Bead Displacement Research Articles

Nuclear deformation and dynamics of migrating cells in 3D confinement reveal adaptation of pulling and pushing forces.

Eukaryotic cells show an astounding ability to remodel their shape and cytoskeleton and to migrate through pores and constrictions smaller than their nuclear diameter. However, the relation of nuclear deformation and migration dynamics in confinement remains unclear. Here, we study the mechanics and dynamics of mesenchymal cancer cell nuclei transitioning through three-dimensional compliant hydrogel channels. We find a biphasic dependence of migration speed and transition frequency on channel width, peaking at widths comparable to the nuclear diameter. Using confocal imaging and hydrogel bead displacement, we determine nuclear deformations and corresponding forces during confined migration. The nucleus deforms reversibly with a reduction in volume during confinement. With decreasing channel width, the nuclear shape during transmigration changes biphasically, concomitant with the transitioning dynamics. Our proposed physical model explains the observed nuclear shapes and transitioning dynamics in terms of the cytoskeletal force generation adapting from purely pulling-based to a combined pulling- and pushing-based mechanism with increasing nuclear confinement.

Bayesian traction force estimation using cell boundary-dependent force priors

Understanding the principles of cell migration necessitates measurements of the forces generated by cells. In traction force microscopy (TFM), fluorescent beads are placed on a substrate’s surface and the substrate strain caused by the cell traction force is observed as displacement of the beads. Mathematical analysis can estimate traction force from bead displacement. However, most algorithms estimate substrate stresses independently of cell boundary, which results in poor estimation accuracy in low-density bead environments. To achieve accurate force estimation at low density, we proposed a Bayesian traction force estimation (BTFE) algorithm that incorporates cell-boundary-dependent force as a prior. We evaluated the performance of the proposed algorithm using synthetic data generated with mathematical models of cells and TFM substrates. BTFE outperformed other methods, especially in low-density bead conditions. In addition, the BTFE algorithm provided a reasonable force estimation using TFM images from the experiment.

Simulational Tests of the Rouse Model.

An extensive review of literature simulations of quiescent polymer melts is given, considering results that test aspects of the Rouse model in the melt. We focus on Rouse model predictions for the mean-square amplitudes ⟨(Xp(0))2⟩ and time correlation functions ⟨Xp(0)Xp(t)⟩ of the Rouse mode Xp(t). The simulations conclusively demonstrate that the Rouse model is invalid in polymer melts. In particular, and contrary to the Rouse model, (i) mean-square Rouse mode amplitudes ⟨(Xp(0))2⟩ do not scale as sin-2(pπ/2N), N being the number of beads in the polymer. For small p (say, p≤3) ⟨(Xp(0))2⟩ scales with p as p-2; for larger p, it scales as p-3. (ii) Rouse mode time correlation functions ⟨Xp(t)Xp(0)⟩ do not decay with time as exponentials; they instead decay as stretched exponentials exp(-αtβ). β depends on p, typically with a minimum near N/2 or N/4. (iii) Polymer bead displacements are not described by independent Gaussian random processes. (iv) For p≠q, ⟨Xp(t)Xq(0)⟩ is sometimes non-zero. (v) The response of a polymer coil to a shear flow is a rotation, not the affine deformation predicted by Rouse. We also briefly consider the Kirkwood-Riseman polymer model.

Measuring tension states of hiPSC-cardiomyocytes via traction force microscopy.

Cardiomyocytes (CMs), the cells responsible for the contraction of the heart, experience mechanical forces such as active and resting tension as the cell contracts and relaxes. Active tension is generated by the contractile cytoskeleton, which is composed of actin-myosin arrays called sarcomeres. Resting tension is the tension that results when spring-like elements are stretched beyond their resting length, and this resting tension determines how forceful a contraction will be. The ability to investigate how resting tension is regulated has been a challenge due to the lack of a robust method to measure resting tension at the cell level. Previous methods involve stretching an individual cell and measuring the resulting resting tension; however, this method is low throughput and invasive. Single-cell Traction Force Microscopy (TFM) serves as a promising alternative method to measure resting tension due to the ability to measure the tension states of individual CMs with high throughput. We culture human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) on polyacrylamide (PA) gels with stiffness of healthy myocardium (10 kPa) patterned with islands of adhesive protein (Matrigel). We use TFM video analysis of beating cells to measure the traction forces when the hiPSC-CM is contracted and relaxed. We measure bead positions again after treatment with blebbistatin, and finally after the cell is dissociated from the gel. We calculate resting tension by calculating the TFM bead displacements from their positions under a relaxed cell to their positions after blebbistatin and after the cell is dissociated. To probe how resting tension is regulated by increased loading, we culture hiPSC-CMs on PA gels of increasing stiffness to mimic fibrotic tissue. These results provide insight into how the resting tension of a CM is regulated and how the CM adapts in response to pathophysiological conditions.

Non-equilibrium tracer dynamics in oscillating active gel

Non-equilibrium features in active gels are commonly assessed by the van Hove distribution (VHD) of tracer bead displacement. For instance, pulsed stress is shown to cause side peaks in the VHD such as those induced by Myosin-II motors. Here, we report the same side peaks in the VHD of tracer beads that are periodically displaced by the shuttle streaming of Physarum polycephalum. On the other hand, the mean square displacement (MSD) of tracer trajectory includes oscillatory components, which are not expected for a pulsed stress source. Thus, the VHD and a dynamical measurement are needed to fully characterize tracer dynamics. We present a modified active trap model that fully captures the experimental data for this class of oscillating active gel.

Slope position and biochar influence soil properties and seed displacement in a tropical agroecosystem

AbstractSoil erosion due to land use change is a major concern in agricultural areas. There is an urgent need to develop appropriate agricultural practices that promote higher biodiversity and improve soil properties. Little is known about the processes of natural vegetation recovery after disturbance by human activities in tropical humid climates. This is particularly the case for dispersion by water (hydrochory) that affects seeds. The displacement of native plant seeds on the soil surface by runoff may be influenced by soil properties and by the use of biochar in agriculture. As these effects have not been documented in tropical agroecosystems and on steep slopes, we present here an in‐field experiment on a steeply sloping maize field affected by rainfall and tillage erosion in Northern Thailand. Our objectives were to test the effect of catena position and biochar use on seed displacement, soil loss and nutrient losses. We used 24 plots of 1 m2 located at four positions in the catena. Two treatments were tested: (1) conventional system with tillage and (2) biochar incorporated into the soil. We measured the displacement of plastic beads (to imitate round seeds), runoff volume, soil and nutrient losses and soil surface features during in‐situ rainfall simulations. After two simulated rainfall events and four natural rainfall events, total bead exportation was 7.2%. We found that catena position and biochar had an impact on runoff, soil and nutrient losses. In this study, previously incorporated biochar reappeared at the soil surface after 8 months under natural conditions and acted like a crust at the soil surface. This biochar crust increased runoff, increased potassium concentration and decreased phosphorus concentration in runoff, decreased bead displacement and had no effect on soil loss. This study, under tropical climate and steep slope conditions, highlights differences in soil surface features and runoff along the catena. We should consider catena position for improving soil management and using appropriate agroecological practices.Highlights The effect of catena position and biochar use on seed displacement, soil loss and nutrient losses were tested. These effects have not been documented in tropical agroecosystems and steep slopes. Catena position and biochar use had an impact on runoff, soil and nutrient losses. Catena position for improving soil management and using agroecological practices should be considered.

Effects of chain length on Rouse modes and non-Gaussianity in linear and ring polymer melts.

The dynamics of ring polymer melts are studied via molecular dynamics simulations of the Kremer-Grest bead-spring model. Rouse mode analysis is performed in comparison with linear polymers by changing the chain length. Rouse-like behavior is observed in ring polymers by quantifying the chain length dependence of the Rouse relaxation time, whereas a crossover from Rouse to reptation behavior is observed in linear polymers. Furthermore, the non-Gaussian parameters of the monomer bead displacement and chain center-of-mass displacement are analyzed. It is found that the non-Gaussianity of ring polymers is remarkably suppressed with slight growth for the center-of-mass dynamics at long chain length, which is in contrast to the growth in linear polymers for both the monomer bead and center-of-mass dynamics.

Characterization of the hydrodynamics within a toroid wear tester

AbstractThe toroid wear tester (TWT) is a lab‐scale device used for the assessment of slurry erosion in pipelines. Historically, its application has been limited to the relative ranking of material performance under different slurry flow conditions; however, recent studies have indicated that TWT tests could be predictive and directly applied to slurry pipeline design—provided that the flow inside a TWT is better characterized. In the present study, air‐liquid multiphase flow inside the TWT was investigated. Torque measurements were taken to characterize friction loss for different air‐liquid combinations. A visualization experiment was also conducted to evaluate flow patterns within the TWT. In the experiment, the displacements of spherical glass beads were used to estimate velocity vector fields for different TWT rotational speeds. A computational fluid dynamics (CFD) analysis was also conducted to complement the experimental measurements. A 3D transient analysis using the volume of fluid (VOF) approach was used to model the system. The simulation results agreed closely with the experimental findings. Furthermore, the simulations revealed that strong secondary flows (back flow, rotation) exist in the TWT. These type of flows do not occur in horizontal pipelines. Therefore, to use the TWT as a tool for slurry pipeline wear assessment, the differences in the flow field between the two systems must be properly quantified.

Quantifying the Compressive Force of 3D Cardiac Tissues via Calculating the Volumetric Deformation of Built-In Elastic Gelatin Microspheres.

Quantifying cardiac contractile force is of paramount important in studying mechanical heart failure and screening therapeutic drugs. However, most existing methods can only measure the in-plane component of twitch force of cardiomyocytes, such that mismatching the centripetal compressive stress of heart beating in physiology. Here, a non-destructive method is developed for quantifying the compressive stress and mapping the distribution of the local stress within the 3D cardiac tissues. In detail, elastic gelatin microspheres labeled with fluorescence beads are fabricated by microfluidic chips with high throughput, and they serve as built-in pressure sensors which are wrapped by cardiomyocytes in 3D tissues. The deformation of microspheres and the displacements of fluorescent beads induced by the contraction of cardiomyocytes are demonstrated to characterize the amount and distribution of the centripetal compressive stress. Further, the method shows a potent capability to locally quantify contractile force variation of 3D cardiac tissues, which is induced by agonist (norepinephrine) and inhibitor (blebbistatin). On the whole, the method significantly improves the 3D measurement of mechanical force in vitro and provides a solution for locally quantifying the compressive stress within engineered cardiac tissues.

A new 3D finite element-based approach for computing cell surface tractions assuming nonlinear conditions.

Advances in methods for determining the forces exerted by cells while they migrate are essential for attempting to understand important pathological processes, such as cancer or angiogenesis, among others. Precise data from three-dimensional conditions are both difficult to obtain and manipulate. For this purpose, it is critical to develop workflows in which the experiments are closely linked to the subsequent computational postprocessing. The work presented here starts from a traction force microscopy (TFM) experiment carried out on microfluidic chips, and this experiment is automatically joined to an inverse problem solver that allows us to extract the traction forces exerted by the cell from the displacements of fluorescent beads embedded in the extracellular matrix (ECM). Therefore, both the reconstruction of the cell geometry and the recovery of the ECM displacements are used to generate the inputs for the resolution of the inverse problem. The inverse problem is solved iteratively by using the finite element method under the hypothesis of finite deformations and nonlinear material formulation. Finally, after mathematical postprocessing is performed, the traction forces on the surface of the cell in the undeformed configuration are obtained. Therefore, in this work, we demonstrate the robustness of our computational-based methodology by testing it under different conditions in an extreme theoretical load problem and then by applying it to a real case based on experimental results. In summary, we have developed a new procedure that adds value to existing methodologies for solving inverse problems in 3D, mainly by allowing for large deformations and not being restricted to any particular material formulation. In addition, it automatically bridges the gap between experimental images and mechanical computations.

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo.

Rouse model in crowded environment modeled by “diffusing diffusivity”

We derive exact results for the dynamics of Rouse model in crowded environment modeled by stochastically varying diffusivity, the concept of “diffusing diffusivity”. We specifically generalize the initial analytical formulation of this concept of diffusivity in single particle diffusion to many particle systems with localized interactions, particularly to chains of connected beads, the Rouse model. Independent Rouse modes are allowed to diffuse with stochastically varying time dependent diffusivities to model diffusion in the crowded rearranging environment, where zeroth mode represents the chain center of mass coordinate. We develop an analytical formalism to calculate the probability distribution of these individual mode displacements and subsequently average bead displacement distribution of the Rouse chain and its moments in terms of these modes. The resulting diffusion behavior of thus modified Rouse model is a function of mode resolved parameters which model local interactions and crowded environment collectively. Our analytically tractable implementation of the “diffusing diffusivity” concept to Rouse model is one of its kind that yields non-Gaussian diffusion with the scope of being anomalous and plausibly applied to dynamics in polymeric liquids.

Bayesian Cell Force Estimation Introducing Cell Shape Prior

Cell morphogenesis and migration are essential for various physiological and pathological processes. To elucidate their mechanisms, it is necessary to observe the mechanical force generated by the cells. Traction force microscopy (TFM) uses fluorescent nanobeads that are embedded in the surface layer of an elastic gel substrate. When cells cultured on the substrate generate traction force, the beads change their locations due to the force-induced deformation of the gel substrate. Various algorithms for estimating traction force from bead displacement have been proposed. Higher bead density is better for accurate force estimation. However, substrates containing high-density beads have several problems such as heterogeneous distribution of beads, fusions of condensed beads on the image, and influence on the effective Young's modulus of the substrate. Therefore, it is important to develop an algorithm to estimate accurate traction forces under the condition of a small number of beads. In this study, we proposed the traction force estimation algorithm introducing the force priors: force is more likely to occur near the edge of a cell and is directed from the edge to the center of the cell. Using synthetic condition with 3∼8 beads per one um2, we estimated traction force by employing Bayesian framework. We also estimated the minimum density of the beads against the cell size that guarantees the accuracy of force estimation. The proposed algorithm showed better performance than other regression-based methods such as Ridge regression or LASSO. Furthermore, we applied the algorithm to the traction force estimation of neuronal growth cone.

Application of Mechanical Forces on Drosophila Embryos by Manipulation of Microinjected Magnetic Particles.

Cells generate mechanical forces to shape tissues during morphogenesis. These forces can activate several biochemical pathways and trigger diverse cellular responses by mechano-sensation, such as differentiation, division, migration and apoptosis. Assessing the mechano-responses of cells in living organisms requires tools to apply controlled local forces within biological tissues. For this, we have set up a method to generate controlled forces on a magnetic particle embedded within a chosen tissue of Drosophila embryos. We designed a protocol to inject an individual particle in early embryos and to position it, using a permanent magnet, within the tissue of our choice. Controlled forces in the range of pico to nanonewtons can be applied on the particle with the use of an electromagnet that has been previously calibrated. The bead displacement and the epithelial deformation upon force application can be followed with live imaging and further analyzed using simple analysis tools. This method has been successfully used to identify changes in mechanics in the blastoderm before gastrulation. This protocol provides the details, (i) for injecting a magnetic particle in Drosophila embryos, (ii) for calibrating an electromagnet and (iii) to apply controlled forces in living tissues.

Surface deformation and displacement of bed elements during splash – Model tests

The phenomenon of splash is connected with soil erosion, occurs commonly, and has a complex nature. The study presents the results of research in which glass beads (dry and saturated beds) with a diameter size range of 0.425–0.6 mm were used as a soil model. Patterns and layers of glass particles (beads), distinguished by their colour were placed on the surface of the samples. Displacements of the marked beads were used for the analysis of movement caused by splash. The measurements were supplemented with recordings made with high-speed cameras and photographs from an X-ray microtomograph. Three types of displacement for dry bed elements were distinguished: (i) displacement inside the surface area wetted with a falling drop of water, (ii) ejection and (iii) placement on the crater rim, as well as two types of displacement for elements from saturated beds: (i) displacement inside the deformation and (ii) ejection. The distance travelled by the beads was related to the specificity their displacement, which was associated with the initial location of the beads on the sample surface. In the case of dry beds, glass beads with an initial location equal to 14 mm or more from the centre of the drop impact were not moved. In the case of saturated beds, this distance was about 10 mm. More than 95% of the beads ejected from dry beds came from the surface layer of the sample. All the measurement methods applied show that the deformations of the dry bed surfaces were greater and more diversified than those of the saturated beds. The results presented in the study are important for understanding the mechanisms of transport of bacteria, fungi, and pollutants transferred with ejected soil particles during splash. The results can also be used for a physical description of the phenomenon for the computer modelling of both splash and water erosion.

In Vivo Force Application Reveals a Fast Tissue Softening and External Friction Increase during Early Embryogenesis

SummaryDuring development, cell-generated forces induce tissue-scale deformations to shape the organism [1, 2]. The pattern and extent of these deformations depend not solely on the temporal and spatial profile of the generated force fields but also on the mechanical properties of the tissues that the forces act on. It is thus conceivable that, much like the cell-generated forces, the mechanical properties of tissues are modulated during development in order to drive morphogenesis toward specific developmental endpoints. Although many approaches have recently emerged to assess effective mechanical parameters of tissues [3, 4, 5, 6, 7, 8], they could not quantitatively relate spatially localized force induction to tissue-scale deformations in vivo. Here, we present a method that overcomes this limitation. Our approach is based on the application of controlled forces on a single microparticle embedded in an individual cell of an embryo. Combining measurements of bead displacement with the analysis of induced deformation fields in a continuum mechanics framework, we quantify material properties of the tissue and follow their changes over time. In particular, we uncover a rapid change in tissue response occurring during Drosophila cellularization, resulting from a softening of the blastoderm and an increase of external friction. We find that the microtubule cytoskeleton is a major contributor to epithelial mechanics at this stage. We identify developmentally controlled modulations in perivitelline spacing that can account for the changes in friction. Overall, our method allows for the measurement of key mechanical parameters governing tissue-scale deformations and flows occurring during morphogenesis.

Recurrent venous thromboembolism patients form clots with lower elastic modulus than those formed by patients with non‐recurrent disease

Essentials Venous thromboembolism (VTE) recurrence leads to decreased clot elastic modulus in plasma.Recurrent VTE is not linked to changes in clot structure, fiber radius, or factor XIII activity.Other plasma components may play a role in VTE recurrence.Prospective studies should resolve if clot stiffness can be used as predictor for recurrent VTE. SummaryBackgroundVenous thromboembolism (VTE) is associated with a high risk of recurrent events after withdrawal of anticoagulation.ObjectivesTo determine the difference in plasma clot mechanical properties between patients with recurrent VTE (rVTE) and those with non‐recurrent VTE (nrVTE).MethodsWe previously developed a system for determining clot mechanical properties by use of an in‐house magnetic tweezers system. This system was used to determine the mechanical properties of clots made from plasma of 11 patients with rVTE and 33 with nrVTE. Plasma was mixed with micrometer‐sized beads, and thrombin and calcium were added to induce clotting; the mixture was then placed in small capillary tubes, and clotting was allowed to proceed overnight. Bead displacements upon manipulation with magnetic forces were analyzed to determine clot elastic and viscous moduli. Fibrin clot structure was analyzed with turbidimetry and confocal microscopy. Factor XIII was measured by pentylamine incorporation into fibrin.ResultsClots from rVTE patients showed nearly two‐fold less elastic and less viscous moduli than clots from nrVTE patients, regardless of male sex, unprovoked events, family history of VTE, fibrinogen concentration, or body mass index. No differences were observed in clot structure, fibrinolysis rates, or FXIII levels.ConclusionUsing magnetic tweezers for the first time in patient samples, we found that plasma clots from rVTE patients showed a reduced elastic modulus and a reduced viscous modulus as compared with clots from nrVTE patients. These data indicate a possible role for fibrin clot viscoelastic properties in determining VTE recurrence.

Design and characterization of a hydrodynamically confined microflow device for applying controlled loads to investigate single-cell mechanics

Single-cell mechanics measurements are crucial in understanding mechanotransduction and cellular properties, such as adhesion and stiffness. Here, we present a microfluidic probe device that can generate controlled hydrodynamic loads on single cells in an open cell culture environment. The device is optimized to produce uniform stresses across the area of a cell for cell adhesion measurements. Microfluidic probe (MFP) devices that can be used to create hydrodynamically confined flows (HCMs) have emerged as a unique device for selectively treating cells and surfaces. Typical MFP devices generate complex-shaped flows and non-uniform hydrodynamic loads on the surface beneath the device. We have used computational fluid dynamics to optimize the port geometry of the MFP device to generate an HCM with uniform shear stresses in a region beneath the device. The devices were fabricated from a combination of silicon and PDMS and characterized through flow experiments above a polyacrylamide gel seeded with fluorescent beads. Bead displacements were measured as a function of flow conditions and general agreement with the model was obtained. Finally, we have used the devices to characterize the adhesion strength of patterned fibroblast cells adhered to a collagen-coated substrate. The results presented establish a design for an MFP device that can apply controlled mechanical forces to cells in open liquid environments.

Abstract 17205: Contractility in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes is Highly Dependent on Stochastic Variability in Myofilament Development

Introduction: Modeling heart diseases using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) enables direct study of cellular and molecular mechanisms. However, the variable phenotypes of individual cells has limited the reproducibility of contractile measurements. We hypothesized that variability in myofibrillar development in hiPSC-CMs influences the contractile kinetics and could be controlled for by visualizing and quantifying myofilaments in vitro . Methods and Results: Control hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) and purified. hiPSC-CMs were replated at day ~25 onto 8.7 kPa polyacrylamide gels containing fluorescent microbeads and micropatterned with fibronectin in 7:1 aspect ratio rectangles (1750 um 2 ). Single iPSC-CMs grew into the rectangular shapes and exhibited spontaneous contractions by day 2 of replating. hiPSC-CMs were stained with live-cell actin dye and imaged on day 5-7 for fluorescent bead displacements and actin videos depicting myofilaments. Traction force analysis of bead displacements was performed to calculate whole-cell force vs. time curves at 33 msec temporal resolution. Myofilament area was measured by thresholding a binary mask on the actin live cell images. Myofilament number varied markedly among individual iPSC-CMs. iPSC-CMs with few myofilaments (myofilament area / cell area <30%) developed low force (<400 uN per cell) and were not included in subsequent analyses. Among cells with myofilament area >30%, the mean traction force was 1368±357 uN and the myofilament area demonstrated a linear correlation with maximum force (R 2 =0.19, runs test p=0.8) with slope of 0.8±0.2 uN/um 2 (p=0.002 vs zero slope). Maximum contraction velocity strongly correlated with maximum force (R 2 =0.31, runs test p=0.4) with slope of 3.9±0.9 sec -1 (p<0.0001 vs zero slope), as did maximum relaxation velocity (R 2 =0.27, runs test p=0.6, slope 4.1+1.0 sec -1 , p=0.0003 vs zero slope). Similar results were observed in two control cell lines and with two different purification methods. Conclusion: Variability in myofilament development is a critical determinant of contractile force and kinetics in individual iPSC-CMs that should be accounted for in iPSC-CM contractile experiments.

Co-Entangled Actin-Microtubule Composites Exhibit Tunable Stiffness and Power-Law Stress Relaxation

We use optical tweezers microrheology and fluorescence microscopy to characterize the nonlinear mesoscale mechanics and mobility of in vitro co-entangled actin-microtubule composites. We create a suite of randomly oriented, well-mixed networks of actin and microtubules by co-polymerizing varying ratios of actin and tubulin in situ. To perturb each composite far from equilibrium, we use optical tweezers to displace an embedded microsphere a distance greater than the lengths of the filaments at a speed much faster than their intrinsic relaxation rates. We simultaneously measure the force the filaments exert on the bead and the subsequent force relaxation. We find that the presence of a large fraction of microtubules (>0.7) is needed to substantially increase the measured force, which is accompanied by large heterogeneities in force response. Actin minimizes these heterogeneities by reducing the mesh size of the composites and supporting microtubules against buckling. Composites also undergo a sharp transition from strain softening to stiffening when the fraction of microtubules (ϕT) exceeds 0.5, which we show arises from faster poroelastic relaxation and suppressed actin bending fluctuations. The force after bead displacement relaxes via power-law decay after an initial period of minimal relaxation. The short-time relaxation profiles (t < 0.06 s) arise from poroelastic and bending contributions, whereas the long-time power-law relaxation is indicative of filaments reptating out of deformed entanglement constraints. The scaling exponents for the long-time relaxation exhibit a nonmonotonic dependence on ϕT, reaching a maximum for equimolar composites (ϕT = 0.5), suggesting that reptation is fastest in ϕT = 0.5 composites. Corresponding mobility measurements of steady-state actin and microtubules show that both filaments are indeed the most mobile in ϕT = 0.5 composites. This nonmonotonic dependence of mobility on ϕT demonstrates the important interplay between mesh size and filament rigidity in polymer networks and highlights the surprising emergent properties that can arise in composites.