Despite their overwhelming complexity, living cells display a high degree of internal mechanical and functional organization which can largely be attributed to the intracellular biopolymer scaffold, the cytoskeleton. Being a very complex system far from thermodynamic equilibrium, the cytoskeleton's ability to organize is at the same time challenging and fascinating. The extensive amounts of frequently interacting cellular building blocks and their inherent multifunctionality permits highly adaptive behavior and obstructs a purely reductionist approach. Nevertheless (and despite the field's relative novelty), the physics approach has already proved to be extremely successful in revealing very fundamental concepts of cytoskeleton organization and behavior. This review aims at introducing the physics of the cytoskeleton ranging from single biopolymer filaments to multicellular organisms. Throughout this wide range of phenomena, the focus is set on the intertwined nature of the different physical scales (levels of complexity) that give rise to numerous emergent properties by means of self-organization or self-assembly.

The mechanics of complex soft matter often cannot be understood in the classical physical frame of flexible polymers or rigid rods. The underlying constituents are semiflexible polymers, whose finite bending stiffness (κ) leads to non-trivial mechanical responses. A natural model for such polymers is the protein actin. Experimental studies of actin networks, however, are limited since the persistence length (lp ∝ κ) cannot be tuned. Here, we experimentally characterize this parameter for the first time in entangled networks formed by synthetically produced, structurally tunable DNA nanotubes. This material enabled the validation of characteristics inherent to semiflexible polymers and networks thereof, i.e., persistence length, inextensibility, reptation and mesh size scaling. While the scaling of the elastic plateau modulus with concentration G0 ∝ c 7/5 is consistent with previous measurements and established theories, the emerging persistence length scaling G0 ∝ lp opposes predominant theoretical predictions.

F-actin bundles are prominent cytoskeletal structures in eukaryotes. They provide mechanical stability in stereocilia, microvilli, filopodia, stress fibers and the sperm acrosome. Bundles are typically stabilized by a wide range of specific crosslinking proteins, most of which exhibit off-rates on the order of 1s(-1). Yet F-actin bundles exhibit structural and mechanical integrity on time scales that are orders of magnitude longer. By applying large deformations to reconstituted F-actin bundles using optical tweezers, we provide direct evidence of their differential mechanical response in vitro: bundles exhibit fully reversible, elastic response on short time scales and irreversible, elasto-plastic response on time scales that are long compared to the characteristic crosslink dissociation time. Our measurements show a broad range of characteristic relaxation times for reconstituted F-actin bundles. This can be reconciled by considering that bundle relaxation behavior is also modulated by the number of filaments, crosslinking type and occupation number as well as the consideration of defects due to filament ends.

Actin networks are adaptive materials enabling dynamic and static functions of living cells. A central element for tuning their underlying structural and mechanical properties is the ability to reversibly connect, i.e., transiently crosslink, filaments within the networks. Natural crosslinkers, however, vary across many parameters. Therefore, systematically studying the impact of their fundamental properties like size and binding strength is unfeasible since their structural parameters cannot be independently tuned. Herein, this problem is circumvented by employing a modular strategy to construct purely synthetic actin crosslinkers from DNA and peptides. These crosslinkers mimic both intuitive and noncanonical mechanical properties of their natural counterparts. By isolating binding affinity as the primary control parameter, effects on structural and dynamic behaviors of actin networks are characterized. A concentration-dependent triphasic behavior arises from both strong and weak crosslinkers due to emergent structural polymorphism. Beyond a certain threshold, strong binding leads to a nonmonotonic elastic pulse, which is a consequence of self-destruction of the mechanical structure of the underlying network. The modular design also facilitates an orthogonal regulatory mechanism based on enzymatic cleaving. This approach can be used to guide the rational design of further biomimetic components for programmable modulation of the properties of biomaterials and cells.

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell's response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

Composite networks of actin and vimentin filaments can be described by a superposition via an inelastic glassy wormlike chain model.

Biopolymer networks contribute mechanical integrity as well as functional organization to living cells. One of their major constituents, the protein actin, is present in a large variety of different network architectures, ranging from extensive networks to densely packed bundles. The shape of the network is directly linked to its mechanical properties and essential physiological functions. However, a profound understanding of architecture-determining mechanisms and their physical constraints remains elusive. We use experimental bottom-up systems to study the formation of confined actin networks by entropic forces. Experiments based on molecular crowding as well as counterion condensation reveal a generic tendency of homogeneous filament solutions to aggregate into regular actin bundle networks connected by aster-like centers. The network architecture is found to critically rely on network formation history. Starting from identical biochemical compositions, we observe drastic changes in network architecture as a consequence of initially biased filament orientation or mixing-induced perturbations. Our experiments suggest that the tendency to form regularly spaced bundle networks is a rather general feature of isotropic, homogeneous filament solutions subject to uniform attractive interactions. Due to the fundamental nature of the considered interactions, we expect that the investigated type of network formation further implies severe physical constraints for cytoskeleton self-organization on the more complex level of living cells.

Semiflexible polymers form central structures in biological material. Modeling approaches usually neglect influences of polymer-specific molecular features aiming to describe semiflexible polymers universally. Here, we investigate the influence of molecular details on networks assembled from filamentous actin, intermediate filaments, and synthetic DNA nanotubes. In contrast to prevalent theoretical assumptions, we find that bulk properties are affected by various inter-filament interactions. We present evidence that these interactions can be merged into a single parameter in the frame of the glassy wormlike chain model. The interpretation of this parameter as a polymer specific stickiness is consistent with observations from macro-rheological measurements and reptation behavior. Our findings demonstrate that stickiness should generally not be ignored in semiflexible polymer models. MATERIALS AND METHODS KeratinRecombinant human keratins K8 and K18 were expressed, purified and prepared as described in [50,51]. Briefly, proteins were expressed in E. coli, purified and stored in 8 M urea at −80 • C. Before use, K8 and K18 were mixed in equimolar ratios and renatured by dialysis against 8 M urea, 2 mM Tris-HCl (pH 9.0) and 1 mM DTT with stepwise reduction of the urea concentration (6 M, 4 M, 2 M, 1 M, 0 M). Each dialysis step was done for 20 min at room temperature, then the dialysis was continued overnight against 2 mM Tris-HCl, pH 9.0, 1 mM DTT at 4 • C. The dialyzed protein was kept on ice for a maximum of four days. The final protein concentration was determined by measuring the absorption at 280 nm using a DU 530 UV/Vis Spectrophotometer (Beckman Coulter Inc., USA). Assembly of keratin was initiated by addition of an equal volume of 18 mM Tris-HCl buffer (pH 7.0) to renatured keratins resulting in a final buffer condition of 10 mM Tris-HCl (pH 7.4). Double-Crossover DNA NanotubesAll oligomers for hybridization of the DNA nanotubes were adapted from [Table SII] and purchased from Biomers.net with HPLC purification. In order to assemble a nanotube network of a desired concentration the required strands (SE1-SE5) were mixed in equimolar concentration in an assembly buffer containing 40 mM Tris-acetate, 1 mM EDTA and 12.5 mM Mg 2+ (pH 8.3). The concentration of each stock solution was confirmed spectrophotometrically by a NanoDrop 1000 (Thermo Fisher Scientifc Inc., USA) at a wavelength of 260 nm. These strands were hybridized in a TProfessional Standard PCR Thermocycler (Core Life Sciences Inc.,USA) by denaturation for 10 min at 90 • C and complementary base pairing for 20 h between 80 • C and 20 • C by lowering the temperature by 0.5 K every 10 min. After hybridization DNA nanotubes were stored at room temperature. For visualization the oligomer SE3 was modified with the fluorescent Cyanine dye 3 with two additional spacer thymine bases in between. DNA nanotubes were labeled by partially or fully replacing the unlabeled oligo SE3 by SE3-Cy3. Actin