Transcription inhibition suppresses nuclear blebbing and rupture independently of nuclear rigidity.

Chromatin plays an essential role in the nuclear mechanical response and determining nuclear shape, which maintain nuclear compartmentalization and function. However, major genomic functions, such as transcription activity, might also impact cell nuclear shape via blebbing and rupture through their effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with several RNA polymerase II inhibitors in wild-type cells and perturbed cells that presented increased nuclear blebbing. Transcription inhibition suppressed nuclear blebbing for several cell types, nuclear perturbations and transcription inhibitors. Furthermore, transcription inhibition suppressed nuclear bleb formation, bleb stabilization and bleb-based nuclear ruptures. Interestingly, transcription inhibition did not alter the histone H3 lysine 9 (H3K9) modification state, nuclear rigidity, and actin compression and contraction, which typically control nuclear blebbing. Polymer simulations suggested that RNA polymerase II motor activity within chromatin could drive chromatin motions that deform the nuclear periphery. Our data provide evidence that transcription inhibition suppresses nuclear blebbing and rupture, in a manner separate and distinct from chromatin rigidity.

Emergence of tissue-like mechanics from fibrous networks confined by close-packed cells.

The viscoelasticity of the crosslinked semiflexible polymer networks-such as the internal cytoskeleton and the extracellular matrix-that provide shape and mechanical resistance against deformation is assumed to dominate tissue mechanics. However, the mechanical responses of soft tissues and semiflexible polymer gels differ in many respects. Tissues stiffen in compression but not in extension1-5, whereas semiflexible polymer networks soften in compression and stiffen in extension6,7. In shear deformation, semiflexible polymer gels stiffen with increasing strain, but tissues do not1-8. Here we use multiple experimental systems and a theoretical model to show that a combination of nonlinear polymer network elasticity and particle (cell) inclusions is essential to mimic tissue mechanics that cannot be reproduced by either biopolymer networks or colloidal particle systems alone. Tissue rheology emerges from an interplay between strain-stiffening polymer networks and volume-conserving cells within them. Polymer networks that soften in compression but stiffen in extension can be converted to materials that stiffen in compression but not in extension by including within the network either cells or inert particles to restrict the relaxation modes of the fibrous networks that surround them. Particle inclusions also suppress stiffening in shear deformation; when the particle volume fraction is low, they have little effect on the elasticity of the polymer networks. However, as the particles become more closely packed, the material switches from compression softening to compression stiffening. The emergence of an elastic response in these composite materials has implications for how tissue stiffness is altered in disease and can lead to cellular dysfunction9-11. Additionally, the findings could be used in the design of biomaterials with physiologically relevant mechanical properties.

Extracellular vimentin is sufficient to promote cell attachment, spreading, and motility by a mechanism involving N-acetyl glucosamine-containing structures.

Vimentin intermediate filaments form part of the cytoskeleton of mesenchymal cells, but under pathological conditions often associated with inflammation, vimentin filaments depolymerize as the result of phosphorylation or citrullination, and vimentin oligomers are secreted or released into the extracellular environment. In the extracellular space, vimentin can bind surfaces of cells and the extracellular matrix, and the interaction between extracellular vimentin and cells can trigger changes in cellular functions, such as activation of fibroblasts to a fibrotic phenotype. The mechanism by which extracellular vimentin binds external cell membranes and whether vimentin alone can act as an adhesive anchor for cells is largely uncharacterized. Here, we show that various cell types (normal and vimentin null fibroblasts, mesenchymal stem cells, and A549 lung carcinoma cells) attach to and spread on polyacrylamide hydrogel substrates covalently linked to vimentin. Using traction force microscopy and spheroid expansion assays, we characterize how different cell types respond to extracellular vimentin. Cell attachment to and spreading on vimentin-coated surfaces is inhibited by hyaluronic acid degrading enzymes, hyaluronic acid synthase inhibitors, soluble heparin or N-acetyl glucosamine, all of which are treatments that have little or no effect on the same cell types binding to collagen-coated hydrogels. These studies highlight the effectiveness of substrate-bound vimentin as a ligand for cells and suggest that carbohydrate structures, including the glycocalyx and glycosylated cell surface proteins that contain N-acetyl glucosamine, form a novel class of adhesion receptors for extracellular vimentin that can either directly support cell adhesion to a substrate or fine-tune the glycocalyx adhesive properties.

Vimentin tunes cell migration on collagen by controlling ß1 integrin activation and clustering.

Vimentin is a structural protein that is required for mesenchymal cell migration and directly interacts with actin, ß1 integrin and paxillin. We examined how these interactions enable vimentin to regulate cell migration on collagen. In fibroblasts, depletion of vimentin increased talin-dependent activation of ß1 integrin by more than 2-fold. Loss of vimentin was associated with reduction of ß1 integrin clustering by 50% and inhibition of paxillin recruitment to focal adhesions by more than 60%, which was restored by vimentin expression. This reduction of paxillin was associated with 65% lower Cdc42 activation, a 60% reduction of cell extension formation and a greater than 35% decrease in cell migration on collagen. The activation of PAK1, a downstream effector of Cdc42, was required for vimentin phosphorylation and filament maturation. We propose that vimentin tunes cell migration through collagen by acting as an adaptor protein for focal adhesion proteins, thereby regulating ß1 integrin activation, resulting in well-organized, mature integrin clusters.This article has an associated First Person interview with the first author of the paper.

Unique Role of Vimentin Networks in Compression Stiffening of Cells and Protection of Nuclei from Compressive Stress.

In this work, we investigate whether stiffening in compression is a feature of single cells and whether the intracellular polymer networks that comprise the cytoskeleton (all of which stiffen with increasing shear strain) stiffen or soften when subjected to compressive strains. We find that individual cells, such as fibroblasts, stiffen at physiologically relevant compressive strains, but genetic ablation of vimentin diminishes this effect. Further, we show that unlike networks of purified F-actin or microtubules, which soften in compression, vimentin intermediate filament networks stiffen in both compression and extension, and we present a theoretical model to explain this response based on the flexibility of vimentin filaments and their surface charge, which resists volume changes of the network under compression. These results provide a new framework by which to understand the mechanical responses of cells and point to a central role of intermediate filaments in response to compression.

Antibacterial and Cytocompatible pH-Responsive Peptide Hydrogel.

A short peptide, FHHF-11, was designed to change stiffness as a function of pH due to changing degree of protonation of histidines. As pH changes in the physiologically relevant range, G' was measured at 0 Pa (pH 6) and 50,000 Pa (pH 8). This peptide-based hydrogel is antimicrobial and cytocompatible with skin cells (fibroblasts). It was demonstrated that the incorporation of unnatural AzAla tryptophan analog residue improves the antimicrobial properties of the hydrogel. The material developed can have a practical application and be a paradigm shift in the approach to wound treatment, and it will improve healing outcomes for millions of patients each year.

Extracellular Vimentin as a Target Against SARS-CoV-2 Host Cell Invasion.

Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. The primary receptor for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2), yet new studies reveal the importance of additional extracellular co-receptors that mediate binding and host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens' cellular uptake. Biophysical and cell infection studies are done to determine whether vimentin might bind SARS-CoV-2 and facilitate its uptake. Dynamic light scattering shows that vimentin binds to pseudovirus coated with the SARS-CoV-2 spike protein, and antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cells. The results are consistent with a model in which extracellular vimentin acts as a co-receptor for SARS-CoV-2 spike protein with a binding affinity less than that of the spike protein with ACE2. Extracellular vimentin may thus serve as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry, and vimentin-targeting agents may yield new therapeutic strategies for preventing and slowing SARS-CoV-2 infection.

Mechanical and Non-Mechanical Functions of Filamentous and Non-Filamentous Vimentin.

Intermediate filaments (IFs) formed by vimentin are less understood than their cytoskeletal partners, microtubules and F-actin, but the unique physical properties of IFs, especially their resistance to large deformations, initially suggest a mechanical function. Indeed, vimentin IFs help regulate cell mechanics and contractility, and in crowded 3D environments they protect the nucleus during cell migration. Recently, a multitude of studies, often using genetic or proteomic screenings show that vimentin has many non-mechanical functions within and outside of cells. These include signaling roles in wound healing, lipogenesis, sterol processing, and various functions related to extracellular and cell surface vimentin. Extracellular vimentin is implicated in marking circulating tumor cells, promoting neural repair, and mediating the invasion of host cells by viruses, including SARS-CoV, or bacteria such as Listeria and Streptococcus. These findings underscore the fundamental role of vimentin in not only cell mechanics but also a range of physiological functions. Also see the video abstract here https://youtu.be/YPfoddqvz-g.

Dynamic Nuclear Structure Emerges from Chromatin Cross-Links and Motors.

The cell nucleus houses the chromosomes, which are linked to a soft shell of lamin protein filaments. Experiments indicate that correlated chromosome dynamics and nuclear shape fluctuations arise from motor activity. To identify the physical mechanisms, we develop a model of an active, cross-linked Rouse chain bound to a polymeric shell. System-sized correlated motions occur but require both motor activity and cross-links. Contractile motors, in particular, enhance chromosome dynamics by driving anomalous density fluctuations. Nuclear shape fluctuations depend on motor strength, cross-linking, and chromosome-lamina binding. Therefore, complex chromosome dynamics and nuclear shape emerge from a minimal, active chromosome-lamina system.

A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling.

Abnormal human trabecular meshwork (HTM) cell function and extracellular matrix (ECM) remodeling contribute to HTM stiffening in primary open-angle glaucoma (POAG). Most current cellular HTM model systems do not sufficiently replicate the complex native three dimensional (3D) cell-ECM interface, limiting their use for investigating POAG pathology. Tissue-engineered hydrogels are ideally positioned to overcome shortcomings of current models. Here, we report a novel biomimetic HTM hydrogel and test its utility as a POAG disease model. HTM hydrogels were engineered by mixing normal donor-derived HTM cells with collagen type I, elastin-like polypeptide and hyaluronic acid, each containing photoactive functional groups, followed by UV crosslinking. Glaucomatous conditions were induced with dexamethasone (DEX), and effects of the Rho-associated kinase (ROCK) inhibitor Y27632 on cytoskeletal organization and tissue-level function, contingent on HTM cell-ECM interactions, were assessed. DEX exposure increased HTM hydrogel contractility, f-actin and alpha smooth muscle actin abundance and rearrangement, ECM remodeling, and fibronectin deposition - all contributing to HTM hydrogel condensation and stiffening consistent with glaucomatous HTM tissue behavior. Y27632 treatment produced precisely the opposite effects and attenuated the DEX-induced pathologic changes, resulting in HTM hydrogel relaxation and softening. For model validation, confirmed glaucomatous HTM (GTM) cells were encapsulated; GTM hydrogels showed increased contractility, fibronectin deposition, and stiffening vs. normal HTM hydrogels despite reduced GTM cell proliferation. We have developed a biomimetic HTM hydrogel model for detailed investigation of 3D cell-ECM interactions under normal and simulated glaucomatous conditions. Its bidirectional responsiveness to pharmacological challenge and rescue suggests promising potential to serve as screening platform for new POAG treatments with focus on HTM biomechanics.

Bacterial activity hinders particle sedimentation.

Sedimentation in active fluids has come into focus due to the ubiquity of swimming micro-organisms in natural and industrial processes. Here, we investigate sedimentation dynamics of passive particles in a fluid as a function of bacteria E. coli concentration. Results show that the presence of swimming bacteria significantly reduces the speed of the sedimentation front even in the dilute regime, in which the sedimentation speed is expected to be independent of particle concentration. Furthermore, bacteria increase the dispersion of the passive particles, which determines the width of the sedimentation front. For short times, particle sedimentation speed has a linear dependence on bacterial concentration. Mean square displacement data shows, however, that bacterial activity decays over long experimental (sedimentation) times. An advection-diffusion equation coupled to bacteria population dynamics seems to capture concentration profiles relatively well. A single parameter, the ratio of single particle speed to the bacteria flow speed can be used to predict front sedimentation speed.

The role of vimentin-nuclear interactions in persistent cell motility through confined spaces.

The ability of cells to move through small spaces depends on the mechanical properties of the cellular cytoskeleton and on nuclear deformability. In mammalian cells, the cytoskeleton is composed of three interacting, semi-flexible polymer networks: actin, microtubules, and intermediate filaments (IF). Recent experiments of mouse embryonic fibroblasts with and without vimentin have shown that the IF vimentin plays a role in confined cell motility. Here, we develop a minimal model of a cell moving through a microchannel that incorporates explicit effects of actin and vimentin and implicit effects of microtubules. Specifically, the model consists of a cell with an actomyosin cortex and a deformable cell nucleus and mechanical linkages between the two. By decreasing the amount of vimentin, we find that the cell speed increases for vimentin-null cells compared to cells with vimentin. The loss of vimentin increases nuclear deformation and alters nuclear positioning in the cell. Assuming nuclear positioning is a read-out for cell polarity, we propose a new polarity mechanism which couples cell directional motion with cytoskeletal strength and nuclear positioning and captures the abnormally persistent motion of vimentin-null cells, as observed in experiments. The enhanced persistence indicates that the vimentin-null cells are more controlled by the confinement and so less autonomous, relying more heavily on external cues than their wild-type counterparts. Our modeling results present a quantitative interpretation for recent experiments and have implications for understanding the role of vimentin in the epithelial-mesenchymal transition.

The vimentin cytoskeleton: when polymer physics meets cell biology.

The proper functions of tissues depend on the ability of cells to withstand stress and maintain shape. Central to this process is the cytoskeleton, comprised of three polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). IF proteins are among the most abundant cytoskeletal proteins in cells; yet they remain some of the least understood. Their structure and function deviate from those of their cytoskeletal partners, F-actin and microtubules. IF networks show a unique combination of extensibility, flexibility and toughness that confers mechanical resilience to the cell. Vimentin is an IF protein expressed in mesenchymal cells. This review highlights exciting new results on the physical biology of vimentin intermediate filaments and their role in allowing whole cells and tissues to cope with stress.

Loss of Vimentin Enhances Cell Motility through Small Confining Spaces.

The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F-actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin-driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild-type and vimentin-null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto-myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.

Particle diffusion in active fluids is non-monotonic in size.

We experimentally investigate the effect of particle size on the motion of passive polystyrene spheres in suspensions of Escherichia coli. Using particles covering a range of sizes from 0.6 to 39 microns, we probe particle dynamics at both short and long time scales. In all cases, the particles exhibit super-diffusive ballistic behavior at short times before eventually transitioning to diffusive behavior. Surprisingly, we find a regime in which larger particles can diffuse faster than smaller particles: the particle long-time effective diffusivity exhibits a peak in particle size, which is a deviation from classical thermal diffusion. We also find that the active contribution to particle diffusion is controlled by a dimensionless parameter, the Péclet number. A minimal model qualitatively explains the existence of the effective diffusivity peak and its dependence on bacterial concentration. Our results have broad implications on characterizing active fluids using concepts drawn from classical thermodynamics.

Bacteria Colonies Modify Their Shear and Compressive Mechanical Properties in Response to Different Growth Substrates.

Bacteria build multicellular communities termed biofilms, which are often encased in a self-secreted extracellular matrix that gives the community mechanical strength and protection against harsh chemicals. How bacteria assemble distinct multicellular structures in response to different environmental conditions remains incompletely understood. Here, we investigated the connection between bacteria colony mechanics and the colony growth substrate by measuring the oscillatory shear and compressive rheology of bacteria colonies grown on agar substrates. We found that bacteria colonies modify their own mechanical properties in response to shear and uniaxial compression in a manner that depends on the concentration of agar in their growth substrate. These findings highlight that mechanical interactions between bacteria and their microenvironments are an important element in bacteria colony development, which can aid in developing strategies to disrupt or reduce biofilm growth.

Switchable microscale stress response of actin-vimentin composites emerges from scale-dependent interactions.

The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and longtime stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.

Vimentin promotes collective cell migration through collagen networks via increased matrix remodeling and spheroid fluidity.

The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin's role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. We find that loss of vimentin significantly impairs the ability of the spheroid to collectively expand through collagen networks and remodel the collagen network. Traction force analysis reveals that vimentin null spheroids exert less contractile force than their wild-type counterparts. In addition, spheroids made of mEFs with only vimentin unit length filaments (ULFs) exhibit similar behavior as vimentin-null spheroids, suggesting filamentous vimentin is required to promote 3D collective cell migration. We find the vimentin-mediated collective cell expansion is dependent on matrix metalloproteinase (MMP) degradation of the collagen matrix. Further, 3D vertex model simulation of spheroid and embedded ECM indicates that wild-type spheroids behave more fluid-like, enabling more active pulling and reconstructing the surrounding collagen network. Altogether, these results signify that VIF plays a critical role in enhancing migratory persistence in 3D matrix environments through MMP transportation and tissue fluidity.

Enhanced extracellular matrix remodeling due to embedded spheroid fluidization.

Tumor spheroids are in vitro three-dimensional, cellular collectives consisting of cancerous cells. Embedding these spheroids in an in vitro fibrous environment, such as a collagen network, to mimic the extracellular matrix (ECM) provides an essential platform to quantitatively investigate the biophysical mechanisms leading to tumor invasion of the ECM. To understand the mechanical interplay between tumor spheroids and the ECM, we computationally construct and study a three-dimensional vertex model for a tumor spheroid that is mechanically coupled to a cross-linked network of fibers. In such a vertex model, cells are represented as deformable polyhedrons that share faces. Some fraction of the boundary faces of the tumor spheroid contain linker springs connecting the center of the boundary face to the nearest node in the fiber network. As these linker springs actively contract, the fiber network remodels. By toggling between fluid-like and solid-like spheroids via changing the dimensionless cell shape index, we find that the spheroid rheology affects the remodeling of the fiber network. More precisely, fluid-like spheroids displace the fiber network more on average near the vicinity of the spheroid than solid-like spheroids. We also find more densification of the fiber network near the spheroid for the fluid-like spheroids. These spheroid rheology-dependent effects are the result of cellular motility due to active cellular rearrangements that emerge over time in the fluid-like spheroids to generate spheroid shape fluctuations. Our results uncover intricate morphological-mechanical interplay between an embedded spheroid and its surrounding fiber network with both spheroid contractile strength and spheroid shape fluctuations playing important roles in the pre-invasion stages of tumor invasion.