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

The cytoskeleton is a major determinant of cell mechanics, and alterations in the central mechanical aspects of cells are observed during many pathological situations. Therefore, it is essential to investigate...

Networks of crosslinked keratin filaments are abundant in epithelial cells and tissues, providing resilience against mechanical forces and ensuring cellular integrity. Although studies of in vitro models of reconstituted keratin networks have revealed important mechanical aspects, the mechanical properties of crosslinked keratin structures remain poorly understood. Here, we exploited the power of electron beam irradiation (EBI) to crosslink in vitro networks of soft epithelial keratins 8 and 18 (k8–k18) filaments with different irradiation doses (30 kGy, 50 kGy, 80 kGy, 100 kGy, and 150 kGy). We combined bulk shear rheology with confocal microscopy to investigate the impact of crosslinking on the mechanical and structural properties of the resultant keratin gels. We found that irradiated keratin gels display higher linear elastic modulus than the unirradiated, entangled networks at all doses tested. However, at the high doses (80 kGy, 100 kGy, and 150 kGy), we observed a remarkable drop in the elastic modulus compared to 50 kGy. Intriguingly, the irradiation drastically changed the behavior for large, nonlinear deformations. While untreated keratin networks displayed a strong strain stiffening, increasing irradiation doses shifted the system to a strain softening behavior. In agreement with the rheological behavior in the linear regime, the confocal microscopy images revealed fully isotropic networks with high percolation in 30 kGy and 50 kGy-treated keratin samples, while irradiation with 100 kGy induced the formation of thick bundles and clusters. Our results demonstrate the impact of permanent crosslinking on k8–k18 mechanics and provide new insights into the potential contribution of intracellular covalent crosslinking to the loss of mechanical resilience in some human keratin diseases. These insights will also provide inspiration for the synthesis of new keratin-based biomaterials.