Origin of slow stress relaxation in the cytoskeleton

Dynamically crosslinked semiflexible biopolymers such as the actin cytoskeleton govern the mechanical behavior of living cells. Semiflexible biopolymers nonlinearly stiffen in response to mechanical loads, whereas the crosslinker dynamics allow for stress relaxation over time. Here we show, through rheology and theoretical modeling, that the combined nonlinearity in time and stress leads to an unexpectedly slow stress relaxation, similar to the dynamics of disordered systems close to the glass transition. Our work suggests that transient crosslinking combined with internal stress can explain prior reports of soft glassy rheology of cells, in which the shear modulus increases weakly with frequency.Comment: 6 pages, 4 figure

Uncovering the dynamic precursors to motor-driven contraction of active gels

Cells and tissues have the remarkable ability to actively generate the forces required to change their shape. This active mechanical behavior is largely mediated by the actin cytoskeleton, a crosslinked network of actin filaments that is contracted by myosin motors. Experiments and active gel theories have established that the length scale over which gel contraction occurs is governed by a balance between molecular motor activity and crosslink density. By contrast, the dynamics that govern the contractile activity of the cytoskeleton remain poorly understood. Here we investigate the microscopic dynamics of reconstituted actin-myosin networks using simultaneous real-space video microscopy and Fourier-space dynamic light scattering. Light scattering reveals rich and unanticipated microscopic dynamics that evolve with sample age. We uncover two dynamical precursors that precede macroscopic gel contraction. One is characterized by a progressive acceleration of stress-induced rearrangements, while the other consists of sudden rearrangements that depend on network adhesion to the boundaries and are highly heterogeneous. Our findings reveal an intriguing analogy between self-driven rupture and collapse of active gels to the delayed rupture of passive gels under external loads

Stress management in composite biopolymer networks

Living tissues show an extraordinary adaptiveness to strain, which is crucial for their proper biological functioning. The physical origin of this mechanical behaviour has been widely investigated using reconstituted networks of collagen fibres, the principal load-bearing component of tissues. However, collagen fibres in tissues are embedded in a soft hydrated polysaccharide matrix which generates substantial internal stresses whose effect on tissue mechanics is unknown. Here, by combining mechanical measurements and computer simulations, we show that networks composed of collagen fibres and a hyaluronan matrix exhibit synergistic mechanics characterized by an enhanced stiffness and delayed strain-stiffening. We demonstrate that the polysaccharide matrix has a dual effect on the composite response involving both internal stress and elastic reinforcement. Our findings elucidate how tissues can tune their strain-sensitivity over a wide range and provide a novel design principle for synthetic materials with programmable mechanical properties

Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly

International audienceSeptins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of pu- rified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy

Cytoplasmic diffusion: molecular motors mix it up

Random motion within the cytoplasm gives rise to molecular diffusion; this motion is essential to many biological processes. However, in addition to thermal Brownian motion, the cytoplasm also undergoes constant agitation caused by the activity of molecular motors and other nonequilibrium cellular processes. Here, we discuss recent work that suggests this activity can give rise to cytoplasmic motion that has the appearance of diffusion but is significantly enhanced in its magnitude and which can play an important biological role, particularly in cytoskeletal assembly

Normal stresses in semiflexible polymer hydrogels

Biopolymer gels such as fibrin and collagen networks are known to develop tensile axial stress when subject to torsion. This negative normal stress is opposite to the classical Poynting effect observed for most elastic solids including synthetic polymer gels, where torsion provokes a positive normal stress. As recently shown, this anomalous behavior in fibrin gels depends on the open, porous network structure of biopolymer gels, which facilitates interstitial fluid flow during shear and can be described by a phenomenological two-fluid model with viscous coupling between network and solvent. Here we extend this model and develop a microscopic model for the individual diagonal components of the stress tensor that determine the axial response of semi-flexible polymer hydrogels. This microscopic model predicts that the magnitude of these stress components depends inversely on the characteristic strain for the onset of nonlinear shear stress, which we confirm experimentally by shear rheometry on fibrin gels. Moreover, our model predicts a transient behavior of the normal stress, which is in excellent agreement with the full time-dependent normal stress we measure.Comment: 12 pages, 8 figure

Non-equilibrium microtubule fluctuations in a model cytoskeleton

Biological activity gives rise to non-equilibrium fluctuations in the cytoplasm of cells; however, there are few methods to directly measure these fluctuations. Using a reconstituted actin cytoskeleton, we show that the bending dynamics of embedded microtubules can be used to probe local stress fluctuations. We add myosin motors that drive the network out of equilibrium, resulting in an increased amplitude and modified time-dependence of microtubule bending fluctuations. We show that this behavior results from step-like forces on the order of 10 pN driven by collective motor dynamics