Elastocapillary effects determine early matrix deformation by glioblastoma cell spheroids.

During cancer pathogenesis, cell-generated mechanical stresses lead to dramatic alterations in the mechanical and organizational properties of the extracellular matrix (ECM). To date, contraction of the ECM is largely attributed to local mechanical stresses generated during cell invasion, but the impact of "elastocapillary" effects from surface tension on the tumor periphery has not been examined. Here, we embed glioblastoma cell spheroids within collagen gels, as a model of tumors within the ECM. We then modulate the surface tension of the spheroids, such that the spheroid contracts or expands. Surprisingly, in both cases, at the far-field, the ECM is contracted toward the spheroids prior to cellular migration from the spheroid into the ECM. Through computational simulation, we demonstrate that contraction of the ECM arises from a balance of spheroid surface tension, cell-ECM interactions, and time-dependent, poroelastic effects of the gel. This leads to the accumulation of ECM near the periphery of the spheroid and the contraction of the ECM without regard to the expansion or contraction of the spheroid. These results highlight the role of tissue-level surface stresses and fluid flow within the ECM in the regulation of cell-ECM interactions.

Growth-induced collective bending and kinetic trapping of cytoskeletal filaments.

Growth and turnover of actin filaments play a crucial role in the construction and maintenance of actin networks within cells. Actin filament growth occurs within limited space and finite subunit resources in the actin cortex. To understand how filament growth shapes the emergent architecture of actin networks, we developed a minimal agent-based model coupling filament mechanics and growth in a limiting subunit pool. We find that rapid filament growth induces kinetic trapping of highly bent actin filaments. Such collective bending patterns are long-lived, organized around nematic defects, and arise from competition between filament polymerization and bending elasticity. The stability of nematic defects and the extent of kinetic trapping are amplified by an increase in the abundance of the actin pool and by crosslinking the network. These findings suggest that kinetic trapping is a robust consequence of growth in crowded environments, providing a route to program shape memory in actin networks.

F-actin architecture determines the conversion of chemical energy into mechanical work.

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

Cofilin-mediated actin filament network flexibility facilitates 2D to 3D actomyosin shape change.

The organization of actin filaments (F-actin) into crosslinked networks determines the transmission of mechanical stresses within the cytoskeleton and subsequent changes in cell and tissue shape. Principally mediated by proteins such as α-actinin, F-actin crosslinking increases both network connectivity and rigidity, thereby facilitating stress transmission at low crosslinking yet attenuating transmission at high crosslinker concentration. Here, we engineer a two-dimensional model of the actomyosin cytoskeleton, in which myosin-induced mechanical stresses are controlled by light. We alter the extent of F-actin crosslinking by the introduction of oligomerized cofilin. At pH 6.5, F-actin severing by cofilin is weak, but cofilin bundles and crosslinks filaments. Given its effect of lowering the F-actin bending stiffness, cofilin- crosslinked networks are significantly more flexible and softer in bending than networks crosslinked by α-actinin. Thus, upon local activation of myosin-induced contractile stress, the network bends out-of-plane in contrast to the in-plane compression as observed with networks crosslinked by α-actinin. Here, we demonstrate that local effects on filament mechanics by cofilin introduces novel large-scale network material properties that enable the sculpting of complex shapes in the cell cytoskeleton.

Growth-induced collective bending and kinetic trapping of cytoskeletal filaments.

Growth and turnover of actin filaments play a crucial role in the construction and maintenance of actin networks within cells. Actin filament growth occurs within limited space and finite subunit resources in the actin cortex. To understand how filament growth shapes the emergent architecture of actin networks, we developed a minimal agent-based model coupling filament mechanics and growth in a limiting subunit pool. We find that rapid filament growth induces kinetic trapping of highly bent actin filaments. Such collective bending patterns are long-lived, organized around nematic defects, and arises from competition between filament polymerization and bending elasticity. The stability of nematic defects and the extent of kinetic trapping are amplified by an increase in the abundance of the actin pool and by crosslinking the network. These findings suggest that kinetic trapping is a robust consequence of growth in crowded environments, providing a route to program shape memory in actin networks.

Intracellular tension sensor reveals mechanical anisotropy of the actin cytoskeleton.

The filamentous actin (F-actin) cytoskeleton is a composite material consisting of cortical actin and bundled F-actin stress fibers, which together mediate the mechanical behaviors of the cell, from cell division to cell migration. However, as mechanical forces are typically measured upon transmission to the extracellular matrix, the internal distribution of forces within the cytoskeleton is unknown. Likewise, how distinct F-actin architectures contribute to the generation and transmission of mechanical forces is unclear. Therefore, we have developed a molecular tension sensor that embeds into the F-actin cytoskeleton. Using this sensor, we measure tension within stress fibers and cortical actin, as the cell is subject to uniaxial stretch. We find that the mechanical response, as measured by FRET, depends on the direction of applied stretch relative to the cell's axis of alignment. When the cell is aligned parallel to the direction of the stretch, stress fibers and cortical actin both accumulate tension. By contrast, when aligned perpendicular to the direction of stretch, stress fibers relax tension while the cortex accumulates tension, indicating mechanical anisotropy within the cytoskeleton. We further show that myosin inhibition regulates this anisotropy. Thus, the mechanical anisotropy of the cell and the coordination between distinct F-actin architectures vary and depend upon applied load.

SPAK-dependent cotransporter activity mediates capillary adhesion and pressure during glioblastoma migration in confined spaces.

The invasive potential of glioblastoma cells is attributed to large changes in pressure and volume, driven by diverse elements, including the cytoskeleton and ion cotransporters.  However, how the cell actuates changes in pressure and volume in confinement, and how these changes contribute to invasive motion is unclear. Here, we inhibited SPAK activity, with known impacts on the cytoskeleton and cotransporter activity and explored its role on the migration of glioblastoma cells in confining microchannels to model invasive spread through brain tissue. First, we found that confinement altered cell shape, inducing a transition in morphology that resembled droplet interactions with a capillary vessel, from "wetting" (more adherent) at low confinement, to "nonwetting" (less adherent) at high confinement. This transition was marked by a change from negative to positive pressure by the cells to the confining walls, and an increase in migration speed. Second, we found that the SPAK pathway impacted the migration speed in different ways dependent upon the extent of wetting. For nonwetting cells, SPAK inhibition increased cell-surface tension and cotransporter activity. By contrast, for wetting cells, it also reduced myosin II and YAP phosphorylation. In both cases, membrane-to-cortex attachment is dramatically reduced. Thus, our results suggest that SPAK inhibition differentially coordinates cotransporter and cytoskeleton-induced forces, to impact glioblastoma migration depending on the extent of confinement.

Membrane tension induces F-actin reorganization and flow in a biomimetic model cortex.

The accumulation and transmission of mechanical stresses in the cell cortex and membrane determines the mechanics of cell shape and coordinates essential physical behaviors, from cell polarization to cell migration. However, the extent that the membrane and cytoskeleton each contribute to the transmission of mechanical stresses to coordinate diverse behaviors is unclear. Here, we reconstitute a minimal model of the actomyosin cortex within liposomes that adheres, spreads and ultimately ruptures on a surface. During spreading, accumulated adhesion-induced (passive) stresses within the membrane drive changes in the spatial assembly of actin. By contrast, during rupture, accumulated myosin-induced (active) stresses within the cortex determine the rate of pore opening. Thus, in the same system, devoid of biochemical regulation, the membrane and cortex can each play a passive or active role in the generation and transmission of mechanical stress, and their relative roles drive diverse biomimetic physical behaviors.

Dysregulation of TSP2-Rac1-WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction.

Fibroblasts are a major cell population that perform critical functions in the wound healing process. In response to injury, they proliferate and migrate into the wound space, engaging in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were employed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived matrix (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblasts. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were due, in part, to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the TSP2-Rac1-WAVE2-actin axis, and describing a new function of TSP2 in regulating cytoskeleton organization.

F-actin architecture determines constraints on myosin thick filament motion.

Active stresses are generated and transmitted throughout diverse F-actin architectures within the cell cytoskeleton, and drive essential behaviors of the cell, from cell division to migration. However, while the impact of F-actin architecture on the transmission of stress is well studied, the role of architecture on the ab initio generation of stresses remains less understood. Here, we assemble F-actin networks in vitro, whose architectures are varied from branched to bundled through F-actin nucleation via Arp2/3 and the formin mDia1. Within these architectures, we track the motions of embedded myosin thick filaments and connect them to the extent of F-actin network deformation. While mDia1-nucleated networks facilitate the accumulation of stress and drive contractility through enhanced actomyosin sliding, branched networks prevent stress accumulation through the inhibited processivity of thick filaments. The reduction in processivity is due to a decrease in translational and rotational motions constrained by the local density and geometry of F-actin.

Interplay between substrate rigidity and tissue fluidity regulates cell monolayer spreading.

Coordinated and cooperative motion of cells is essential for embryonic development, tissue morphogenesis, wound healing and cancer invasion. A predictive understanding of the emergent mechanical behaviors in collective cell motion is challenging due to the complex interplay between cell-cell interactions, cell-matrix adhesions and active cell behaviors. To overcome this challenge, we develop a predictive cellular vertex model that can delineate the relative roles of substrate rigidity, tissue mechanics and active cell properties on the movement of cell collectives. We apply the model to the specific case of collective motion in cell aggregates as they spread into a two-dimensional cell monolayer adherent to a soft elastic matrix. Consistent with recent experiments, we find that substrate stiffness regulates the driving forces for the spreading of cellular monolayer, which can be pressure-driven or crawling-based depending on substrate rigidity. On soft substrates, cell monolayer spreading is driven by an active pressure due to the influx of cells coming from the aggregate, whereas on stiff substrates, cell spreading is driven primarily by active crawling forces. Our model predicts that cooperation of cell crawling and tissue pressure drives faster spreading, while the spreading rate is sensitive to the mechanical properties of the tissue. We find that solid tissues spread faster on stiff substrates, with spreading rate increasing with tissue tension. By contrast, the spreading of fluid tissues is independent of substrate stiffness and is slower than solid tissues. We compare our theoretical results with experimental results on traction force generation and spreading kinetics of cell monolayers, and provide new predictions on the role of tissue fluidity and substrate rigidity on collective cell motion.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles.

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

A molecular clock controls periodically driven cell migration in confined spaces.

Navigation through a dense, physically confining extracellular matrix is common in invasive cell spread and tissue reorganization but is still poorly understood. Here, we show that this migration is mediated by cyclic changes in the activity of a small GTPase RhoA, which is dependent on the oscillatory changes in the activity and abundance of the RhoA guanine nucleotide exchange factor, GEF-H1, and triggered by a persistent increase in the intracellular Ca2+ levels. We show that the molecular clock driving these cyclic changes is mediated by two coupled negative feedback loops, dependent on the microtubule dynamics, with a frequency that can be experimentally modulated based on a predictive mathematical model. We further demonstrate that an increasing frequency of the clock translates into a faster cell migration within physically confining spaces. This work lays the foundation for a better understanding of the molecular mechanisms dynamically driving cell migration in complex environments.

Correction: The structural, vibrational, and mechanical properties of jammed packings of deformable particles in three dimensions.

Correction for 'The structural, vibrational, and mechanical properties of jammed packings of deformable particles in three dimensions' by Dong Wang et al., Soft Matter, 2021, 17, 9901-9915, DOI: 10.1039/D1SM01228B.

The structural, vibrational, and mechanical properties of jammed packings of deformable particles in three dimensions.

We investigate the structural, vibrational, and mechanical properties of jammed packings of deformable particles with shape degrees of freedom in three dimensions (3D). Each 3D deformable particle is modeled as a surface-triangulated polyhedron, with spherical vertices whose positions are determined by a shape-energy function with terms that constrain the particle surface area, volume, and curvature, and prevent interparticle overlap. We show that jammed packings of deformable particles without bending energy possess low-frequency, quartic vibrational modes, whose number decreases with increasing asphericity and matches the number of missing contacts relative to the isostatic value. In contrast, jammed packings of deformable particles with non-zero bending energy are isostatic in 3D, with no quartic modes. We find that the contributions to the eigenmodes of the dynamical matrix from the shape degrees of freedom are significant over the full range of frequency and shape parameters for particles with zero bending energy. We further show that the ensemble-averaged shear modulus 〈G〉 scales with pressure P as 〈G〉 ∼ Pß, with ß ≈ 0.75 for jammed packings of deformable particles with zero bending energy. In contrast, ß ≈ 0.5 for packings of deformable particles with non-zero bending energy, which matches the value for jammed packings of soft, spherical particles with fixed shape. These studies underscore the importance of incorporating particle deformability and shape change when modeling the properties of jammed soft materials.

Detailed Balance Broken by Catch Bond Kinetics Enables Mechanical-Adaptation in Active Materials.

Unlike nearly all engineered materials which contain bonds that weaken under load, biological materials contain "catch" bonds which are reinforced under load. Consequently, materials, such as the cell cytoskeleton, can adapt their mechanical properties in response to their state of internal, non-equilibrium (active) stress. However, how large-scale material properties vary with the distance from equilibrium is unknown, as are the relative roles of active stress and binding kinetics in establishing this distance. Through course-grained molecular dynamics simulations, the effect of breaking of detailed balance by catch bonds on the accumulation and dissipation of energy within a model of the actomyosin cytoskeleton is explored. It is found that the extent to which detailed balance is broken uniquely determines a large-scale fluid-solid transition with characteristic time-reversal symmetries. The transition depends critically on the strength of the catch bond, suggesting that active stress is necessary but insufficient to mount an adaptive mechanical response.

Irreversibility in dynamical phases and transitions.

Living and non-living active matter consumes energy at the microscopic scale to drive emergent, macroscopic behavior including traveling waves and coherent oscillations. Recent work has characterized non-equilibrium systems by their total energy dissipation, but little has been said about how dissipation manifests in distinct spatiotemporal patterns. We introduce a measure of irreversibility we term the entropy production factor to quantify how time reversal symmetry is broken in field theories across scales. We use this scalar, dimensionless function to characterize a dynamical phase transition in simulations of the Brusselator, a prototypical biochemically motivated non-linear oscillator. We measure the total energetic cost of establishing synchronized biochemical oscillations while simultaneously quantifying the distribution of irreversibility across spatiotemporal frequencies.

Endothelial cell secreted metalloproteinase-2 enhances neural stem cell N-cadherin expression, clustering, and migration.

Neuroblasts have a clustered phenotype critical for their unidirectional migration, which in part is dependent on signaling from microvascular endothelial cells (EC) and pericytes (PC). Diffusible signals secreted by vascular cells have been demonstrated to increase survival, proliferation, and differentiation of subventricular zone resident neural stem cells (NSC); however, the signals that promote the necessary initiating step of NSC clustering are undefined. To investigate the role of vascular cells in promoting NSC clustering and directing migration, we created a 3-D hydrogel that mimics the biomechanics, biochemistry, and architectural complexity of brain tissue. We demonstrate that EC, and not PC, have a crucial role in NSC clustering and migration, further verified through microfluidic chamber systems and traction force microscopy. Ablation of the extended NSC aggregate arm halts aggregate movement, suggesting that clustering is a prerequisite for migration. When cultured with EC, NSC clustering occurs and NSC coincidentally increase their expression of N-cadherin, as compared to NSC cultured alone. NSC-presented N-cadherin expression was increased following exposure to EC secreted metalloproteinase-2 (MMP2). We demonstrate that inhibition of MMP2 prevented NSC N-cadherin surface expression and subsequent NSC clustering, even when NSC were in direct contact with EC. Furthermore, with exogenous activation of EGFR, which serves as a downstream activator of N-cadherin cleavage, NSC form clusters. Our results suggest that EC secretion of MMP2 promotes NSC clustering through N-cadherin expression. The insight gained about the mechanisms by which EC promote NSC migration may enhance NSC therapeutic response to sites of injury.