Semiflexible polymer networks are ubiquitous in biological systems, including a scaffolding structure within cells called the actin cytoskeleton. The polymers in these networks are interconnected by transient bonds. For example, actin filaments in the cytoskeleton are physically connected via cross-linker proteins. The mechanical and kinetic properties of the cross-linkers significantly affect the rheological properties of the actin cytoskeleton. Here, we employed an agent-based model to elucidate how the force-dependent behaviors of the cross-linkers determine the material properties of passive networks without molecular motors and the force generation of active networks with molecular motors. The cross-linkers are assumed to behave either as a slip bond, whose dissociation rate increases with forces, or as a catch-slip bond, whose dissociation rate decreases with forces at low force level but increases with forces at high force level. We found that catch-slip-bond cross-linkers can simultaneously increase both the stress and the strain at the yield point. Through a systematic variation in the force dependence of the catch-slip bonds, we identified the specific parameter regimes that enable network reinforcement and enhanced extensibility simultaneously. Specifically, we found that a sufficiently large force threshold for the catch-slip transition is essential for maintaining dynamic force-bearing elements that turnover continuously—a mechanism not achievable with slip bonds. Additionally, we demonstrate that such force-dependent redistribution of the catch-slip bonds substantially enhances internal contractile forces generated by a motor in active networks. Statement of significance: Polymer networks are ubiquitous in industrial and biological systems. The polymers in these networks are often interconnected by transient bonds. The transient bonds behave as a slip bond whose dissociation rate is proportional to forces or as a catch-slip bond whose dissociation rate decreases with increased force (catch) at low force level but increases with increased force (slip) at high force level. In this study, we computationally tested different types of catch-slip bonds to define how the material properties of polymer networks are fine-tuned by each property of molecular bonds. We found that catch-slip bonds can increase both stress and strain at a yield point, which is impossible to achieve without the catch-slip bonds.

Collagen networks form the structural backbone of the extracellular matrix in both healthy and cancerous tissues, exhibiting nonlinear mechanical properties that crucially regulate tissue mechanics and cell behavior. Here, we investigate how the presence of invasive breast cancer cells (MDA-MB-231) influences the polymerization kinetics and mechanics of collagen networks using bulk shear rheology and rheo-confocal microscopy. We show that embedded cancer cells delay the onset of collagen polymerization due to volume exclusion effects. During polymerization, the cells (at 4% volume fraction) cause an unexpected time-dependent softening of the network. We show that this softening effect arises from active remodeling via adhesion and contractility rather than from proteolytic degradation. At higher cell volume fractions, the dominant effect of the cells shifts to volume exclusion, causing a two-fold reduction of network stiffness. Additionally, we demonstrate that cancer cells suppress the characteristic stress-stiffening response of collagen. This effect (partially) disappears when cell adhesion and contractility are inhibited, and it is absent when the cells are replaced by passive hydrogel particles. These findings provide new insights into how active inclusions modify the mechanics of fibrous networks, contributing to a better understanding of the role of cells in the mechanics of healthy and diseased tissues like invasive tumors. Statement of significance: Understanding how cells influence tissue mechanics is crucial to unravel disease progression. While fibroblasts are known to stiffen tissues, the role of invasive cancer cells is less clear. Using collagen-based tissue models, we reveal that cancer cells unexpectedly soften the collagen matrix and disrupt its stress-stiffening response. By comparing active cells to passive particles and selectively blocking cell functions, we show that volume exclusion, adhesion, and contractility each play distinct roles in shaping tissue mechanics. This work sheds light on the physical impact of cancer cells on their environment, advancing our understanding on how cells dynamically alter the mechanical properties of tissues.

Collagen is the most abundant protein in the human body and plays an essential role in determining the mechanical properties of the tissues. Both as a monomeric protein and in fibrous assemblies, collagen interacts with its surrounding molecules, in particular with water. Interestingly, while it is well established that the interaction with water strongly influences the molecular and mechanical properties of collagen and its assemblies, the underlying mechanisms remain largely unknown. Here, we review the research conducted over the past 30 years on the interplay between water and collagen and its relevance for tissue properties. We discuss the water–collagen interaction on relevant time- and length scales, ranging from the vital role of water in stabilizing the characteristic triple helix structure to the negative impact of dehydration on the mechanical properties of tissues. A better understanding of the water–collagen interaction will help to unravel the effect of mutations and defective collagen production in collagen-related diseases and to pinpoint the key design features required to synthesize collagen-based biomimetic tissues with tailored mechanical properties.

Complex morphogenetic processes such as cell division require a tight coordination of the activities of microtubules and actin filaments. There is evidence that anillin, conventionally known as an actin-binding and -bundling protein, regulates microtubule/actin crosstalk during cell division. However, it is unknown whether anillin binds directly to microtubules and whether it is sufficient to establish crosslinking between microtubules and actin filaments. Here we address both questions by developing an in vitro system for observing anillin-mediated interactions with actin filaments and dynamic microtubules via total internal-reflection fluorescence microscopy. We find that anillin can interact directly with microtubules and promote microtubule bundling. We confirm that anillin binds and bundles actin filaments, and find that it has a strong preference for actin bundles over individual filaments. Moreover, we show that anillin can directly crosslink microtubules and actin filaments, cause sliding of actin filaments on the microtubule lattice, and transport actin filaments by the growing microtubule tip. Our findings indicate that anillin can potentially serve as a direct regulator of microtubule/actin crosstalk, e.g., during cell division.

The extracellular matrix (ECM) provides structural support to cells, thereby forming a functional tissue. In cancer, the growth of the tumor creates internal mechanical stress, which, together with the remodeling activity of tumor cells and fibroblasts, alters the ECM structure, leading to an increased stiffness of the pathological ECM. The enhanced ECM stiffness, in turn, stimulates tumor growth and activates tumor-promoting fibroblasts and tumor cell migration, leading to metastasis and increased therapy resistance. While the relationship between matrix stiffness and migration has been studied before, their connection to internal tumor stress remains unresolved. Here we used 3D ECM-embedded spheroids and hydrogel particle stress sensors to quantify and correlate internal tumor spheroid pressure, ECM stiffness, ECM remodeling, and tumor cell migration. We note that 4T1 breast cancer spheroids and SV80 fibroblast spheroids showed increased invasion—described by area, complexity, number of branches, and branch area—in a stiffer, cross-linked ECM. On the other hand, changing ECM stiffness only minimally changed the radial alignment of fibers but highly changed the amount of fibers. For both cell types, the pressure measured in spheroids gradually decreased as the distance into the ECM increased. For 4T1 spheroids, increased ECM stiffness resulted in a further reach of mechanical stress into the ECM, which, together with the invasive phenotype, was reduced by inhibition of ROCK-mediated contractility. By contrast, such correlation between ECM stiffness and stress-reach was not observed for SV80 spheroids. Our findings connect ECM stiffness with tumor invasion, ECM remodeling, and the reach of tumor-induced mechanical stress into the ECM. Such mechanical connections between tumor and ECM are expected to drive early steps in cancer metastasis.

Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but it reduced cell stiffness. Mitochondria and endoplasmic reticulum (ER) sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

The single most common microbe causing cardiovascular infections is Staphylococcus aureus (S. aureus). S. aureus produces coagulase that converts fibrinogen to fibrin, which is incorporated into biofilms. This process aids in adherence to intravascular structures, defense against the host immune system, and resistance to antimicrobial treatment. Despite its significance, fibrin formation in S. aureus biofilms remains poorly understood. Therefore, this study aimed to elucidate the early development of cardiovascular biofilms. Clinically isolated coagulase-positive S. aureus and coagulase-negative Staphylococcus lugdunensis (S. lugdunensis) from patients with cardiovascular infections, and a coagulase mutant S. aureus Δcoa, were grown in tryptic soy broth (TSB), Iscove's Modified Dulbecco's Medium (IMDM), and pooled human plasma, with or without porcine heart valves. Bacterial growth, metabolic activity, and bacterial fibrinogen utilization were measured over 24 h at 37 °C. Time-lapse confocal microscopy was used to visualize and track biofilm development. S. aureus exhibited more growth in TSB and human plasma than S. lugdunensis and S. aureus Δcoa, but showed similar growth in IMDM after 24 h. Peak metabolic activity for all isolates was highest in TSB and lowest in human plasma. The presence of porcine valves caused strain-dependent alterations in time to peak metabolic activity. Confocal imaging revealed fibrin-based biofilm development exclusively in the coagulase-producing S. aureus strains. Between 2 and 6 h of biofilm development, 74.9 % (p = 0.034) of the fibrinogen from the medium was converted to fibrin. Variations in fibrin network porosity and density were observed among different coagulase-producing S. aureus strains. Fibrin formation is mediated by S. aureus coagulase and first strands occurred within 3 h for clinical strains after exposure to human plasma. This study stresses the importance of experimental design given the bacterial changes due to different media and substrates and provides insights into the early pathogenesis of S. aureus cardiovascular biofilms.

The biofabrication of recombinant structural proteins with a range of mechanical or structural features usually relies on the generation of protein libraries displaying variations in terms of amino acid composition, block structure, molecular weight, or physical/chemical cross-linking sites. This approach, while highly successful in generating a wealth of knowledge regarding the links between design features and material properties, has some inherent limitations related to its low throughput. This slows down the pace of the development of de novo recombinant structural proteins. Here, we propose an approach to tune the viscoelastic properties of temperature-responsive hydrogels made of silk-elastin-like polypeptides (SELPs) without modifying their sequence. To do so, we subject purified SELPs to two different postprocessing methods─water annealing or EtOH annealing─that alter the topology of highly disordered SELP networks via the formation of ordered intermolecular β-sheet physical cross-links. Combining different analytical techniques, we connect the order/disorder balance in SELPs with their gelling behavior. Furthermore, we show that introducing a functional block (in this case, a biomineralizing peptide) in the sequence of SELPs can disrupt its self-assembly and that such disruption can only be overcome by EtOH annealing. Our results suggest that postprocessing of as-purified SELPs might be a simple approach to tune the self-assembly of SELPs into biomaterials with bespoke viscoelastic properties beyond the traditional approach of developing SELP libraries via genetic engineering.

Plectin is a giant protein of the plakin family that cross-links the cytoskeleton of mammalian cells. It is expressed in virtually all tissues, and its dysfunction is associated with various diseases such as skin blistering. There is evidence that plectin regulates the mechanical integrity of the cytoskeleton in diverse cell and tissue types. However, it is unknown how plectin modulates the mechanical response of cells depending on the frequency and amplitude of mechanical loading. Here we demonstrate the role of plectin in the viscoelastic properties of fibroblasts at small and large deformations by quantitative single-cell compression measurements. To identify the importance of plectin, we compared the mechanical properties of wild-type (Plec^+/+) fibroblasts and plectin knockout (Plec^−/−) fibroblasts. We show that plectin knockout cells are nearly twofold softer than wild-type cells, but their strain-stiffening behavior is similar. Plectin deficiency also caused faster viscoelastic stress relaxation at long times. Fluorescence recovery after photobleaching experiments indicated that this was due to threefold faster actin turnover. Short-time poroelastic relaxation was also faster in Plec^−/− cells compared with Plec^+/+ cells, suggesting a more sparse cytoskeletal network. Confocal imaging indicated that this was due to a marked change in the architecture of the vimentin network, from a fine meshwork in wild-type cells to a bundled network in the plectin knockout cells. Our findings therefore indicate that plectin is an important regulator of the organization and viscoelastic properties of the cytoskeleton in fibroblasts. Our findings emphasize that mechanical integration of the different cytoskeletal networks present in cells is important for regulating the versatile mechanical properties of cells.

Cancer cells can utilize different invasion strategies to overcome physical arrest during confined migration through tissues with small pores. Cancer cell plasticity allows switches between different migration modes and transitions between single-cell and collective migration. The biophysical parameters that guide these decisions are poorly understood. In this work, we investigated the link between cell deformability and migration efficacy in constrictions of two mesenchymal cancer cell-types with similar invasion strategies: HT1080 fibrosarcoma cells and MV3 melanoma cells. To this end, we designed microfluidic platforms for (1) high-throughput cell deformability measurements and (2) migration through a variety of confining geometries. We measured different deformabilities for HT1080 and MV3 cells and correlated this with their migration efficacy through confinements. However, higher deformability and improved squeezing ability did not impact path selection at junctions of channels of different widths. Our findings show that cell deformability correlates with better squeezing abilities through confinements, but minimally impacts confinement directionality.