Networks of cross-linked and bundled actin filaments are ubiquitous in the cellular cytoskeleton, but their elasticity remains poorly understood. We show that these networks exhibit exceptional elastic behavior that reflects the mechanical properties of individual filaments. There are two distinct regimes of elasticity, one reflecting bending of single filaments and a second reflecting stretching of entropic fluctuations of filament length. The mechanical stiffness can vary by several decades with small changes in cross-link concentration, and can increase markedly upon application of external stress. We parameterize the full range of behavior in a state diagram and elucidate its origin with a robust model.

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity.

Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells.

The linear and nonlinear viscoelastic response of networks of cross-linked and bundled cytoskeletal filaments demonstrates remarkable scaling with both frequency and applied prestress, which helps elucidate the origins of the viscoelasticity. The frequency dependence of the shear modulus reflects the underlying single-filament relaxation dynamics for 0.1-10 rad/sec. Moreover, the nonlinear strain stiffening of such networks exhibits a universal form as a function of prestress; this is quantitatively explained by the full force-extension relation of single semiflexible filaments.

The organization of individual actin filaments into higher-order structures is controlled by actin-binding proteins (ABPs). Although the biological significance of the ABPs is well documented, little is known about how bundling and cross-linking quantitatively affect the microstructure and mechanical properties of actin networks. Here we quantify the effect of the ABP scruin on actin networks by using imaging techniques, cosedimentation assays, multiparticle tracking, and bulk rheology. We show how the structure of the actin network is modified as the scruin concentration is varied, and we correlate these structural changes to variations in the resultant network elasticity.F -actin is one of the most important participants in maintaining the mechanical integrity of eukaryotic cells. In vivo, actin filaments rarely exist as isolated single filaments but instead associate into bundles or networks, in concert with Ͼ60 different actin binding proteins (ABPs), to influence cell shape, division, adhesion, and motility (1-4). The elastic modulus of cytoplasmic actin gels is estimated to be of order 100-1,000 Pa (5), and the gel must be able to sustain shear stresses of up to 1,000 Pa for proper cell functions (6). This large elasticity cannot result exclusively from a network of actin alone; in vitro, solutions of entangled actin filaments are weak elastic solids. For example, a solution of actin filaments at a concentration of 24 M has an elastic modulus of only 0.1 Pa and breaks under a shear stress of Ͻ0.1 Pa (7,8). Therefore, the properties of the actin cytoskeleton must be regulated predominantly by ABPs. Modest changes in the concentration of ABPs can significantly modify the structure of the network because they can both bundle and cross-link the actin filaments. These structural changes can lead to concomitant change in the mechanical properties, dramatically enhancing the mechanical rigidity (9-13). The changes in structure occur over a large range of length scales, ranging from a few nanometers, the size of an ABP, to several micrometers, the length of an individual actin filament. The dearth of techniques that probe the structure and properties over this range of length scales has limited our ability to determine the modifications caused by the ABPs and to identify their critical contributions. As a result, a quantitative understanding of how the changes in mechanical stiffness are correlated with structure remains elusive.In this study, we probe the changes in structure and mechanical properties of a F-actin network as a function of ABP concentration. We use electron microscopy (EM) to measure structural changes on the nanometer scale and confocal microscopy to measure structural changes on the micrometer scale. We exploit the technique of multiparticle tracking (MPT), by using small nonbinding particles to directly probe the variations in mesh size with changing ABP concentration, and compare the results with the analysis of images obtained with confocal microscopy. Moreover, we employ MPT to probe the loc...

In two experiments, we examined two related conditioning problems previously investigated by Redhead and Pearce (1995a) and Pearce, Aydin, and Redhead (1997). Experiment 1 involved an A+, B+, C+, AB+, AC+, BC+, ABC2 discrimination. The Rescorla-Wagner model predicts that response to AB, AC, and BC will be greater than that to A, B, and C at asymptote, whereas the Pearce model makes the opposite prediction. In Experiment 2, we investigated the responding to a novel ABC compound in groups trained with either A+, B+, C+ or AB+, AC+, BC+. The Rescorla-Wagner model predicts greater response to ABC in the group trained with A+, B+, C+ than in the group trained with AB+, AC+, BC+, whereas the Pearce model makes the opposite prediction. In contrast to the findings of Redhead and Pearce (1995a) and Pearce et al. (1997) in pigeon autoshaping, our findings in rabbit eyelid conditioning support the Rescorla-Wagner model.

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Insulin secretion is elaborately modulated in pancreatic ß cells within islets of three-dimensional (3D) structures. Using human pluripotent stem cells (hPSCs) to develop islet-like structures with insulin-producing ß cells for the treatment of diabetes is challenging. Here, we report that pancreatic islet-like clusters derived from hESCs are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects. Pancreatic hormone-expressing endocrine cells (ECs) were differentiated from hESCs using a step-wise protocol. The hESC-derived ECs expressed pancreatic endocrine hormones, such as insulin, somatostatin, and pancreatic polypeptide. Notably, dissociated ECs autonomously aggregated to form islet-like, 3D structures of consistent sizes (100–150 μm in diameter). These EC clusters (ECCs) enhanced insulin secretion in response to glucose stimulus and potassium channel inhibition in vitro. Furthermore, ß cell-deficient mice transplanted with ECCs survived for more than 40 d while retaining a normal blood glucose level to some extent. The expression of pancreatic endocrine hormones was observed in tissues transplanted with ECCs. In addition, ECCs could be generated from human induced pluripotent stem cells. These results suggest that hPSC-derived, islet-like clusters may be alternative therapeutic cell sources for treating diabetes.