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...

Cells make use of semiflexible biopolymers such as actin or intermediate filaments to control their local viscoelastic response by dynamically adjusting the concentration and type of cross-linking molecules. The microstructure of the resulting networks mainly determines their mechanical properties. It remains an important challenge to relate structural transitions to both the molecular properties of the cross-linking molecules and the mechanical response of the network. This can be achieved best by well defined in vitro model systems in combination with microscopic techniques. Here, we show that with increasing concentrations of the cross-linker heavy meromyosin, a transition in the mechanical network response occurs. At low cross-linker densities the network elasticity is dominated by the entanglement length l(e) of the polymer, whereas at high heavy meromyosin densities the cross-linker distance l(c) determines the elastic behavior. Using microrheology the formation of heterogeneous networks is observed at low cross-linker concentrations. Micro- and macrorheology both report the same transition to a homogeneous cross-linked phase. This transition is set by a constant average cross-linker distance l(c) approximately 15 microm. Thus, the micro- and macromechanical properties of isotropically cross-linked in vitro actin networks are determined by only one intrinsic network parameter.

Natural killer (NK) cells are important early responders against viral infections. Changes in metabolism are crucial to fuel NK cell responses, and altered metabolism is linked to NK cell dysfunction in obesity and cancer. However, very little is known about the metabolic requirements of NK cells during acute retroviral infection and their importance for antiviral immunity. Here, using the Friend retrovirus mouse model, we show that following infection NK cells increase nutrient uptake, including amino acids and iron, and reprogram their metabolic machinery by increasing glycolysis and mitochondrial metabolism. Specific deletion of the amino acid transporter Slc7a5 has only discrete effects on NK cells, but iron deficiency profoundly impaires NK cell antiviral functions, leading to increased viral loads. Our study thus shows the requirement of nutrients and metabolism for the antiviral activity of NK cells, and has important implications for viral infections associated with altered iron levels such as HIV and SARS-CoV-2.

A hexathymidine-DNA “hexT” tolerates acid and coinage metal catalysts, and can be ligated to coding DNA sequences.

BackgroundDespite the high level of protection against severe COVID-19 provided by the currently available vaccines some breakthrough infections occur. Until now, there is no information whether a potential risk of a breakthrough infection can be inferred from the level of antibodies after booster vaccination.MethodsLevels of binding antibodies and neutralization capacity after the first, one and six month after the second, and one month after the third (booster) vaccination against COVID-19 were measured in serum samples from 1391 healthcare workers at the University Hospital Essen. Demographics, vaccination scheme, pre-infection antibody titers and neutralization capacity were compared between individuals with and without breakthrough infections.ResultsThe risk of developing an Omicron breakthrough infection was independent of vaccination scheme, sex, body mass index, smoking status or pre-existing conditions. In participants with low pre-infection anti-spike antibodies (≤ 2641.0 BAU/ml) and weaker neutralization capacity (≤ 65.9%) against Omicron one month after the booster vaccination the risk for developing an Omicron infection was 10-fold increased (P = 0.001; 95% confidence interval, 2.36 - 47.55).ConclusionRoutine testing of anti-SARS-CoV-2 IgG antibodies and surrogate virus neutralization can quantify vaccine-induced humoral immune response and may help to identify subjects who are at risk for a breakthrough infection. The establishment of thresholds for SARS-CoV-2 IgG antibody levels identifying “non”-, “low” and “high”-responders may be used as an indication for re-vaccination.