Cholesterol is an essential component of eukaryotic cell membranes, influencing membrane packing, fluidity, and domain formation. Replicating these properties in model membranes is critical for reconstitution studies, but common emulsion-based methods for producing giant unilamellar vesicles (GUVs) fail to incorporate cholesterol efficiently. Here, we use methyl-β-cyclodextrin–cholesterol (MβCD–CL) complexes to deliver cholesterol into GUVs produced by the emulsion droplet interface crossing encapsulation (eDICE) method and demonstrate a convenient way to quantify the degree of cholesterol incorporation using fluorescent membrane biosensors. Spectral imaging of NR12A as well as fluorescence lifetime imaging of Flipper-TR revealed dose-dependent increases in cholesterol content for DOPC GUVs upon MβCD–CL addition, consistent with increased membrane order. By calibrating these effects against GUVs with defined cholesterol contents prepared via gel-assisted swelling, we found that the cholesterol content of eDICE vesicles can be increased to at least 40 mol%. Binary mixtures of DOPC with saturated lipids (DMPC and PC (18 : 0–14 : 0)) showed a similar trend as pure DOPC GUVs. Interestingly, we could trigger liquid-ordered domain formation by adding cholesterol to DOPC : DMPC vesicles. Our findings provide a quantitative and non-disruptive method to modulate and assess cholesterol content in emulsion-based GUVs, advancing their use in bottom-up synthetic biology and membrane biophysics.

Background: Arterial thrombosis arises under high shear conditions and is a leading cause of ischemic stroke and myocardial infarction. The local hemodynamic environment during the formation of the thrombus influences its final composition, which in turn influences its mechanical characteristics and the associated risk of embolization. However, how arterial shear rates determine the local mechanical properties, structure, and composition of clots remains poorly understood. Objectives: This study investigated how physiological and pathologic arterial shear rates shape blood clot composition, formation dynamics, and regional mechanical properties in a microfluidic flow model. Methods: We used a microfluidic flow model that allows real-time imaging of blood clot formation under controlled arterial shear rates (300/s, 1000/s, and 3500/s). We visualized platelets and fibrin deposition using confocal microscopy and quantified composition-dependent spatial heterogeneities in clot stiffness with microindentation. Results: Pathologically high (3500/s) arterial shear rates promoted more rapid platelet aggregation and the formation of taller platelet-rich clots. Fibrin deposition started after ∼6 minutes in all shear conditions and coincided with platelet aggregate stabilization at higher shear rates. Microindentation revealed that platelet- and fibrin-rich regions were significantly stiffer than red blood cell–rich regions. Clots formed at 3500/s had a >3-fold higher average stiffness than those formed at 300/s, reflecting their higher platelet content. Conclusion: This study demonstrates that arterial shear rate governs clot growth and composition, translating into shear-dependent microscale mechanical properties. These findings provide new mechanistic insights into clot properties that are relevant for clot stability and vulnerability to embolization.

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.

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.

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.

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.

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.

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.

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.

Abstract – Introduction: Arterial thrombosis is a multifaceted process characterized by platelet aggregation and fibrin deposition, leading to the occlusion of blood vessels. It plays a central role in cardiovascular conditions such as myocardial infarction and ischemic stroke. Gaining insight into the mechanisms underlying arterial thrombosis is essential for developing effective treatments aimed at preventing thrombotic events and reducing associated health burdens. In vitro and ex vivo models serve as critical tools for investigating the pathophysiology of arterial thrombosis by providing controlled environments to study thrombus formation and characteristics. This systematic review provides a comprehensive overview of in vitro and ex vivo flow-based models used to study arterial thrombosis, classifying them by scale (macro vs. micro) and evaluating their design principles, physiological relevance, and experimental utility. Methods: A systematic search of Medline, Embase, and Web of Science was conducted using broad and specific terms related to arterial thrombosis models incorporating flow or shear stress. Articles were screened by two independent reviewers. Studies were included if they described in vitro or ex vivo models with dynamic flow; models limited to static or venous conditions or in vivo studies were excluded. In total, 82 studies met the inclusion criteria. Results: Macro-scale models can mimic complex flow patterns in larger arterial conditions and enable the formation of thrombi comparable in size to clinical specimens. Microfluidic models allow precise control over shear conditions and geometry with minimal blood volumes and are suitable for high-resolution imaging and customization, including endothelialization and patient-specific designs. While, both model types present limitations in replicating complex in vivo hemodynamics, standardization, and scalability, they offer valuable, controllable platforms for mechanistic studies and drug testing in arterial thrombosis. Conclusions: While no single model fully recapitulates the in vivo environment, ongoing innovations, particularly in microfabrication and model standardization, continue to improve physiological relevance and clinical translatability.

Cell migration is a fundamental process for life and is highly dependent on the dynamical and mechanical properties of the cytoskeleton. Intensive physical and biochemical crosstalk among actin, microtubules, and intermediate filaments ensures their coordination to facilitate and enable migration. In this review, we discuss the different mechanical aspects that govern cell migration and provide, for each mechanical aspect, a novel perspective by juxtaposing two complementary approaches to the biophysical study of cytoskeletal crosstalk: live-cell studies (often referred to as top-down studies) and cell-free studies (often referred to as bottom-up studies). We summarize the main findings from both experimental approaches, and we provide our perspective on bridging the two perspectives to address the open questions of how cytoskeletal crosstalk governs cell migration and makes cells move.

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE), which make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating >30 μm-sized droplets and only in some cases GUV-sized (∼15 μm) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.