which the contribution of the support was found to be negligible. The obtained Young's modulus (E) of 19.3 MPa and 28.1 MPa allowed us to estimate the area stretch modulus (k(A)) as 106 pN/nm and 199 pN/nm and the bending stiffness (k(c)) as 18 k(B)T and 57 k(B)T for the liquid and gel phases, respectively.

Mechanical mapping of single membrane proteins at submolecular resolution.

The capacity of proteins to carry out different functions is related to their ability to undergo conformation changes, which depends on the flexibility of protein structures. In this work, we applied a novel imaging mode based on indentation force spectroscopy to map quantitatively the flexibility of individual membrane proteins in their native, folded state at unprecedented submolecular resolution. Our results enabled us to correlate protein flexibility with crystal structure and showed that α-helices are stiff structures that may contribute importantly to the mechanical stability of membrane proteins, while interhelical loops appeared more flexible, allowing conformational changes related to function.