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Organoids are transformative in vitro model systems that mimic features of the corresponding tissue in vivo. However, across tissue types and species, organoids still often fail to reach full maturity and function because biochemical cues cannot be provided from within the organoid to guide their development. Here we introduce nanoengineered DNA microbeads with tissue mimetic tunable stiffness for implementing spatio-temporally controlled morphogen gradients inside of organoids at any point in their development. Using medaka retinal organoids and early embryos, we show that DNA microbeads can be integrated into embryos and organoids by microinjection and erased in a non-invasive manner with light. Coupling a recombinant surrogate Wnt to the DNA microbeads, we demonstrate the spatio-temporally controlled morphogen release from the microinjection site, which leads to morphogen gradients resulting in the formation of retinal pigmented epithelium while maintaining neuroretinal cell types. Thus, we bioengineered retinal organoids to more closely mirror the cell type diversity of in vivo retinae. Owing to the facile, one-pot fabrication process, the DNA microbead technology can be adapted to other organoid systems for improved tissue mimicry., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Clathrin-mediated endocytosis is the main pathway used by eukaryotic cells to take up extracellular material, but the dominant physical mechanisms driving this process are still elusive. Recently, several high-resolution imaging techniques have been used on different cell lines to measure the geometrical properties of clathrin-coated pits over their whole lifetime. Here, we first show that the combination of all datasets with the recently introduced cooperative curvature model defines a consensus pathway, which is characterized by a flat-to-curved transition at finite area, followed by linear growth and subsequent saturation of curvature. We then apply an energetic model for the composite of the plasma membrane and clathrin coat to this consensus pathway to show that the dominant mechanism for invagination could be coat stiffening, which might originate from cooperative interactions between the different clathrin molecules and progressively drives the system toward its intrinsic curvature. Our theory predicts that two length scales determine the invagination pathway, namely the patch size at which the flat-to-curved transition occurs and the final pit radius.

Competing Interests: Declaration of interests The author declares no competing interests.

Liquid crystalline (LC) materials are especially suited for the preparation of active three-dimensional (3D) and 4D microstructures using two-photon laser printing. To achieve the desired actuation, the alignment of the LCs has to be controlled during the printing process. In most cases studied before, the alignment relied on surface modifications and complex alignment patterns and concomitant actuation were not possible. Here, we introduce a strategy for spatially aligning LC domains in three-dimensional space by using 3D-printed polydimethylsiloxane-based microscaffolds as confinement barriers, which induce the desired director field. The director field resulting from the boundary conditions is calculated with Landau de Gennes theory and validated by comparing experimentally measured and theoretically predicted birefringence patterns. We demonstrate our procedures for structures of varying complexity and then employed them to fabricate 4D microstructures that show the desired actuation. Overall, we obtain excellent agreement between theory and experiment. This opens the door for rational design of functional materials for 4D (micro)printing in the future.

Gliding motility proceeds with little changes in cell shape and often results from actively driven surface flows of adhesins binding to the extracellular environment. It allows for fast movement over surfaces or through tissue, especially for the eukaryotic parasites from the phylum apicomplexa, which includes the causative agents of the widespread diseases malaria and toxoplasmosis. We have developed a fully three-dimensional active particle theory which connects the self-organized, actively driven surface flow over a fixed cell shape to the resulting global motility patterns. Our analytical solutions and numerical simulations show that straight motion without rotation is unstable for simple shapes and that straight cell shapes tend to lead to pure rotations. This suggests that the curved shapes of Plasmodium sporozoites and Toxoplasma tachyzoites are evolutionary adaptations to avoid rotations without translation. Gliding motility is also used by certain myxo- or flavobacteria, which predominantly move on flat external surfaces and with higher control of cell surface flow through internal tracks. We extend our theory for these cases. We again find a competition between rotation and translation and predict the effect of internal track geometry on overall forward speed. While specific mechanisms might vary across species, in general, our geometrical theory predicts and explains the rotational, circular, and helical trajectories which are commonly observed for microgliders. Our theory could also be used to design synthetic microgliders., Competing Interests: Competing interests statement:The authors declare no competing interest.

The nucleus of eukaryotic cells typically makes up around 30% of the cell volume and has significantly different mechanics, which can make it effectively up to ten times stiffer than the surrounding cytoplasm. Therefore it is an important element for cell mechanics, but a quantitative understanding of its mechanical role during whole cell dynamics is largely missing. Here we demonstrate that elastic phase fields can be used to describe dynamical cell processes in adhesive or confining environments in which the nucleus acts as a stiff inclusion in an elastic cytoplasm. We first introduce and verify our computational method and then study several prevalent cell-mechanical measurement methods. For cells on adhesive patterns, we find that nuclear stress is shielded by the adhesive pattern. For cell compression between two parallel plates, we obtain force-compression curves that allow us to extract an effective modulus for the cell-nucleus composite. For micropipette aspiration, the effect of the nucleus on the effective modulus is found to be much weaker, highlighting the complicated interplay between extracellular geometry and cell mechanics that is captured by our approach. We also show that our phase field approach can be used to investigate the effects of Kelvin-Voigt-type viscoelasticity and cortical tension.

Cell shape plays a fundamental role in many biological processes, including adhesion, migration, division and development, but it is not clear which shape model best predicts three-dimensional cell shape in structured environments. Here, we compare different modelling approaches with experimental data. The shapes of single mesenchymal cells cultured in custom-made 3D scaffolds were compared by a Fourier method with surfaces that minimize area under the given adhesion and volume constraints. For the minimized surface model, we found marked differences to the experimentally observed cell shapes, which necessitated the use of more advanced shape models. We used different variants of the cellular Potts model, which effectively includes both surface and bulk contributions. The simulations revealed that the Hamiltonian with linear area energy outperformed the elastic area constraint in accurately modelling the 3D shapes of cells in structured environments. Explicit modelling the nucleus did not improve the accuracy of the simulated cell shapes. Overall, our work identifies effective methods for accurately modelling cellular shapes in complex environments., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Link et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 β 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Fusion of biological membranes is fundamental in various physiological events. The fusion process involves several intermediate stages with energy barriers that are tightly dependent on the mechanical and physical properties of the system, one of which is membrane tension. As previously established, the late stages of fusion, including hemifusion diaphragm and pore expansions, are favored by membrane tension. However, a current understanding of how the energy barrier of earlier fusion stages is affected by membrane tension is lacking. Here, we apply a newly developed experimental approach combining micropipette-aspirated giant unilamellar vesicles and optically trapped membrane-coated beads, revealing that membrane tension inhibits lipid mixing. We show that lipid mixing is 6 times slower under a tension of 0.12 mN/m compared with tension-free membranes. Furthermore, using continuum elastic theory, we calculate the dependence of the hemifusion stalk formation energy on membrane tension and intermembrane distance and find the increase in the corresponding energy barrier to be 1.6 k B T in our setting, which can explain the increase in lipid mixing time delay. Finally, we show that tension can be a significant factor in the stalk energy if the pre-fusion intermembrane distance is on the order of several nanometers, while for membranes that are tightly docked, tension has a negligible effect.

Blood platelets are central elements of the blood clotting response after wounding. Upon vessel damage, they bind to the surrounding matrix and contract the forming thrombus, thus helping to restore normal blood circulation. The hemostatic function of platelets is directly connected to their mechanics and cytoskeletal organization. The reorganization of the platelet cytoskeleton during spreading occurs within minutes and leads to the formation of contractile actomyosin bundles, but it is not known if there is a direct correlation between the emerging actin structures and the force field that is exerted to the environment. In this study, we combine fluorescence imaging of the actin structures with simultaneous traction force measurements in a time-resolved manner. In addition, we image the final states with superresolution microscopy. We find that both the force fields and the cell shapes have clear geometrical patterns defined by stress fibers. Force generation is localized in a few hotspots, which appear early during spreading, and, in the mature state, anchor stress fibers in focal adhesions. Moreover, we show that, for a gel stiffness in the physiological range, force generation is a very robust mechanism and we observe no systematic dependence on the amount of added thrombin in solution or fibrinogen coverage on the substrate, suggesting that force generation after platelet activation is a threshold phenomenon that ensures reliable thrombus contraction in diverse environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: AR, DW, VM, MK, IW, PM, AM, JR, GC, GC, TB, US, MB No competing interests declared, (© 2023, Ruppel, Wörthmüller et al.)

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., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The fusion of lipid membranes progresses through a series of hemifusion intermediates with two significant energy barriers related to the formation of stalk and fusion pore, respectively. These energy barriers determine the speed and success rate of many critical biological processes, including the fusion of highly curved membranes, for example synaptic vesicles and enveloped viruses. Here we use continuum elastic theory of lipid monolayers to determine the relationship between membrane shape and energy barriers to fusion. We find that the stalk formation energy decreases with curvature by up to 31 k B T in a 20-nm-radius vesicle compared with planar membranes and by up to 8 k B T in the fusion of highly curved, long, tubular membranes. In contrast, the fusion pore formation energy barrier shows a more complicated behavior. Immediately after stalk expansion to the hemifusion diaphragm, the fusion pore formation energy barrier is low (15-25 k B T) due to lipid stretching in the distal monolayers and increased tension in highly curved vesicles. Therefore, the opening of the fusion pore is faster. However, these stresses relax over time due to lipid flip-flop from the proximal monolayer, resulting in a larger hemifusion diaphragm and a higher fusion pore formation energy barrier, up to 35 k B T. Therefore, if the fusion pore fails to open before significant lipid flip-flop takes place, the reaction proceeds to an extended hemifusion diaphragm state, which is a dead-end configuration in the fusion process and can be used to prevent viral infections. In contrast, in the fusion of long tubular compartments, the surface tension does not accumulate due to the formation of the diaphragm, and the energy barrier for pore expansion increases with curvature by up to 11 k B T. This suggests that inhibition of polymorphic virus infection could particularly target this feature of the second barrier., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane., (© 2023 Mund, Tschanz, et al.)

The Spindle Assembly Abnormal Protein 6 (SAS-6) forms dimers, which then self-assemble into rings that are critical for the nine-fold symmetry of the centriole organelle. It has recently been shown experimentally that the self-assembly of SAS-6 rings is strongly facilitated on a surface, shifting the reaction equilibrium by four orders of magnitude compared to the bulk. Moreover, a fraction of non-canonical symmetries (i.e., different from nine) was observed. In order to understand which aspects of the system are relevant to ensure efficient self-assembly and selection of the nine-fold symmetry, we have performed Brownian dynamics computer simulation with patchy particles and then compared our results with the experimental ones. Adsorption onto the surface was simulated by a grand canonical Monte Carlo procedure and random sequential adsorption kinetics. Furthermore, self-assembly was described by Langevin equations with hydrodynamic mobility matrices. We find that as long as the interaction energies are weak, the assembly kinetics can be described well by coagulation-fragmentation equations in the reaction-limited approximation. By contrast, larger interaction energies lead to kinetic trapping and diffusion-limited assembly. We find that the selection of nine-fold symmetry requires a small value for the angular interaction range. These predictions are confirmed by the experimentally observed reaction constant and angle fluctuations. Overall, our simulations suggest that the SAS-6 system works at the crossover between a relatively weak binding energy that avoids kinetic trapping and a small angular range that favors the nine-fold symmetry.

Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Computer simulations have become a widely used method for the field of mechanobiology. An important question is whether one can predict the shape and forces of cells as a function of the extracellular environment. Different types of models have been described before to simulate cell and tissue shapes in structured environments. In this chapter, we give a brief overview of commonly used models and then describe the Cellular Potts Model, a lattice-based modelling framework, in more detail. We provide a hands-on guide on how to build a model that simulates the shape of a single cell on a micropattern in three dimensions in different open source software packages using the Cellular Potts framework. A simulation is set up with an initial configuration of generalized cells that change shape and position due to an energy function that incorporates cellular volume and surface area constraints as well as interaction energies between the generalized cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: The authors have declared that no competing interests exist.

Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2 n progression, and each proliferative cycle produces a variable number of progeny. Here, by tracking nuclei and DNA replication, we show that individual nuclei replicate their DNA at different times, despite residing in a shared cytoplasm. Extrapolating from experimental data using mathematical modeling, we provide strong indication that a limiting factor exists, which slows down the nuclear multiplication rate. Consistent with this prediction, our data show that temporally overlapping DNA replication events were significantly slower than partially overlapping or nonoverlapping events. Our findings suggest the existence of evolutionary pressure that selects for asynchronous DNA replication, balancing available resources with rapid pathogen proliferation.

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II paralogs A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modeling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between paralogs A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-localizes with pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two paralogs form a balanced system for regulated force generation., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells., (© 2021. The Author(s).)

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., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

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

Essential cellular processes such as cell adhesion, migration and division strongly depend on mechanical forces. The standard method to measure cell forces is traction force microscopy (TFM) on soft elastic substrates with embedded marker beads. While in 2D TFM one only reconstructs tangential forces, in 2.5D TFM one also considers normal forces. Here we present a systematic comparison between two fundamentally different approaches to 2.5D TFM, which in particular require different methods to deal with noise in the displacement data. In the direct method, one calculates strain and stress tensors directly from the displacement data, which in principle requires a divergence correction. In the inverse method, one minimizes the difference between estimated and measured displacements, which requires some kind of regularization. By calculating the required Green's functions in Fourier space from Boussinesq-Cerruti potential functions, we first derive a new variant of 2.5D Fourier Transform Traction Cytometry (FTTC). To simulate realistic traction patterns, we make use of an analytical solution for Hertz-like adhesion patches. We find that FTTC works best if only tangential forces are reconstructed, that 2.5D FTTC is more precise for small noise, but that the performance of the direct method approaches the one of 2.5D FTTC for larger noise, before both fail for very large noise. Moreover we find that a divergence correction is not really needed for the direct method and that it profits more from increased resolution than the inverse method., Competing Interests: The authors have declared that no competing interests exist.

Immuno-electron microscopy can detect and localize antigens in cells or tissues at a resolution of several nanometers. In the case of P. falciparum-infected erythrocytes, immuno-EM studies are frequently hampered by the electron-dense nature of the hemoglobin and access of antibodies to antigenic sites, particularly if the targeted protein is presented on the host cell surface or lies in proximity to the host cell cytoskeleton. Here, we describe an improved immuno-EM protocol that overcomes these problems. The improved signal to noise ratio and the enhanced access to antigenic sites now allows one to obtain information regarding target density and distribution and, hence, additional insights into the architecture and function of parasite-induced, or -affected, structures., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Discovering mechanisms governing organelle assembly is a fundamental pursuit in biology. The centriole is an evolutionarily conserved organelle with a signature 9-fold symmetrical chiral arrangement of microtubules imparted onto the cilium it templates. The first structure in nascent centrioles is a cartwheel, which comprises stacked 9-fold symmetrical SAS-6 ring polymers emerging orthogonal to a surface surrounding each resident centriole. The mechanisms through which SAS-6 polymerization ensures centriole organelle architecture remain elusive. We deploy photothermally-actuated off-resonance tapping high-speed atomic force microscopy to decipher surface SAS-6 self-assembly mechanisms. We show that the surface shifts the reaction equilibrium by ~10 4 compared to solution. Moreover, coarse-grained molecular dynamics and atomic force microscopy reveal that the surface converts the inherent helical propensity of SAS-6 polymers into 9-fold rings with residual asymmetry, which may guide ring stacking and impart chiral features to centrioles and cilia. Overall, our work reveals fundamental design principles governing centriole assembly., (© 2021. The Author(s).)

In multicellular organisms, antiviral defense mechanisms evoke a reliable collective immune response despite the noisy nature of biochemical communication between tissue cells. A molecular hub of this response, the interferon I receptor (IFNAR), discriminates between ligand types by their affinity regardless of concentration. To understand how ligand type can be decoded robustly by a single receptor, we frame ligand discrimination as an information-theoretic problem and systematically compare the major classes of receptor architectures: allosteric, homodimerizing, and heterodimerizing. We demonstrate that asymmetric heterodimers achieve the best discrimination power over the entire physiological range of local ligand concentrations. This design enables sensing of ligand presence and type, and it buffers against moderate concentration fluctuations. In addition, receptor turnover, which drives the receptor system out of thermodynamic equilibrium, allows alignment of activation points for ligands of different affinities and thereby makes ligand discrimination practically independent of concentration. IFNAR exhibits this optimal architecture, and our findings thus suggest that this specialized receptor can robustly decode digital messages carried by its different ligands., Competing Interests: The authors declare no competing interest.

The pathology of Plasmodium falciparum malaria is largely defined by the cytoadhesion of infected erythrocytes to the microvascular endothelial lining. The complexity of the endothelial surface and the large range of interactions available for the infected erythrocyte via parasite-encoded adhesins make analysis of critical contributions during cytoadherence challenging to define. Here, we have explored supported membranes functionalized with two important adhesion receptors, ICAM1 or CD36, as a quantitative biomimetic surface to help understand the processes involved in cytoadherence. Parasitized erythrocytes bound to the receptor-functionalized membranes with high efficiency and selectivity under both static and flow conditions, with infected wild-type erythrocytes displaying a higher binding capacity than do parasitized heterozygous sickle cells. We further show that the binding efficiency decreased with increasing intermolecular receptor distance and that the cell-surface contacts were highly dynamic and increased with rising wall shear stress as the cell underwent a shape transition. Computer simulations using a deformable cell model explained the wall-shear-stress-induced dynamic changes in cell shape and contact area via the specific physical properties of erythrocytes, the density of adhesins presenting knobs, and the lateral movement of receptors in the supported membrane., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: KW, JG, MH, MF, MT, KS, US, MB No competing interests declared, (© 2021, Weißenbruch et al.)

The actin cytoskeleton of cells is in continuous motion due to both polymerization of new filaments and their contraction by myosin II molecular motors. Through adhesion to the substrate, such intracellular flow can be converted into cell migration. Recently, optogenetics has emerged as a new powerful experimental method to control both actin polymerization and myosin II contraction. While optogenetic control of polymerization can initiate cell migration by generating protrusion, it is less clear if and how optogenetic control of contraction can also affect cell migration. Here we analyze the latter situation using a minimal variant of active gel theory into which we include optogenetic activation as a spatiotemporally constrained perturbation. The model can describe the symmetrical flow of the actomyosin system observed in optogenetic experiments, but not the long-lasting polarization required for cell migration. Motile solutions become possible if cytoskeletal polymerization is included through the boundary conditions. Optogenetic activation of contraction can then initiate locomotion in a symmetrically spreading cell and strengthen motility in an asymmetrically polymerizing one. If designed appropriately, it can also arrest motility even for protrusive boundaries.

Centrosome positioning is essential for many processes in animal cells, in particular during development and migration. A new study using quantitative analysis of enucleated cells plated on adhesive micropatterns reveals that microtubules position the centrosome in the geometric center of the intracellular space defined by the actin cytoskeleton., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Clathrin-mediated endocytosis is the major pathway by which eukaryotic cells take up extracellular material, but it is still elusive which physical pathways are being taken during membrane invagination. From a continuum point of view, it can be driven by increases in coat stiffness, preferred curvature or line tension. Here we develop a comprehensive theoretical framework that can be solved analytically and that predicts the consequences of these different scenarios. We find that for the case of increasing stiffness or preferred curvature, curvature will be acquired gradually with growth, while for increasing line tension, the lattice must have grown to a certain size before a flat-to-curved transition can occur. At low membrane tension, the critical value for coat stiffness is 30 kBT, for preferred curvature it is 200 nm, and for line tension it is 6 pN. For high membrane tension, critical coat stiffness is 150 kBT and critical preferred curvature is 70 nm. In the mixed case when a coat with finite rigidity but increasing line tension is considered, a cup-to-sphere transition can occur for a line tension of 6 pN. The flat-to-curved and the cup-to-sphere transitions driven by line tension are both suppressed by high membrane tension.

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.

Recent experiments with super-resolution live cell microscopy revealed that nonmuscle myosin II minifilaments are much more dynamic than formerly appreciated, often showing plastic processes such as splitting, concatenation and stacking. Here we combine sequence information, electrostatics and elasticity theory to demonstrate that the parallel staggers at 14.3, 43.2 and 72 nm have a strong tendency to splay their heads away from the minifilament, thus potentially initiating the diverse processes seen in live cells. In contrast, the straight antiparallel stagger with an overlap of 43 nm is very stable and likely initiates minifilament nucleation. Using stochastic dynamics in a newly defined energy landscape, we predict that the optimal parallel staggers between the myosin rods are obtained by a trial-and-error process in which two rods attach and re-attach at different staggers by rolling and zipping motion. The experimentally observed staggers emerge as the configurations with the largest contact times. We find that contact times increase from isoforms C to B to A, that A-B-heterodimers are surprisingly stable and that myosin 18A should incorporate into mixed filaments with a small stagger. Our findings suggest that nonmuscle myosin II minifilaments in the cell are first formed by isoform A and then convert to mixed A-B-filaments, as observed experimentally., Competing Interests: The authors have declared that no competing interests exist.

Actin is the main protein used by biological cells to adapt their structure and mechanics to their needs. Cellular adaptation is made possible by molecular processes that strongly depend on mechanics. The actin cytoskeleton is also an active material that continuously consumes energy. This allows for dynamical processes that are possible only out of equilibrium and opens up the possibility for multiple layers of control that have evolved around this single protein.Here we discuss the actin cytoskeleton from the viewpoint of physics as an active adaptive material that can build structures superior to man-made soft matter systems. Not only can actin be used to build different network architectures on demand and in an adaptive manner, but it also exhibits the dynamical properties of feedback systems, like excitability, bistability, or oscillations. Therefore, it is a prime example of how biology couples physical structure and information flow and a role model for biology-inspired metamaterials.

Self-assembly and force generation are two central processes in biological systems that usually are considered in separation. However, the signals that activate nonmuscle myosin II molecular motors simultaneously lead to self-assembly into myosin II minifilaments as well as progression of the motor heads through the cross-bridge cycle. Here we investigate theoretically the possible effects of coupling these two processes. Our assembly model, which builds on a consensus architecture of the minifilament, predicts a critical aggregation concentration at which the assembly kinetics slows down dramatically. The combined model predicts that increasing actin filament concentration and force both lead to a decrease in the critical aggregation concentration. We suggest that due to these effects, myosin II minifilaments in a filamentous context might be in a critical state that reacts faster to varying conditions than in solution. We finally compare our model to experiments by simulating fluorescence recovery after photobleaching.

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.

Receptor-mediated endocytosis requires that the energy of adhesion overcomes the deformation energy of the plasma membrane. The resulting driving force is balanced by dissipative forces, leading to deterministic dynamical equations. While the shape of the free membrane does not play an important role for tensed and loose membranes, in the intermediate regime it leads to an important energy barrier. Here we show that this barrier is similar to but different from an effective line tension and suggest a simple analytical approximation for it. We then explore the rich dynamics of uptake for particles of different shapes and present the corresponding dynamical state diagrams. We also extend our model to include stochastic fluctuations, which facilitate uptake and lead to corresponding changes in the phase diagrams.

During intraerythrocytic development, the human malaria parasite Plasmodium falciparum alters the mechanical deformability of its host cell. The underpinning biological processes involve gain in parasite mass, changes in the membrane protein compositions, reorganization of the cytoskeletons and its coupling to the plasma membrane, and formation of membrane protrusions, termed knobs. The hemoglobinopathies S and C are known to partially protect carriers from severe malaria, possibly through additional changes in the erythrocyte biomechanics, but a detailed quantification of cell mechanics is still missing. Here, we combined flicker spectroscopy and a mathematical model and demonstrated that knob formation strongly suppresses membrane fluctuations by increasing membrane-cytoskeleton coupling. We found that the confinement increased with hemoglobin S but decreases with hemoglobin C in spite of comparable knob densities and diameters. We further found that the membrane bending modulus strongly depends on the hemoglobinopathetic variant, suggesting increased amounts of irreversibly oxidized hemichromes bound to membranes., Competing Interests: Competing interestsThe authors declare no competing interests.

Microtubules are cytoskeletal filaments essential for many cellular processes, including establishment and maintenance of polarity, intracellular transport, division and migration. In most metazoan cells, the number and length of microtubules are highly variable, while they can be precisely defined in some protozoan organisms. However, in either case the significance of these two key parameters for cells is not known. Here, we quantitatively studied the impact of modulating microtubule number and length in Plasmodium, the protozoan parasite causing malaria. Using a gene deletion and replacement strategy targeting one out of two α-tubulin genes, we show that chromosome segregation proceeds in the oocysts even in the absence of microtubules. However, fewer and shorter microtubules severely impaired the formation, motility and infectivity of Plasmodium sporozoites, the forms transmitted by the mosquito, which usually contain 16 microtubules. We found that α-tubulin expression levels directly determined the number of microtubules, suggesting a high nucleation barrier as supported by a mathematical model. Infectious sporozoites were only formed in parasite lines featuring at least 10 microtubules, while parasites with 9 or fewer microtubules failed to transmit., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Red blood cells in shear flow show a variety of different shapes due to the complex interplay between hydrodynamics and membrane elasticity. Malaria-infected red blood cells become generally adhesive and less deformable. Adhesion to a substrate leads to a reduction in shape variability and to a flipping motion of the non-spherical shapes during the mid-stage of infection. Here, we present a complete state diagram for wall adhesion of red blood cells in shear flow obtained by simulations, using a particle-based mesoscale hydrodynamics approach, multiparticle collision dynamics. We find that cell flipping at a substrate is replaced by crawling beyond a critical shear rate, which increases with both membrane stiffness and viscosity contrast between the cytosol and suspending medium. This change in cell dynamics resembles the transition between tumbling and tank-treading for red blood cells in free shear flow. In the context of malaria infections, the flipping-crawling transition would strongly increase the adhesive interactions with the vascular endothelium, but might be suppressed by the combined effect of increased elasticity and viscosity contrast.

Pathogens face varying microenvironments in vivo, but suitable experimental systems and analysis tools to dissect how three-dimensional (3D) tissue environments impact pathogen spread are lacking. Here we develop an Integrative method to Study Pathogen spread by Experiment and Computation within Tissue-like 3D cultures (INSPECT-3D), combining quantification of pathogen replication with imaging to study single-cell and cell population dynamics. We apply INSPECT-3D to analyze HIV-1 spread between primary human CD4 T-lymphocytes using collagen as tissue-like 3D-scaffold. Measurements of virus replication, infectivity, diffusion, cellular motility and interactions are combined by mathematical analyses into an integrated spatial infection model to estimate parameters governing HIV-1 spread. This reveals that environmental restrictions limit infection by cell-free virions but promote cell-associated HIV-1 transmission. Experimental validation identifies cell motility and density as essential determinants of efficacy and mode of HIV-1 spread in 3D. INSPECT-3D represents an adaptable method for quantitative time-resolved analyses of 3D pathogen spread.

PfEMP1 (erythrocyte membrane protein 1) adhesins play a pivotal role in the pathophysiology of falciparum malaria, by mediating sequestration of Plasmodium falciparum -infected erythrocytes in the microvasculature. PfEMP1 variants are expressed by var genes and are presented on membrane elevations, termed knobs. However, the organization of PfEMP1 on knobs is largely unclear. Here, we use super-resolution microscopy and genetically altered parasites expressing a modified var2cs a gene in which the coding sequence of the photoactivatable mEOS2 was inserted to determine the number and distribution of PfEMP1 on single knobs. The data were verified by quantitative fluorescence-activated cell sorting analysis and immuno-electron microscopy together with stereology methods. We show that knobs contain 3.3 ± 1.7 and 4.3 ± 2.5 PfEMP1 molecules, predominantly placed on the knob tip, in parasitized erythrocytes containing wild type and sickle haemoglobin, respectively. The ramifications of our findings for cytoadhesion and immune evasion are discussed., Competing Interests: The authors declare no competing interests.

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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The cellular uptake of nanoparticles or viruses requires that the gain of adhesion energy overcomes the cost of plasma membrane bending. It is well known that this leads to a minimal particle size for uptake. Using a simple deterministic theory for this process, we first show that, for the same radius and volume, cylindrical particles should be taken up faster than spherical particles, both for normal and parallel orientations. We then address stochastic effects, which are expected to be relevant due to small system size, and show that, now, spherical particles can have a faster uptake because the mean first passage time profits from the multiplicative noise induced by the spherical geometry. We conclude that stochastic effects are strongly geometry dependent and may favor spherical shapes during adhesion-driven particle uptake.

Sickle cell trait, a common hereditary blood disorder, protects carriers from severe disease in infections with the human malaria parasite Plasmodium falciparum . Protection is associated with a reduced capacity of parasitized erythrocytes to cytoadhere to the microvascular endothelium and cause vaso-occlusive events. However, the underpinning cellular and biomechanical processes are only partly understood and the impact on endothelial cell activation is unclear. Here, we show, by combining quantitative flow chamber experiments with multiscale computer simulations of deformable cells in hydrodynamic flow, that parasitized erythrocytes containing the sickle cell haemoglobin displayed altered adhesion dynamics, resulting in restricted contact footprints on the endothelium. Main determinants were cell shape, knob density and membrane bending. As a consequence, the extent of endothelial cell activation was decreased. Our findings provide a quantitative understanding of how the sickle cell trait affects the dynamic cytoadhesion behavior of parasitized erythrocytes and, in turn, endothelial cell activation., Competing Interests: The authors declare no competing interests.

Cytoplasmic flows are an ubiquitous feature of biological systems, in particular in large cells, such as oocytes and eggs in early animal development. Here we show that cytoplasmic flows in starfish oocytes, which can be imaged well with transmission light microscopy, are fully determined by the cortical dynamics during surface contraction waves. We first show that the dynamics of the oocyte surface is highly symmetric around the animal-vegetal axis. We then mathematically solve the Stokes equation for flows inside a deforming sphere using the measured surface displacements as boundary conditions. Our theoretical predictions agree very well with the intracellular flows quantified by particle image velocimetry, proving that during this stage the starfish cytoplasm behaves as a simple Newtonian fluid on the micrometer scale. We calculate the pressure field inside the oocyte and find that its gradient is too small as to explain polar body extrusion, in contrast to earlier suggestions. Myosin II inhibition by blebbistatin confirms this conclusion, because it diminishes cell shape changes and hydrodynamic flow, but does not abolish polar body formation., Competing Interests: The authors have declared that no competing interests exist.