Condensate interfacial forces reposition DNA loci and probe chromatin viscoelasticity.

Biomolecular condensates assemble in living cells through phase separation and related phase transitions. An underappreciated feature of these dynamic molecular assemblies is that they form interfaces with other cellular structures, including membranes, cytoskeleton, DNA and RNA, and other membraneless compartments. These interfaces are expected to give rise to capillary forces, but there are few ways of quantifying and harnessing these forces in living cells. Here, we introduce viscoelastic chromatin tethering and organization (VECTOR), which uses light-inducible biomolecular condensates to generate capillary forces at targeted DNA loci. VECTOR can be utilized to programmably reposition genomic loci on a timescale of seconds to minutes, quantitatively revealing local heterogeneity in the viscoelastic material properties of chromatin. These synthetic condensates are built from components that naturally form liquid-like structures in living cells, highlighting the potential role for native condensates to generate forces and do work to reorganize the genome and impact chromatin architecture.

Hepatic Spheroid Formation on Carbohydrate-Functionalized Supramolecular Hydrogels.

Two synthetic supramolecular hydrogels, formed from bis-urea amphiphiles containing lactobionic acid (LBA) and maltobionic acid (MBA) bioactive ligands, are applied as cell culture matrices in vitro. Their fibrillary and dynamic nature mimics essential features of the extracellular matrix (ECM). The carbohydrate amphiphiles self-assemble into long supramolecular fibers in water, and hydrogels are formed by physical entanglement of fibers through bundling. Gels of both amphiphiles exhibit good self-healing behavior, but remarkably different stiffnesses. They display excellent bioactive properties in hepatic cell cultures. Both carbohydrate ligands used are proposed to bind to asialoglycoprotein receptors (ASGPRs) in hepatic cells, thus inducing spheroid formation when seeding hepatic HepG2 cells on both supramolecular hydrogels. Ligand nature, ligand density, and hydrogel stiffness influence cell migration and spheroid size and number. The results illustrate the potential of self-assembled, carbohydrate-functionalized hydrogels as matrices for liver tissue engineering.

Transient response and domain formation in electrically deforming liquid crystal networks.

Recently, three distinct, well-separated transient regimes were discovered in the dynamics of the volume expansion of shape-shifting liquid crystal network films in response to the switching on of an alternating electric field [Van der Kooij et al., Nat. Commun., 2019, 10, 1]. Employing a spatially resolved, time-dependent Landau theory that couples local volume generation to the degree of orientational order of mesogens that are part of a viscoelastic network, we are able to offer a physical explanation for the existence of three time scales. We find that the initial response is dominated by overcoming the impact of thermal noise, after which the top of the film expands, followed by a permeation of this response into the bulk region. An important signature of our predictions is a significant dependence of the three time scales on the film thickness, where we observe a clear thin-film-to-bulk transition. The point of transition coincides with the emergence of spatial inhomogeneities in the bulk of the film in the form of domains separated by regions of suppressed expansion. This ultimately gives rise to variations in the steady-state overall expansion of the film and may lead to uncontrolled patterning. According to our model, domain formation can be suppressed by (1) decreasing the thickness of the as-prepared film, (2) increasing the linear dimensions of the mesogens, or (3) their degree of orientational order when cross-linked into the network. Our findings provide a handle to achieve finer control over the actuation of smart liquid crystal network coatings.

Enhanced persistence and collective migration in cooperatively aligning cell clusters.

Most cells possess the capacity to locomote. Alone or collectively, this allows them to adapt, to rearrange, and to explore their surroundings. The biophysical characterization of such motile processes, in health and in disease, has so far focused mostly on two limiting cases: single-cell motility on the one hand and the dynamics of confluent tissues such as the epithelium on the other. The in-between regime of clusters, composed of relatively few cells moving as a coherent unit, has received less attention. Such small clusters are, however, deeply relevant in development but also in cancer metastasis. In this work, we use cellular Potts models and analytical active matter theory to understand how the motility of small cell clusters changes with N, the number of cells in the cluster. Modeling and theory reveal our two main findings: cluster persistence time increases with N, whereas the intrinsic diffusivity decreases with N. We discuss a number of settings in which the motile properties of more complex clusters can be analytically understood, revealing that the focusing effects of small-scale cooperation and cell-cell alignment can overcome the increased bulkiness and internal disorder of multicellular clusters to enhance overall migrational efficacy. We demonstrate this enhancement for small-cluster collective durotaxis, which is shown to proceed more effectively than for single cells. Our results may provide some novel, to our knowledge, insights into the connection between single-cell and large-scale collective motion and may point the way to the biophysical origins of the enhanced metastatic potential of small tumor cell clusters.

Combined Force-Torque Spectroscopy of Proteins by Means of Multiscale Molecular Simulation.

Assessing the structural properties of large proteins is important to gain an understanding of their function in, e.g., biological systems or biomedical applications. We propose a method to examine the mechanical properties of proteins subject to applied forces by means of multiscale simulation. Both stretching and torsional forces are considered, and these may be applied independently of each other. As a proof of principle, we apply torsional forces to a coarse-grained continuum model of the antibody protein immunoglobulin G using fluctuating finite element analysis and use it to identify the area of strongest deformation. This region is essential to the torsional properties of the molecule as a whole because it represents the softest, most deformable domain. Zooming in, this part of the molecule is subjected to torques and stretching forces using molecular dynamics simulations on an atomistically resolved level to investigate its torsional properties. We calculate the torsional resistance as a function of the rotation of the domain while subjecting it to various stretching forces. From this, we assess how the measured twist-torque profiles develop with increasing stretching force and show that they exhibit torsion stiffening, in qualitative agreement with experimental findings. We argue that combining the twist-torque profiles for various stretching forces effectively results in a combined force-torque spectroscopy analysis, which may serve as a mechanical signature for a biological macromolecule.

Mimicking Active Biopolymer Networks with a Synthetic Hydrogel.

Stiffening due to internal stress generation is of paramount importance in living systems and is the foundation for many biomechanical processes. For example, cells stiffen their surrounding matrix by pulling on collagen and fibrin fibers. At the subcellular level, molecular motors prompt fluidization and actively stiffen the cytoskeleton by sliding polar actin filaments in opposite directions. Here, we demonstrate that chemical cross-linking of a fibrous matrix of synthetic semiflexible polymers with thermoresponsive poly( N-isopropylacrylamide) (PNIPAM) produces internal stress by induction of a coil-to-globule transition upon crossing the lower critical solution temperature of PNIPAM, resulting in a macroscopic stiffening response that spans more than 3 orders of magnitude in modulus. The forces generated through collapsing PNIPAM are sufficient to drive a fluid material into a stiff gel within a few seconds. Moreover, rigidified networks dramatically stiffen in response to applied shear stress featuring power law rheology with exponents that match those of reconstituted collagen and actomyosin networks prestressed by molecular motors. This concept holds potential for the rational design of synthetic materials that are fluid at room temperature and rapidly rigidify at body temperature to form hydrogels mechanically and structurally akin to cells and tissues.

Nonaffinity and Fluid-Coupled Viscoelastic Plateau for Immersed Fiber Networks.

We employ a matrix-based solver for the linear rheology of fluid-immersed disordered spring networks to reveal four distinct dynamic response regimes. One regime-completely absent in the known vacuum response-exhibits coupled fluid flow and network deformation, with both components responding nonaffinely. This regime contains an additional plateau (peak) in the frequency-dependent storage (loss) modulus-features that vanish without full hydrodynamic interactions. The mechanical response of immersed networks such as biopolymers and hydrogels is thus richer than previously established and offers additional modalities for design and control through fluid interactions.

Harnessing entropy to enhance toughness in reversibly crosslinked polymer networks.

Reversible crosslinking is a design paradigm for polymeric materials, wherein they are microscopically reinforced with chemical species that form transient crosslinks between the polymer chains. Besides the potential for self-healing, recent experimental work suggests that freely diffusing reversible crosslinks in polymer networks, such as gels, can enhance the toughness of the material without substantial change in elasticity. This presents the opportunity for making highly elastic materials that can be strained to a large extent before rupturing. Here, we employ Gaussian chain theory, molecular simulation, and polymer self-consistent field theory for networks to construct an equilibrium picture for how reversible crosslinks can toughen a polymer network without affecting its elasticity. Maximisation of polymer entropy drives the reversible crosslinks to bind preferentially near the permanent crosslinks in the network, leading to local molecular reinforcement without significant alteration of the network topology. In equilibrium conditions, permanent crosslinks share effectively the load with neighbouring reversible crosslinks, forming multi-functional crosslink points. The network is thereby globally toughened, while the linear elasticity is left largely unaltered. Practical guidelines are proposed to optimise this design in experiment, along with a discussion of key kinetic and timescale considerations.

Strain-Stiffening in Dynamic Supramolecular Fiber Networks.

The cytoskeleton is a highly adaptive network of filamentous proteins capable of stiffening under stress even as it dynamically assembles and disassembles with time constants of minutes. Synthetic materials that combine reversibility and strain-stiffening properties remain elusive. Here, strain-stiffening hydrogels that have dynamic fibrous polymers as their main structural components are reported. The fibers form via self-assembly of bolaamphiphiles (BA) in water and have a well-defined cross-section of 9 to 10 molecules. Fiber length recovery after sonication, H/D exchange experiments, and rheology confirm the dynamic nature of the fibers. Cross-linking of the fibers yields strain-stiffening, self-healing hydrogels that closely mimic the mechanics of biological networks, with mechanical properties that can be modulated by chemical modification of the components. Comparison of the supramolecular networks with covalently fixated networks shows that the noncovalent nature of the fibers limits the maximum stress that fibers can bear and, hence, limits the range of stiffening.

Erratum: Persistence-Driven Durotaxis: Generic, Directed Motility in Rigidity Gradients [Phys. Rev. Lett. 118, 078103 (2017)].

This corrects the article DOI: 10.1103/PhysRevLett.118.078103.

Probing the Interplay between Dendritic Spine Morphology and Membrane-Bound Diffusion.

Dendritic spines are protrusions along neuronal dendrites that harbor the majority of excitatory postsynapses. Their distinct morphology, often featuring a bulbous head and small neck that connects to the dendritic shaft, has been shown to facilitate compartmentalization of electrical and cytoplasmic signaling stimuli elicited at the synapse. The extent to which spine morphology also forms a barrier for membrane-bound diffusion has remained unclear. Recent simulations suggested that especially the diameter of the spine neck plays a limiting role in this process. Here, we examine the connection between spine morphology and membrane-bound diffusion through a combination of photoconversion, live-cell superresolution experiments, and numerical simulations. Local photoconversion was used to obtain the timescale of diffusive equilibration in spines and followed by global sparse photoconversion to determine spine morphologies with nanoscopic resolution. These morphologies were subsequently used to assess the role of morphology on the diffusive equilibration. From the simulations, we could determine a robust relation between the equilibration timescale and a generalized shape factor calculated using both spine neck width and neck length, as well as spine head size. Experimentally, we found that diffusive equilibration was often slower, but rarely faster than predicted from the simulations, indicating that other biological confounders further reduce membrane-bound diffusion in these spines. This shape-dependent membrane-bound diffusion in mature spines may contribute to spine-specific compartmentalization of neurotransmitter receptors and signaling molecules and thereby support long-term plasticity of synaptic contacts.

Persistence-Driven Durotaxis: Generic, Directed Motility in Rigidity Gradients.

Cells move differently on substrates with different rigidities: the persistence time of their motion is higher on stiffer substrates. We show that this behavior-in and of itself-results in a net flux of cells directed up a soft-to-stiff gradient. Using simple random walk models with varying persistence and stochastic simulations, we characterize the propensity to move in terms of the durotactic index also measured in experiments. A one-dimensional model captures the essential features and highlights the competition between diffusive spreading and linear, wavelike propagation. Persistence-driven durokinesis is generic and may be of use in the design of instructive environments for cells and other motile, mechanosensitive objects.

Strain Stiffening Hydrogels through Self-Assembly and Covalent Fixation of Semi-Flexible Fibers.

Biomimetic, strain-stiffening materials are reported, made through self-assembly and covalent fixation of small building blocks to form fibrous hydrogels that are able to stiffen by an order of magnitude in response to applied stress. The gels consist of semi-flexible rodlike micelles of bisurea bolaamphiphiles with oligo(ethylene oxide) (EO) outer blocks and a polydiacetylene (PDA) backbone. The micelles are fibers, composed of 9-10 ribbons. A gelation method based on Cu-catalyzed azide-alkyne cycloaddition (CuAAC), was developed and shown to lead to strain-stiffening hydrogels with unusual, yet universal, linear and nonlinear stress-strain response. Upon gelation, the X-ray scattering profile is unchanged, suggesting that crosslinks are formed at random positions along the fiber contour without fiber bundling. The work expands current knowledge about the design principles and chemistries needed to achieve fully synthetic, biomimetic soft matter with on-demand, targeted mechanical properties.

Single-Bond Association Kinetics Determined by Tethered Particle Motion: Concept and Simulations.

Tethered particle motion (TPM), the motion of a micro- or nanoparticle tethered to a substrate by a macromolecule, is a system that has proven to be extremely useful for its ability to reveal physical features of the tether, because the thermal motion of the bound particle reports sensitively on parameters like the length, the rigidity, or the folding state of its tether. In this article, we survey the applicability of TPM to probe the kinetics of single secondary bonds, bonds that form and break between the tethered particle and a substrate due, for instance, to receptor/ligand pairs on particle and substrate. Much like the tether itself affects the motion pattern, so do the presence and absence of such secondary connections. Keeping the tether properties constant, we demonstrate how raw positional TPM data may be parsed to generate detailed insights into the association and dissociation kinetics of single secondary bonds. We do this using coarse-grained molecular dynamics simulations specifically developed to treat the motion of particles close to interfaces.

Confinement without boundaries: anisotropic diffusion on the surface of a cylinder.

Densely packed systems of thermal particles in curved geometries are frequently encountered in biological and microfluidic systems. In 2D systems, at sufficiently high surface coverage, diffusive motion is widely known to be strongly affected by physical confinement, e.g., by the walls. In this work, we explore the effects of confinement by shape, not rigid boundaries, on the diffusion of discs by confining them to the surface of a cylinder. We find that both the magnitude and the directionality of lateral diffusion is strongly influenced by the radius of the cylinder. An anisotropy between diffusion in the longitudinal and circumferential direction of the cylinder develops. We demonstrate that the origin of this effect lies in the fact that screw-like packings of mono- and oligodisperse discs on the surface of a cylinder induce preferential collective motions in the circumferential direction, but also show that even in polydisperse systems lacking such order an intrinsic finite size confinement effect increases diffusivity in the circumferential direction.

The nanoscale architecture of force-bearing focal adhesions.

The combination of micropillar array technology to measure cellular traction forces with super-resolution imaging allowed us to obtain cellular traction force maps and simultaneously zoom in on individual focal adhesions with single-molecule accuracy. We achieved a force detection precision of 500 pN simultaneously with a mean single-molecule localization precision of 30 nm. Key to the achievement was a two-step etching process that provided an integrated spacer next to the micropillar array that permitted stable and reproducible observation of cells on micropillars within the short working distance of a high-magnification, high numerical aperture objective. In turn, we used the technology to characterize the super-resolved structure of focal adhesions during force exertion. Live-cell imaging on MCF-7 cells demonstrated the applicability of the inverted configuration of the micropillar arrays to dynamics measurements. Forces emanated from a molecular base that was localized on top of the micropillars. What appeared as a single adhesion in conventional microscopy were in fact multiple elongated adhesions emanating from only a small fraction of the adhesion on the micropillar surface. Focal adhesions were elongated in the direction of local cellular force exertion with structural features of 100-280 nm in 3T3 Fibroblasts and MCF-7 cells. The combined measure of nanoscale architecture and force exerted shows a high level of stress accumulation at a single site of adhesion.

Contractile fibers and catch-bond clusters: a biological force sensor?

Catch bonds are cellular receptor-ligand pairs whose lifetime, counterintuitively, increases with increasing load. Although their existence was initially pure theoretical speculation, recent years have seen several experimental demonstrations of catch-bond behavior in biologically relevant and functional protein-protein bonds. Particularly notable among these established catch-bond formers is the integrin α5ß1, the primary receptor for fibronectin and, as such, a crucial determinant for the characteristics of the mechanical coupling between cell and matrix. In this work, we explore the implications of single catch-bond characteristics for the behavior of a load-sharing cluster of such bonds: These clusters are shown to possess a regime of strengthening with increasing applied force, similar to the manner in which focal adhesions become selectively reinforced. Our results may shed new light on the fundamental processes that allow cells to sense and respond to the mechanical properties of their environment and in particular show how single focal adhesions may act, autonomously, as local rigidity sensors.

Shape-induced asymmetric diffusion in dendritic spines allows efficient synaptic AMPA receptor trapping.

Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.

Mapping Antibody Domain Exposure on Nanoparticle Surfaces Using DNA-PAINT.

Decorating nanoparticles with antibodies (Ab) is a key strategy for targeted drug delivery and imaging. For this purpose, the orientation of the antibody on the nanoparticle is crucial to maximize fragment antibody-binding (Fab) exposure and thus antigen binding. Moreover, the exposure of the fragment crystallizable (Fc) domain may lead to the engagement of immune cells through one of the Fc receptors. Therefore, the choice of the chemistry for nanoparticle-antibody conjugation is key for the biological performance, and methods have been developed for orientation-selective functionalization. Despite the importance of this issue, there is a lack of direct methods to quantify the antibodies' orientation on the nanoparticle's surface. Here, we present a generic methodology that enables for multiplexed, simultaneous imaging of both Fab and Fc exposure on the surface of nanoparticles, based on super-resolution microscopy. Fab-specific Protein M and Fc-specific Protein G probes were conjugated to single stranded DNAs and two-color DNA-PAINT imaging was performed. Hereby, we quantitatively addressed the number of sites per particle and highlight the heterogeneity in the Ab orientation and compared the results with a geometrical computational model to validate data interpretation. Moreover, super-resolution microscopy can resolve particle size, allowing the study of how particle dimensions affect antibody coverage. We show that different conjugation strategies modulate the Fab and Fc exposure which can be tuned depending on the application of choice. Finally, we explored the biomedical importance of antibody domain exposure in antibody dependent cell mediated phagocytosis (ADCP). This method can be used universally to characterize antibody-conjugated nanoparticles, improving the understanding of relationships between structure and targeting capacities in targeted nanomedicine.

A Single-Molecule View at Nanoparticle Targeting Selectivity: Correlating Ligand Functionality and Cell Receptor Density.

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.