The role of flow in the self-assembly of dragline spider silk proteins.

Hydrodynamic flow in the spider duct induces conformational changes in dragline spider silk proteins (spidroins) and drives their assembly, but the underlying physical mechanisms are still elusive. Here we address this challenging multiscale problem with a complementary strategy of atomistic and coarse-grained molecular dynamics simulations with uniform flow. The conformational changes at the molecular level were analyzed for single-tethered spider silk peptides. Uniform flow leads to coiled-to-stretch transitions and pushes alanine residues into ß sheet and poly-proline II conformations. Coarse-grained simulations of the assembly process of multiple semi-flexible block copolymers using multi-particle collision dynamics reveal that the spidroins aggregate faster but into low-order assemblies when they are less extended. At medium-to-large peptide extensions (50%-80%), assembly slows down and becomes reversible with frequent association and dissociation events, whereas spidroin alignment increases and alanine repeats form ordered regions. Our work highlights the role of flow in guiding silk self-assembly into tough fibers by enhancing alignment and kinetic reversibility, a mechanism likely relevant also for other proteins whose function depends on hydrodynamic flow.

Force propagation between epithelial cells depends on active coupling and mechano-structural polarization.

Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ('cell doublet'). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

The Ebola virus VP40 matrix layer undergoes endosomal disassembly essential for membrane fusion.

Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

A particle-based computational model to analyse remodelling of the red blood cell cytoskeleton during malaria infections.

Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.

KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in Plasmodium falciparum-infected erythrocytes.

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics.

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

Reversible elastic phase field approach and application to cell monolayers.

Motion and generation of forces by single cells and cell collectives are essential elements of many biological processes, including development, wound healing and cancer cell migration. Quantitative wound healing assays have demonstrated that cell monolayers can be both dynamic and elastic at the same time. However, it is very challenging to model this combination with conventional approaches. Here we introduce an elastic phase field approach that allows us to predict the dynamics of elastic sheets under the action of active stresses and localized forces, e.g. from leader cells. Our method ensures elastic reversibility after release of forces. We demonstrate its potential by studying several paradigmatic situations and geometries relevant for single cells and cell monolayers, including elastic bars, contractile discs and expanding monolayers with leader cells.

Forces during cellular uptake of viruses and nanoparticles at the ventral side.

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.

Extracellular Matrix Geometry and Initial Adhesive Position Determine Stress Fiber Network Organization during Cell Spreading.

Three-dimensional matrices often contain highly structured adhesive tracks that require cells to turn corners and bridge non-adhesive areas. Here, we investigate these complex processes using micropatterned cell adhesive frames. Spreading kinetics on these matrices depend strongly on initial adhesive position and are predicted by a cellular Potts model (CPM), which reflects a balance between adhesion and intracellular tension. As cells spread, new stress fibers (SFs) assemble periodically and parallel to the leading edge, with spatial intervals of ∼2.5 µm, temporal intervals of ∼15 min, and characteristic lifetimes of ∼50 min. By incorporating these rules into the CPM, we can successfully predict SF network architecture. Moreover, we observe broadly similar behavior when we culture cells on arrays of discrete collagen fibers. Our findings show that ECM geometry and initial cell position strongly determine cell spreading and that cells encode a memory of their spreading history through SF network organization.

A prospective histomorphometric and cephalometric comparison of bovine bone substitute and autogenous bone grafting in Le Fort I osteotomies.

PURPOSE: The aim of the present study was the histomorphometric and cephalometric comparison of autogenous bone grafting of the anterior iliac crest and the application of bovine bone substitute concerning new bone formation and postoperative stability in patients undergoing orthognathic Le Fort I osteotomy. PATIENTS AND METHODS: Twenty-five patients requiring orthognathic surgery with Le Fort I osteotomy were included in this study. Patients were randomly divided into three groups receiving either autogenous iliac crest BONE grafting (BONE; n = 8) or xenogenic bovine bone grafting (Bio-Oss®) in INTER (n = 12) or in ONLAY (n = 5) position. Histomorphometric analysis was performed using trephine bone biopsies from the autogenous, respectively xenogenic bone grafting region. Postoperative stability was evaluated using teleradiographies of three different timepoints. RESULTS: All groups showed comparable mineralized fractions in bone biopsies of 50.2% (±13.2%) INTER, 46.48% (±12.3%) ONLAY and 57.1% (±20.6%) BONE as well as comparable percentage of connective tissue. Patients in the INTER-group revealed the lowest relapse rate of 20.5% (INTER) compared to 30.3% (ONLAY) and 33.0% (BONE). All groups underwent comparable maxillary advancement and healing time. CONCLUSIONS: Present results indicate that block shaped bovine bone substitute is a promising alternative to autogenous bone grafting to bridge the Le Fort I osteotomy gap in orthognathic surgery.

Mechanical interactions among followers determine the emergence of leaders in migrating epithelial cell collectives.

Regulating the emergence of leaders is a central aspect of collective cell migration, but the underlying mechanisms remain ambiguous. Here we show that the selective emergence of leader cells at the epithelial wound-margin depends on the dynamics of the follower cells and is spatially limited by the length-scale of collective force transduction. Owing to the dynamic heterogeneity of the monolayer, cells behind the prospective leaders manifest locally increased traction and monolayer stresses much before these leaders display any phenotypic traits. Followers, in turn, pull on the future leaders to elect them to their fate. Once formed, the territory of a leader can extend only to the length up-to which forces are correlated, which is similar to the length up-to which leader cells can transmit forces. These findings provide mechanobiological insight into the hierarchy in cell collectives during epithelial wound healing.

Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration.

Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

Physical constraints for pathogen movement.

In this pedagogical review, we discuss the physical constraints that pathogens experience when they move in their host environment. Due to their small size, pathogens are living in a low Reynolds number world dominated by viscosity. For swimming pathogens, the so-called scallop theorem determines which kinds of shape changes can lead to productive motility. For crawling or gliding cells, the main resistance to movement comes from protein friction at the cell-environment interface. Viruses and pathogenic bacteria can also exploit intracellular host processes such as actin polymerization and motor-based transport, if they present the appropriate factors on their surfaces. Similar to cancer cells that also tend to cross various barriers, pathogens often combine several of these strategies in order to increase their motility and therefore their chances to replicate and spread.

Model-based traction force microscopy reveals differential tension in cellular actin bundles.

Adherent cells use forces at the cell-substrate interface to sense and respond to the physical properties of their environment. These cell forces can be measured with traction force microscopy which inverts the equations of elasticity theory to calculate them from the deformations of soft polymer substrates. We introduce a new type of traction force microscopy that in contrast to traditional methods uses additional image data for cytoskeleton and adhesion structures and a biophysical model to improve the robustness of the inverse procedure and abolishes the need for regularization. We use this method to demonstrate that ventral stress fibers of U2OS-cells are typically under higher mechanical tension than dorsal stress fibers or transverse arcs.

Role of dynamic capsomere supply for viral capsid self-assembly.

Many viruses rely on the self-assembly of their capsids to protect and transport their genomic material. For many viral systems, in particular for human viruses like hepatitis B, adeno or human immunodeficiency virus, that lead to persistent infections, capsomeres are continuously produced in the cytoplasm of the host cell while completed capsids exit the cell for a new round of infection. Here we use coarse-grained Brownian dynamics simulations of a generic patchy particle model to elucidate the role of the dynamic supply of capsomeres for the reversible self-assembly of empty T1 icosahedral virus capsids. We find that for high rates of capsomere influx only a narrow range of bond strengths exists for which a steady state of continuous capsid production is possible. For bond strengths smaller and larger than this optimal value, the reaction volume becomes crowded by small and large intermediates, respectively. For lower rates of capsomere influx a broader range of bond strengths exists for which a steady state of continuous capsid production is established, although now the production rate of capsids is smaller. Thus our simulations suggest that the importance of an optimal bond strength for viral capsid assembly typical for in vitro conditions can be reduced by the dynamic influx of capsomeres in a cellular environment.

High-resolution traction force microscopy.

Cellular forces generated by the actomyosin cytoskeleton and transmitted to the extracellular matrix (ECM) through discrete, integrin-based protein assemblies, that is, focal adhesions, are critical to developmental morphogenesis and tissue homeostasis, as well as disease progression in cancer. However, quantitative mapping of these forces has been difficult since there has been no experimental technique to visualize nanonewton forces at submicrometer spatial resolution. Here, we provide detailed protocols for measuring cellular forces exerted on two-dimensional elastic substrates with a high-resolution traction force microscopy (TFM) method. We describe fabrication of polyacrylamide substrates labeled with multiple colors of fiducial markers, functionalization of the substrates with ECM proteins, setting up the experiment, and imaging procedures. In addition, we provide the theoretical background of traction reconstruction and experimental considerations important to design a high-resolution TFM experiment. We describe the implementation of a new algorithm for processing of images of fiducial markers that are taken below the surface of the substrate, which significantly improves data quality. We demonstrate the application of the algorithm and explain how to choose a regularization parameter for suppression of the measurement error. A brief discussion of different ways to visualize and analyze the results serves to illustrate possible uses of high-resolution TFM in biomedical research.

Dynamics of cell shape and forces on micropatterned substrates predicted by a cellular Potts model.

Micropatterned substrates are often used to standardize cell experiments and to quantitatively study the relation between cell shape and function. Moreover, they are increasingly used in combination with traction force microscopy on soft elastic substrates. To predict the dynamics and steady states of cell shape and forces without any a priori knowledge of how the cell will spread on a given micropattern, here we extend earlier formulations of the two-dimensional cellular Potts model. The third dimension is treated as an area reservoir for spreading. To account for local contour reinforcement by peripheral bundles, we augment the cellular Potts model by elements of the tension-elasticity model. We first parameterize our model and show that it accounts for momentum conservation. We then demonstrate that it is in good agreement with experimental data for shape, spreading dynamics, and traction force patterns of cells on micropatterned substrates. We finally predict shapes and forces for micropatterns that have not yet been experimentally studied.

A computational model of nuclear self-organisation in syncytial embryos.

Syncytial embryos develop through cycles of nuclear division and rearrangement within a common cytoplasm. A paradigm example is Drosophila melanogaster in which nuclei form an ordered array in the embryo surface over cell cycles 10-13. This ordering process is assumed to be essential for subsequent cellularisation. Using quantitative tissue analysis, it has previously been shown that the regrowth of actin and microtubule networks after nuclear division generates reordering forces that counteract its disordering effect (Kanesaki et al., 2011). We present here an individual-based computer simulation modelling the nuclear dynamics. In contrast to similar modelling approaches e.g. epithelial monolayers or tumour spheroids, we focus not on the spatial dependence, but rather on the time-dependence of the interaction laws. We show that appropriate phenomenological inter-nuclear force laws reproduce the experimentally observed dynamics provided that the cytoskeletal network regrows sufficiently quickly after mitosis. Then repulsive forces provided by the actin system are necessary and sufficient to regain the observed level of order in the system, after the strong disruption resulting from cytoskeletal network disassembly and spindle formation. We also observe little mixing of nuclei through cell cycles. Our study highlights the importance of the dynamics of cytoskeletal forces during this critical phase of syncytial development and emphasises the need for real-time experimental data at high temporal resolution.

Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.

Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

Oscillations of Min-proteins in micropatterned environments: a three-dimensional particle-based stochastic simulation approach.

The Min-proteins from E. coli and other bacteria are the best characterized pattern forming system in cells and their spatiotemporal oscillations have been successfully reconstituted in vitro. Different mathematical and computational models have been used to better understand these oscillations. Here we use particle-based stochastic simulations to study Min-oscillations in patterned environments. We simulate a rectangular box of length 10 µm and width 5 µm that is filled with grid or checkerboard patterns of different patch sizes and distances. For this geometry, we find different stable oscillation patterns, typically pole-to-pole oscillations along the minor axis and striped oscillations along the major axis. The Min-oscillations can switch from one pattern to the other, either effected by changes in pattern geometry or stochastically. By automatic analysis of large-scale computer simulations, we show quantitatively how the perturbing effect of increased patch distance can be rescued by increased patch size. We also show that striped oscillations occur robustly in arbitrarily shaped filamentous E. coli cells. Our results highlight the robustness and variability of Min-oscillations, put limits on the effect of putative division sites, and provide a powerful computational framework for future studies of protein self-organization in patterned environments.