A minimal physical model for curvotaxis driven by curved protein complexes at the cell's leading edge.

Cells often migrate on curved surfaces inside the body, such as curved tissues, blood vessels, or highly curved protrusions of other cells. Recent in vitro experiments provide clear evidence that motile cells are affected by the curvature of the substrate on which they migrate, preferring certain curvatures to others, termed "curvotaxis." The origin and underlying mechanism that gives rise to this curvature sensitivity are not well understood. Here, we employ a "minimal cell" model which is composed of a vesicle that contains curved membrane protein complexes, that exert protrusive forces on the membrane (representing the pressure due to actin polymerization). This minimal-cell model gives rise to spontaneous emergence of a motile phenotype, driven by a lamellipodia-like leading edge. By systematically screening the behavior of this model on different types of curved substrates (sinusoidal, cylinder, and tube), we show that minimal ingredients and energy terms capture the experimental data. The model recovers the observed migration on the sinusoidal substrate, where cells move along the grooves (minima), while avoiding motion along the ridges. In addition, the model predicts the tendency of cells to migrate circumferentially on convex substrates and axially on concave ones. Both of these predictions are verified experimentally, on several cell types. Altogether, our results identify the minimization of membrane-substrate adhesion energy and binding energy between the membrane protein complexes as key players of curvotaxis in cell migration.

Connectivity and plasticity determine collagen network fracture.

Collagen forms the structural scaffold of connective tissues in all mammals. Tissues are remarkably resistant against mechanical deformations because collagen molecules hierarchically self-assemble in fibrous networks that stiffen with increasing strain. Nevertheless, collagen networks do fracture when tissues are overloaded or subject to pathological conditions such as aneurysms. Prior studies of the role of collagen in tissue fracture have mainly focused on tendons, which contain highly aligned bundles of collagen. By contrast, little is known about fracture of the orientationally more disordered collagen networks present in many other tissues such as skin and cartilage. Here, we combine shear rheology of reconstituted collagen networks with computer simulations to investigate the primary determinants of fracture in disordered collagen networks. We show that the fracture strain is controlled by the coordination number of the network junctions, with less connected networks fracturing at larger strains. The hierarchical structure of collagen fine-tunes the fracture strain by providing structural plasticity at the network and fiber level. Our findings imply that low connectivity and plasticity provide protective mechanisms against network fracture that can optimize the strength of biological tissues.

Effects of Diabetes Mellitus on Fibrin Clot Structure and Mechanics in a Model of Acute Neutrophil Extracellular Traps (NETs) Formation.

Subjects with diabetes mellitus (DM) have an increased risk of arterial thrombosis, to which changes in clot structure and mechanics may contribute. Another contributing factor might be an increased formation of neutrophil extracellular traps (NETs) in DM. NETs are mainly formed during the acute phase of disease and form a network within the fibrin matrix, thereby influencing clot properties. Previous research has shown separate effects of NETs and DM on clot properties, therefore our aim was to study how DM affects clot properties in a model resembling an acute phase of disease with NETs formation. Clots were prepared from citrated plasma from subjects with and without DM with the addition of NETs, induced in neutrophils by S. aureus bacteria or phorbol myristate acetate (PMA). Structural parameters were measured using scanning electron microscopy, mechanical properties using rheology, and sensitivity to lysis using a fluorescence-based fibrinolysis assay. Plasma clots from subjects with DM had significantly thicker fibers and fewer pores and branch points than clots from subjects without DM. In addition, fibrinolysis was significantly slower, while mechanical properties were similar between both groups. In conclusion, in a model of acute NETs formation, DM plasma shows prothrombotic effects on fibrin clots.

Effect of ionic strength on the structure and elongational kinetics of vimentin filaments.

Intermediate filaments are a major structural element in the cytoskeleton of animal cells that mechanically integrate other cytoskeletal components and absorb externally applied stress. Their role is likely to be linked to their complex molecular architecture which is the product of a multi-step assembly pathway. Intermediate filaments form tetrameric subunits which assemble in the presence of monovalent salts to form unit length filaments that subsequently elongate by end-to-end annealing. The present work characterizes this complex assembly process using reconstituted vimentin intermediate filaments with monovalent salts as an assembly trigger. A multi-scale approach is used, comprising static light scattering, dynamic light scattering and quantitative scanning transmission electron microscopy (STEM) mass measurements. Light scattering reveals the radius of gyration (Rg), molecular weight (Mw) and diffusion coefficient (D) of the assembling filaments as a function of time and salt concentration (cS) for the given protein concentration of 0.07 g L-1. At low cS (10 mM KCl) no lateral or elongational growth is observed, whereas at cS = 50-200 mM, the hydrodynamic cross-sectional radius and the elongation rate increases with cS. Rgversus Mw plots suggest that the mass per unit length increases with increasing salt content, which is confirmed by STEM mass measurements. A kinetic model based on rate equations for a two step process is able to accurately describe the variation of mass, length and diffusion coefficient of the filaments with time and provides a consistent description of the elongation accelerated by increasing cS.

Time-lapse scanning surface plasmon microscopy of living adherent cells with a radially polarized beam.

We report on a fibered high-resolution scanning surface plasmon microscope for long term imaging of living adherent cells. The coupling of a high numerical aperture objective lens and a fibered heterodyne interferometer enhances both the sensitivity and the long term stability of this microscope, allowing for time-lapse recording over several days. The diffraction limit is reached with a radially polarized illumination beam. Adherence and motility of living C2C12 myoblast cells are followed for 50 h, revealing that the dynamics of these cells change after 10 h. This plasmon enhanced evanescent wave microscopy is particularly suited for investigating cell adhesion, since it can not only be performed without staining of the sample but it can also capture in real time the exchange of extracellular matrix elements between the substrate and the cells.

Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves.

Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions.

Interplay of fibrinogen αEC globular domains and factor XIIIa cross-linking dictates the extensibility and strain stiffening of fibrin networks.

BACKGROUND: Fibrinogen is a plasma protein forming the fibrin scaffold of blood clots. Its mechanical properties therefore affect the risk of bleeding as well as thrombosis. There has been much recent interest in the biophysical mechanisms controlling fibrin mechanics; however, the role of molecular heterogeneity of the circulating fibrinogen in determining clot mechanical function remains poorly characterized. OBJECTIVES: By comparing 2 fibrinogen variants where the only difference is the Aα-chain length, with one variant having a globular domain at its C-terminus, this study aimed to reveal how the molecular structure impacts the structure and mechanics of fibrin networks. METHODS: We characterized the mechanical response to large shear for networks formed from 2 recombinant fibrinogen variants: the most prevalent variant in circulation with a molecular weight of 340 kDa (recombinant human fibrinogen [rFib] 340) and a minor variant with a molecular weight of 420 kDa (rFib420). RESULTS: We show that the elastic properties of the 2 variants are identical when fibrin is cross-linked with factor XIIIa but differ strongly in its absence. Uncross-linked rFib420 networks are softer and up to 3-fold more extensible than rFib340 networks. Electron microscopy imaging showed that the 2 variants formed networks with a comparable structure, except at 4 mg/mL, where rFib420 formed denser networks. CONCLUSION: We propose that the αEC domains of rFib420 increase the extensibility of uncross-linked fibrin networks by promoting protofibril sliding, which is blocked by FXIIIa cross-linking. Our findings can help explain the functional role of different circulating fibrinogen variants in blood clot mechanics and tissue repair.

Guided wave microscopy: mastering the inverse problem.

Surface plasmon microscopy is widely recognized for its high sensitivity to nanoscale dielectric or metallic structures confined in a close neighborhood of a gold surface. Recently, its coupling to high-numerical-aperture objective lenses pushed its resolution down to the diffraction limit. Here, we show that the same microscope configuration can be used to excite standing guided waves in asymmetric slabs, which definitely extends the range of applications of this type of microscopy from nano- to microscale structure imaging. We demonstrate experimentally on PPMA films that the V(Z) response of a scanning surface plasmon microscope can be Fourier inverted in order to obtain the reflectivity curve R(ν). When the guided waves are excited, R(ν) shows a finite number of sharp peaks corresponding to quantified guiding modes from which one can extract both the refractive index (RI) and the thickness of the layer at the point focused by the microscope. This device can thus be used to reconstruct RI and thickness contours of dielectric samples with a high spatial resolution.

DisGUVery: A Versatile Open-Source Software for High-Throughput Image Analysis of Giant Unilamellar Vesicles.

Giant unilamellar vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVery's three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple predefined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape, and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of nonspherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in synthetic cell research.

Revealing the molecular origins of fibrin's elastomeric properties by in situ X-ray scattering.

Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performing in situ small angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (<5%), followed by progressive fiber alignment (>25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, suggesting that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials. STATEMENT OF SIGNIFICANCE: Fibrin provides structural integrity to blood clots and is also widely used as a scaffold for tissue engineering. To fulfill their biological functions, fibrin networks have to be simultaneously compliant like skin and resilient against rupture. Here, we unravel the structural origin underlying this remarkable mechanical behaviour. To this end, we performed in situ measurements of fibrin structure across multiple length scales by combining X-ray scattering with shear rheology. Our findings show that fibrin sustains large strains by undergoing a sequence of structural changes on different scales with increasing strain levels. This demonstrates new mechanistic aspects of an important biomaterial's structure and its mechanical function, and serves as an example in the design of biomimicking materials.

Revealing the assembly of filamentous proteins with scanning transmission electron microscopy.

Filamentous proteins are responsible for the superior mechanical strength of our cells and tissues. The remarkable mechanical properties of protein filaments are tied to their complex molecular packing structure. However, since these filaments have widths of several to tens of nanometers, it has remained challenging to quantitatively probe their molecular mass density and three-dimensional packing order. Scanning transmission electron microscopy (STEM) is a powerful tool to perform simultaneous mass and morphology measurements on filamentous proteins at high resolution, but its applicability has been greatly limited by the lack of automated image processing methods. Here, we demonstrate a semi-automated tracking algorithm that is capable of analyzing the molecular packing density of intra- and extracellular protein filaments over a broad mass range from STEM images. We prove the wide applicability of the technique by analyzing the mass densities of two cytoskeletal proteins (actin and microtubules) and of the main protein in the extracellular matrix, collagen. The high-throughput and spatial resolution of our approach allow us to quantify the internal packing of these filaments and their polymorphism by correlating mass and morphology information. Moreover, we are able to identify periodic mass variations in collagen fibrils that reveal details of their axially ordered longitudinal self-assembly. STEM-based mass mapping coupled with our tracking algorithm is therefore a powerful technique in the characterization of a wide range of biological and synthetic filaments.

Molecular Origin of the Elastic State of Aqueous Hyaluronic Acid.

The macroscopic mechanical properties of biological hydrogels are broadly studied and successfully mimicked in synthetic materials, but little is known about the molecular interactions that mediate these properties. Here, we use two-dimensional infrared spectroscopy to study the pH-induced gelation of hyaluronic acid, a ubiquitous biopolymer, which undergoes a transition from a viscous to an elastic state in a narrow pH range around 2.5. We find that the gelation originates from the enhanced formation of strong interchain connections, consisting of a double amide-COOH hydrogen bond and an N-D-COO- hydrogen bond on the adjacent sugars of the hyaluronan disaccharide unit. We confirm the enhanced interchain connectivity in the elastic state by atomic force microscopy imaging.

Deciphering the internal complexity of living cells with quantitative phase microscopy: a multiscale approach.

The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

Diffraction phase microscopy: retrieving phase contours on living cells with a wavelet-based space-scale analysis.

We propose a two-dimensional (2-D) space-scale analysis of fringe patterns collected from a diffraction phase microscope based on the 2-D Morlet wavelet transform. We show that the adaptation of a ridge detection method with anisotropic 2-D Morlet mother wavelets is more efficient for analyzing cellular and high refractive index contrast objects than Fourier filtering methods since it can separate phase from intensity modulations. We compare the performance of this ridge detection method on theoretical and experimental images of polymer microbeads and experimental images collected from living myoblasts.