Despite their importance for the proper function of living cells, the physical properties of cross-linked actin networks remain poorly understood as the occurrence of heterogeneities hamper a quantitative physical description. The isotropic homogeneously cross-linked actin network presented here enables us to quantitatively relate the network response to a single filament model by determining the dominating length scale. The frequency dependence of the linear response and nonuniversal form of the nonlinear response reveal the importance of cross-linker unbinding events.

In living cells the mechanical properties of the actin cytoskeleton are defined by the local activation of different actin cross-linking proteins. These proteins consist of actin-binding domains that are separated and geometrically organized by different numbers of rod domains. The detailed molecular structure of the cross-linking molecules determines the structural and mechanical properties of actin networks in vivo. In this study, we systematically investigate the impact of the length of the spacing unit between two actinbinding domains on in vitro actin networks. Such synthetic crosslinkers reveal that the shorter the constructs are, the greater the elastic modulus changes in the linear response regime. Because the same binding domains are used in all constructs, only the differences in the number of rod domains determine their mechanical effectiveness. Structural rearrangements of the networks show that bundling propensity is highest for the shortest construct. The nonlinear mechanical response is affected by the molecular structure of the cross-linker molecules, and the observed critical strains and fracture stress increase proportional to the length of the spacing unit. actin cytoskeleton ͉ cross-linking molecules ͉ mechanical properties F or living cells, tight control of the structure and mechanics of their cytoskeleton is crucial for the cells to function properly. The dynamic and local reorganization of one of the major constituents, the actin network, is coordinated by various actin-binding proteins (ABPs). Cross-linking proteins are a major class of ABPs, consisting of actin-binding domains, which are separated and geometrically organized by different numbers of rod domains. Cross-linker molecules vary (i) in the type of actin-binding affinity caused by specific binding domains used and (ii) in the structure, number, and organization of their spacing rod domains. Both the geometrical structure and actin-binding affinity of ABPs are believed to determine mechanical function in vivo (1). Instead of classifying the ABPs by the mechanical function of the cross-linker molecules, the architecture of actin networks is commonly used for a classification of different cross-linking molecules. Whereas very short cross-linkers, such as plastin or fascin, are generally classified as bundling proteins, longer cross-linkers, such as ␣-actinin or filamin, are thought to induce orthogonal isotropic networks. This qualitative classification does not consider the observed concentration dependence of the structural rearrangements in actin networks (2-4). Different phases, from isotropic cross-linked, composite, or purely bundled networks, have been predicted to occur depending on the interaction potential between rods and the concentrations of linkers and rods (5, 6). Nevertheless, the effect of the structural rearrangements on the mechanical properties of such cross-linked networks is not fully understood. Thus, a correlation of the specific molecular structure of the cross-linker to the resulting network structure and...

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The structure and rheology of cytoskeletal networks are regulated by actin binding proteins. Aside from these specific interactions, depletion forces can also alter the properties of cytoskeletal networks. Here we demonstrate that the addition of poly(ethylene glycol) (PEG) as a depletion agent results not only in severe structural changes, but also in alterations in mechanical properties of actin solutions. In the plateau of the elastic modulus two regimes can be distinguished by micro and macrorheological methods. In the first, the elastic modulus increases only slightly with increasing depletion agent, whereas above a critical concentration c*, a strong increase of cPEG6k3.5 is observed in a distinct second regime. Microrheological data and electron microscopy images show a homogenous network of actin filaments in the first regime, whereas at higher PEG concentrations a network of actin bundles is observed. The concentration dependence of the plateau modulus G0, the shift in entanglement time taue, and the nonlinear response indicate that below c* the network becomes effectively cross-linked, whereas above c* G0(cPEG6k) is primarily determined by the network of bundles that exhibits a linearly increasing bundle thickness.

The cytoskeleton is a complex polymer network that plays an essential role in the functionality of eukaryotic cells. It endows cells with mechanical stability, adaptability, and motility. To identify and understand the mechanisms underlying this large variety of capabilities and to possibly transfer them to engineered networks makes it necessary to have in vitro and in silico model systems of the cytoskeleton. These models must be realistic representatives of the cellular network and at the same time be controllable and reproducible. Here, an approach to design complementary experimental and numerical model systems of the actin cytoskeleton is presented and some of their properties discussed.