Actomyosin Gel Research Articles

Self-assembled active actomyosin gels spontaneously curve and wrinkle similar to biological cells and tissues.

Living systems adopt a diversity of curved and highly dynamic shapes. These diverse morphologies appear on many length scales, from cells to tissues and organismal scales. The common driving force for these dynamic shape changes are contractile stresses generated by myosin motors in the cell cytoskeleton, that converts chemical energy into mechanical work. A good understanding of how contractile stresses in the cytoskeleton arise into different three-dimensional (3D) shapes and what are the shape selection rules that determine their final configurations is still lacking. To obtain insight into the relevant physical mechanisms, we recreate the actomyosin cytoskeleton in vitro, with precisely controlled composition and initial geometry. A set of actomyosin gel discs, intrinsically identical but of variable initial geometry, dynamically self-organize into a family of 3D shapes, such as domes and wrinkled shapes, without the need for specific preprogramming or additional regulation. Shape deformation is driven by the spontaneous emergence of stress gradients driven by myosin and is encoded in the initial disc radius to thickness aspect ratio, which may indicate shaping scalability. Our results suggest that while the dynamical pathways may depend on the detailed interactions between the different microscopic components within the gel, the final selected shapes obey the general theory of elastic deformations of thin sheets. Altogether, our results emphasize the importance for the emergence of active stress gradients for buckling-driven shape deformations and provide insights on the mechanically induced spontaneous shape transitions in contractile active matter, revealing potential shared mechanisms with living systems across scales.

Vascular adaptation model from force balance: Physarum polycephalum as a case study

Understanding vascular adaptation, namely what drives veins to shrink or grow, is key for the self-organization of flow networks and their optimization. From the top–down principle of minimizing flow dissipation at a fixed metabolic cost within flow networks, flow shear rate resulting from the flows pervading veins is hypothesized to drive vein adaptation. Yet, there is no proposed mechanism of how flow forces impact vein dynamics. From the physical principle of force balance, shear rate acts parallel to vein walls, and hence, naively shear rate could only stretch veins and not dilate or shrink them. We, here, resolve this paradox by theoretically investigating force balance on a vein wall in the context of the vascular network of the model organism Physarum polycephalum. We propose, based on previous mechanical studies of cross-linked gels, that shear induces a nonlinear, orthogonal response of the actomyosin gel making up vein walls, that can indeed drive vein dilatation. Furthermore, our force balance approach allows us to identify that shear feedback occurs with a typical timescale and with a typical target shear rate that are not universal properties of the material but instead depend smoothly on the vein’s location within the network. In particular, the target shear rate is related to the vein’s hydrostatic pressure, which highlights the role of pressure in vascular adaptation in this context. Finally, since our derivation is based on force balance and fluid mechanics, we believe our approach can be extended, giving attention to specific differences, to describe vascular adaptation in other organisms.

Two-fluid dynamics and micron-thin boundary layers shape cytoplasmic flows in early Drosophila embryos.

Cytoplasmic flows are widely emerging as key functional players in development. In early Drosophila embryos, flows drive the spreading of nuclei across the embryo. Here, we combine hydrodynamic modeling with quantitative imaging to develop a two-fluid model that features an active actomyosin gel and a passive viscous cytosol. Gel contractility is controlled by the cell cycle oscillator, the two fluids being coupled by friction. In addition to recapitulating experimental flow patterns, our model explains observations that remained elusive and makes a series of predictions. First, the model captures the vorticity of cytosolic flows, which highlights deviations from Stokes' flow that were observed experimentally but remained unexplained. Second, the model reveals strong differences in the gel and cytosol motion. In particular, a micron-sized boundary layer is predicted close to the cortex, where the gel slides tangentially while the cytosolic flow cannot slip. Third, the model unveils a mechanism that stabilizes the spreading of nuclei with respect to perturbations of their initial positions. This self-correcting mechanism is argued to be functionally important for proper nuclear spreading. Fourth, we use our model to analyze the effects of flows on the transport of the morphogen Bicoid and the establishment of its gradients. Finally, the model predicts that the flow strength should be reduced if the shape of the domain is more round, which is experimentally confirmed in Drosophila mutants. Thus, our two-fluid model explains flows and nuclear positioning in early Drosophila, while making predictions that suggest novel future experiments.

The Mechanics of (Poro-)Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton.

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This feature is attributed to the mechanical and dynamic properties of the cell cytoskeleton, notably, the actomyosin cytoskeleton, which is an active gel of polar actin filaments, myosin motors, and accessory proteins that exhibit intrinsic contraction properties. The usually accepted view is that the cytoskeleton behaves as a viscoelastic material. However, this model cannot always explain the experimental results, which are more consistent with a picture describing the cytoskeleton as a poroelastic active material-an elastic network embedded with cytosol. Contractility gradients generated by the myosin motors drive the flow of the cytosol across the gel pores, which infers that the mechanics of the cytoskeleton and the cytosol are tightly coupled. One main feature of poroelasticity is the diffusive relaxation of stresses in the network, characterized by an effective diffusion constant that depends on the gel elastic modulus, porosity, and cytosol (solvent) viscosity. As cells have many ways to regulate their structure and material properties, our current understanding of how cytoskeleton mechanics and cytosol flow dynamics are coupled remains poorly understood. Here, an in vitro reconstitution approach is employed to characterize the material properties of poroelastic actomyosin gels as a model system for the cell cytoskeleton. Gel contraction is driven by myosin motor contractility, which leads to the emergence of a flow of the penetrating solvent. The paper describes how to prepare these gels and run experiments. We also discuss how to measure and analyze the solvent flow and gel contraction both at the local and global scales. The various scaling relations used for data quantification are given. Finally, the experimental challenges and common pitfalls are discussed, including their relevance to cell cytoskeleton mechanics.

Wrinkling and buckling instabilities of contractile suspended actomyosin sheets.

In many biological processes tissues and cells present active shape deformations resulting in wrinkling and buckling through contraction that result from internal forces generated by motor proteins. In many cases, the contracting material is also coupled to an elastic substrate, presenting a major difficulty for studying contraction induced folding. Here, we present the study of contraction and buckling of active, initially homogeneous, thin elastic actomyosin gels isolated from bounding surfaces. We find that the gels exhibit dimensionally dependent contraction that starts at the system boundaries, proceeds into the bulk, and eventually leads to spontaneous buckling of the sheet at the periphery resulting in different three-dimensional shapes at steady state. While thin sheets end up in wrinkled structures of mean negative Gaussian curvature whose configurations are of a single wavy mode that refines with decreasing thickness, thick sheets end up in domes with mean positive Gaussian curvature. Note that regardless of their distinct contraction dynamics and final shape, both thin and thick sheets show similar volume reduction, inferring on similar material properties of the contracted gel. Our system provides a well-controlled platform for studying contraction-induced folding of actively self-organizing gel sheets, in the absence of confinement.

Pulsatile contractions and pattern formation in excitable actomyosin cortex.

The actin cortex is an active adaptive material, embedded with complex regulatory networks that can sense, generate, and transmit mechanical forces. The cortex exhibits a wide range of dynamic behaviours, from generating pulsatory contractions and travelling waves to forming organised structures. Despite the progress in characterising the biochemical and mechanical components of the actin cortex, the emergent dynamics of this mechanochemical system is poorly understood. Here we develop a reaction-diffusion model for the RhoA signalling network, the upstream regulator for actomyosin assembly and contractility, coupled to an active actomyosin gel, to investigate how the interplay between chemical signalling and mechanical forces regulates stresses and patterns in the cortex. We demonstrate that mechanochemical feedback in the cortex acts to destabilise homogeneous states and robustly generate pulsatile contractions. By tuning active stress in the system, we show that the cortex can generate propagating contraction pulses, form network structures, or exhibit topological turbulence.

Tetrasodium pyrophosphate ameliorates oxidative damage to the TGase-catalyzed gelation of actomyosins

The present study attempted to investigate the interactive roles of protein oxidation (0–20 mM H2O2) and tetrasodium pyrophosphate (TSPP) on the crosslinking efficiency of actomyosin mediated by transglutaminase (TGase). Oxidation at 0–20 mM H2O2 was not conducive to TGase-mediated crosslinking as indicated by the relative reduction of free amine consumption from 35.3% to 11.7%, and caused the principle crosslinking sites to progressively convert from myosin subfragment-1 (S1) to subfragment-1 (S2) as evidenced by electrophoresis. However, the binding of TSPP to myosin alleviated oxidation suppression to TGase-catalyzed crosslinking in varying degrees and retarded the migration of crosslinking site from S1 to S2. Moreover, oxidation (especially 20 mM H2O2) decreased the final (90 °C) elasticity index (EI) and water holding capacity of TGase-treated actomyosin gel, while TSPP intensified those of TGase-catalyzed actomyosin gel, indicating that TSPP had a positive effect on ameliorating the oxidative stress to TGase-catalyzed gelation of actomyosin.

Odd Viscosity in Active Matter: Microscopic Origin and 3D Effects.

In common fluids, viscosity is associated with dissipation. However, when time-reversal symmetry is broken a new type of nondissipative "viscosity" emerges. Recent theories and experiments on classical 2D systems with active spinning particles have heightened interest in "odd viscosity," but a microscopic theory for it in active materials is still absent. Here, we present such first-principles microscopic Hamiltonian theory, valid for both 2D and 3D, showing that odd viscosity is present in any system, even at zero temperature, with globally or locally aligned spinning components. Our work substantially extends the applicability of odd viscosity into 3D fluids, and specifically to internally driven active materials, such as living matter (e.g., actomyosin gels). We find intriguing 3D effects of odd viscosity such as propagation of anisotropic bulk shear waves and breakdown of Bernoulli's principle.

Hydrodynamics of contraction-based motility in a compressible active fluid

Cell motility is crucial to biological functions ranging from wound healing to immune response. The physics of cell crawling on a substrate is by now well understood, whilst cell motion in bulk (cell swimming) is far from being completely characterized. We present here a minimal model for pattern formation within a compressible actomyosin gel, in both 2D and 3D, which shows that contractility leads to the emergence of an actomyosin droplet within a low density background. This droplet then becomes self-motile for sufficiently large motor contractility. These results may be relevant to understand the essential physics at play in 3D cell swimming within compressible fluids. We report results of both 2D and 3D numerical simulations, and show that the compressibility of actomyosin plays an important role in the transition to motility.

Living Matter: Mesoscopic Active Materials

An introduction to the physical properties of living active matter at the mesoscopic scale (tens of nanometers to micrometers) and their unique features compared with "dead," nonactive matter is presented. This field of research is increasingly denoted as "biological physics" where physics includes chemical physics, soft matter physics, hydrodynamics, mechanics, and the related engineering sciences. The focus is on the emergent properties of these systems and their collective behavior, which results in active self-organization and how they relate to cellular-level biological function. These include locomotion (cell motility and migration) forces that give rise to cell division, the growth and form of cellular assemblies in development, the beating of heart cells, and the effects of mechanical perturbations such as shear flow (in the bloodstream) or adhesion to other cells or tissues. An introduction to the fundamental concepts and theory with selected experimental examples related to the authors' own research is presented, including red-blood-cell membrane fluctuations, motion of the nucleus within an egg cell, self-contracting acto-myosin gels, and structure and beating of heart cells (cardiomyocytes), including how they can be driven by an oscillating, mechanical probe.

Maximal Fluctuations of Confined Actomyosin Gels: Dynamics of the Cell Nucleus.

We investigate the effect of stress fluctuations on the stochastic dynamics of an inclusion embedded in a viscous gel. We show that, in nonequilibrium systems, stress fluctuations give rise to an effective attraction towards the boundaries of the confining domain, which is reminiscent of an active Casimir effect. We apply this generic result to the dynamics of deformations of the cell nucleus, and we demonstrate the appearance of a fluctuation maximum at a critical level of activity, in agreement with recent experiments [E. Makhija, D. S. Jokhun, and G. V. Shivashankar, Proc. Natl. Acad. Sci. U.S.A. 113, E32 (2016)PNASA60027-842410.1073/pnas.1513189113].

Effect of actomyosin form extracted from skeletal fast muscle on the structural and rheological properties of heat-induced gels

Abstract Little is known about the mechanism of heat-induced gelation of natural actomyosin, or the influence of pyrophosphate on the morphology and physical properties of the product. In the current study, these questions were investigated using scanning and transmission electron microscopy. When using high salt concentrations (0.5 M NaCl), actomyosin lost its “arrowhead” structure after heating for two minutes, and aggregation, including interfilament binding, occurred after six minutes of heating at 60° C. The product in this case exhibited a strand-like gel structure. On the other hand, in the presence of 0.1 M NaCl, cohesion between filaments began after two minutes of heating, and an aggregate-type gel structure was formed. When pyrophosphate was added in the presence of 0.5 M NaCl, marked dissociation of actomyosin into myosin and actin occurred. In the presence of 0.1 M NaCl, the filament structure of the actomyosin bundles became difficult to detect. The addition of pyrophosphate did not improve the physical properties of the resultant heat-induced actomyosin gel, since the elasticity of the actomyosin gel decreased.

Effects of l-lysine on thermal gelation properties of chicken breast actomyosin.

The effects of l-lysine (l-Lys) on the water holding capacity (WHC) and texture of actomyosin (AM) gel and the possible mechanisms were investigated. l-Lys increased the WHC and hardness of the AM gel. These effects may be related to the even and continuous microstructure of the gel according to the scanning electron microscopy analysis. Furthermore, l-Lys increased the surface hydrophobic residues and the reactive sulfhydryl groups. l-Lys decreased the storage modulus at the first transition temperature but increased it at the second transition temperature and the third transition enthalpy. These results suggested that l-Lys varied the thermal behaviors and the microstructure of the AM gel by increasing the surface hydrophobicity and reactive sulfhydryl groups, ultimately contributing to the increased WHC and hardness. The changes in pH did not fully explain the results from the present study. The results were useful for understanding previous findings and may serve as a reference for the preparation of reduced-sodium and phosphate-free meat products.

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

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

Effect of low frequency ultrasonication on biochemical and structural properties of chicken actomyosin

Ultrasonication has been introduced as a promising technique to modify the properties of meat and meat products. This study was carried out to investigate the structural and biochemical properties of actomyosin under the influence of ultrasonication at low frequency (20kHz). CD spectroscopy and second-derivative UV spectra indicated that ultrasonic exposure of 30min causes significant loss of α-helical fraction and marked change in tertiary structure of actomyosin. R-SH content showed maximum amount after 30min of ultrasonic treatment. Additionally, Ca2+-, Mg2+- and K+(EDTA)-ATPase activities were markedly decreased. No fragmentation was observed in SDS–PAGE while transmission electron micrographs showed complete dispersion of aggregates and arrowhead structure of actomyosin. Given that structural properties are closely associated with functional properties, ultrasonication significantly improves the gelling properties of actomyosin. Scanning electron micrographs showed marked improvement in regular three-dimensional networks of actomyosin gels. Concurrently, significant increase in water-holding capacity was also observed.

Theoretical study of actin layers attachment and separation

We use the theory of active gels to study theoretically the merging and separation of two actin dense layers akin to cortical layers of animal cells. The layers bind at a distance equal to twice the thickness of a free layer, thus forming a single dense layer, similar in this sense to a lamellipodium. When that unique layer is stretched apart, it is resilient to break apart up to a critical length larger than twice the thickness of a free layer. We show that this behavior can result from the high contractile properties of the actomyosin gel due to the activity of myosin molecular motors. Furthermore, we establish that the stability of the stretched single layer is highly dependent on the properties of the gel. Indeed, the nematic order of the actin filaments along the polymerizing membranes is a destabilizing factor.

Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting.

Effect of pH on the physicochemical and heat-induced gel properties of scallop Patinopecten yessoensis actomyosin

Gelation is an important functional property of protein in meats. In this study, we prepared actomyosin from scallop Patinopecten yessoensis adductor muscles and studied the effects of pH on the physicochemical properties of the actomyosin preparation and on its heat-induced gel-forming properties. The results showed that the turbidity and surface hydrophobicity of scallop actomyosin increased with increases in the heating temperature, while the α-helical content concomitantly decreased. Higher turbidity and surface hydrophobicity and lower α-helical content were found to be easily obtained at lower pH values. A high water-holding capacity, strong gel strength, fine gel network and uniform ice crystals were all clearly observed at pH 7.0, indicating that a neutral pH was most beneficial for formation of the heat-induced scallop actomyosin gel. We therefore conclude that both the physicochemical properties of scallop actomyosin and its gel-forming ability during the heating process are pH dependent.

Spiral and never-settling patterns in active systems

We present a combined numerical and analytical study of pattern formation in an active system where particles align, possess a density-dependent motility, and are subject to a logistic reaction. The model can describe suspensions of reproducing bacteria, as well as polymerizing actomyosin gels in vitro or in vivo. In the disordered phase, we find that motility suppression and growth compete to yield stable or blinking patterns, which, when dense enough, acquire internal orientational ordering to give asters or spirals. We predict these may be observed within chemotactic aggregates in bacterial fluids. In the ordered phase, the reaction term leads to previously unobserved never-settling patterns which can provide a simple framework to understand the formation of motile and spiral patterns in intracellular actin systems.

Colloids in Active Fluids: Anomalous Microrheology and Negative Drag

We simulate an experiment in which a colloidal probe is pulled through an active nematic fluid. We find that the drag on the particle is non-stokesian (not proportional to its radius). Strikingly, a large enough particle in contractile fluid (such as an actomyosin gel) can show negative viscous drag in steady state: the particle moves in the opposite direction to the externally applied force. We explain this, and the qualitative trends seen in our simulations, in terms of the disruption of orientational order around the probe particle and the resulting modifications to the active stress.