PIEZO1-mediated mechanosensing governs NK-cell killing efficiency and infiltration in three-dimensional matrices.

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK-cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK-cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK-cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK-cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK-cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK-cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK-cell functions.

Cytoskeletal vimentin regulates cell size and autophagy through mTORC1 signaling.

The nutrient-activated mTORC1 (mechanistic target of rapamycin kinase complex 1) signaling pathway determines cell size by controlling mRNA translation, ribosome biogenesis, protein synthesis, and autophagy. Here, we show that vimentin, a cytoskeletal intermediate filament protein that we have known to be important for wound healing and cancer progression, determines cell size through mTORC1 signaling, an effect that is also manifested at the organism level in mice. This vimentin-mediated regulation is manifested at all levels of mTOR downstream target activation and protein synthesis. We found that vimentin maintains normal cell size by supporting mTORC1 translocation and activation by regulating the activity of amino acid sensing Rag GTPase. We also show that vimentin inhibits the autophagic flux in the absence of growth factors and/or critical nutrients, demonstrating growth factor-independent inhibition of autophagy at the level of mTORC1. Our findings establish that vimentin couples cell size and autophagy through modulating Rag GTPase activity of the mTORC1 signaling pathway.

Soft Synthetic Cells with Mobile Membrane Ligands for Ex Vivo Expansion of Therapy-Relevant T Cell Phenotypes.

The expansion of T cells ex vivo is crucial for effective immunotherapy but currently limited by a lack of expansion approaches that closely mimic in vivo T cell activation. Taking inspiration from bottom-up synthetic biology, a new synthetic cell technology is introduced based on dispersed liquid-liquid phase-separated droplet-supported lipid bilayers (dsLBs) with tunable biochemical and biophysical characteristics, as artificial antigen presenting cells (aAPCs) for ex vivo T cell expansion. These findings obtained with the dsLB technology reveal three key insights: first, introducing laterally mobile stimulatory ligands on soft aAPCs promotes expansion of IL-4/IL-10 secreting regulatory CD8+ T cells, with a PD-1 negative phenotype, less prone to immune suppression. Second, it is demonstrated that lateral ligand mobility can mask differential T cell activation observed on substrates of varying stiffness. Third, dsLBs are applied to reveal a mechanosensitive component in bispecific Her2/CD3 T cell engager-mediated T cell activation. Based on these three insights, lateral ligand mobility, alongside receptor- and mechanosignaling, is proposed to be considered as a third crucial dimension for the design of ex vivo T cell expansion technologies.

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells.

IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r -/- and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.

Deterministic actin waves as generators of cell polarization cues.

Dendritic cells "patrol" the human body to detect pathogens. In their search, dendritic cells perform a random walk by amoeboid migration. The efficiency of pathogen detection depends on the properties of the random walk. It is not known how the dendritic cells control these properties. Here, we quantify dendritic cell migration under well-defined 2-dimensional confinement and in a 3-dimensional collagen matrix through recording their long-term trajectories. We find 2 different migration states: persistent migration, during which the dendritic cells move along curved paths, and diffusive migration, which is characterized by successive sharp turns. These states exhibit differences in the actin distributions. Our theoretical and experimental analyses indicate that this kind of motion can be generated by spontaneous actin polymerization waves that contribute to dendritic cell polarization and migration. The relative distributions of persistent and diffusive migration can be changed by modification of the molecular actin filament nucleation and assembly rates. Thus, dendritic cells can control their migration patterns and adapt to specific environments. Our study offers an additional perspective on how dendritic cells tune their searches for pathogens.

Distinct speed and direction memories of migrating dendritic cells diversify their search strategies.

Migrating cells exhibit various motility patterns, resulting from different migration mechanisms, cell properties, or cell-environment interactions. The complexity of cell dynamics is reflected, e.g., in the diversity of the observed forms of velocity autocorrelation function-which has been widely served as a measure of diffusivity and spreading. By analyzing the dynamics of migrating dendritic cells in vitro, we disentangle the contributions of direction θ and speed v to the velocity autocorrelation. We find that the ability of cells to maintain their speed or direction of motion is unequal, reflected in different temporal decays of speed and direction autocorrelation functions, ACv(t)∼t-1.2 and ACθ(t)∼t-0.5, respectively. The larger power-law exponent of ACv(t) indicates that the cells lose their speed memory considerably faster than the direction memory. Using numerical simulations, we investigate the influence of ACθ and ACv as well as the direction-speed cross correlation Cθ-v on the search time of a persistent random walker to find a randomly located target in confinement. Although ACθ and Cθ-v play the major roles, we find that the speed autocorrelation ACv can be also tuned to minimize the search time. Adopting an optimal ACv can reduce the search time even up to 10% compared with uncorrelated spontaneous speeds. Our results suggest that migrating cells can improve their search efficiency, especially in crowded environments, through the directional or speed persistence or the speed-direction correlation.

Ameboid cell migration through regular arrays of micropillars under confinement.

Migrating cells often encounter a wide variety of topographic features-including the presence of obstacles-when navigating through crowded biological environments. Unraveling the impact of topography and crowding on the dynamics of cells is key to better understand many essential physiological processes such as the immune response. We study the impact of geometrical cues on ameboid migration of HL-60 cells differentiated into neutrophils. A microfluidic device is designed to track the cells in confining geometries between two parallel plates with distance h, in which identical micropillars are arranged in regular pillar forests with pillar spacing e. We observe that the cells are temporarily captured near pillars, with a mean contact time that is independent of h and e. By decreasing the vertical confinement h, we find that the cell velocity is not affected, while the persistence reduces; thus, cells are able to preserve their velocity when highly squeezed but lose the ability to control their direction of motion. At a given h, we show that by decreasing the pillar spacing e in the weak lateral confinement regime, the mean escape time of cells from effective local traps between neighboring pillars grows. This effect, together with the increase of cell-pillar contact frequency, leads to the reduction of diffusion constant D. By disentangling the contributions of these two effects on D in numerical simulations, we verify that the impact of cell-pillar contacts on cell diffusivity is more pronounced at smaller pillar spacing.

Effects of vimentin on the migration, search efficiency, and mechanical resilience of dendritic cells.

Dendritic cells use amoeboid migration to pass through narrow passages in the extracellular matrix and confined tissue in search for pathogens and to reach the lymph nodes and alert the immune system. Amoeboid migration is a migration mode that, instead of relying on cell adhesion, is based on mechanical resilience and friction. To better understand the role of intermediate filaments in ameboid migration, we studied the effects of vimentin on the migration of dendritic cells. We show that the lymph node homing of vimentin-deficient cells is reduced in our in vivo experiments in mice. Lack of vimentin also reduces the cell stiffness, the number of migrating cells, and the migration speed in vitro in both 1D and 2D confined environments. Moreover, we find that lack of vimentin weakens the correlation between directional persistence and migration speed. Thus, vimentin-expressing dendritic cells move faster in straighter lines. Our numerical simulations of persistent random search in confined geometries verify that the reduced migration speed and the weaker correlation between the speed and direction of motion result in longer search times to find regularly located targets. Together, these observations show that vimentin enhances the ameboid migration of dendritic cells, which is relevant for the efficiency of their random search for pathogens.

A novel universal algorithm for filament network tracing and cytoskeleton analysis.

The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the mesh hole size, and filament length and connectivity (also known as Coordination Number). In addition, the method allows sub-networks to be distinguished in two-dimensional images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of two-dimensional images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software.

Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome.

Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI ß, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell-extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.

Role of Extracellular Vimentin in Cancer-Cell Functionality and Its Influence on Cell Monolayer Permeability Changes Induced by SARS-CoV-2 Receptor Binding Domain.

The cytoskeletal protein vimentin is secreted under various physiological conditions. Extracellular vimentin exists primarily in two forms: attached to the outer cell surface and secreted into the extracellular space. While surface vimentin is involved in processes such as viral infections and cancer progression, secreted vimentin modulates inflammation through reduction of neutrophil infiltration, promotes bacterial elimination in activated macrophages, and supports axonal growth in astrocytes through activation of the IGF-1 receptor. This receptor is overexpressed in cancer cells, and its activation pathway has significant roles in general cellular functions. In this study, we investigated the functional role of extracellular vimentin in non-tumorigenic (MCF-10a) and cancer (MCF-7) cells through the evaluation of its effects on cell migration, proliferation, adhesion, and monolayer permeability. Upon treatment with extracellular recombinant vimentin, MCF-7 cells showed increased migration, proliferation, and adhesion, compared to MCF-10a cells. Further, MCF-7 monolayers showed reduced permeability, compared to MCF-10a monolayers. It has been shown that the receptor binding domain of SARS-CoV-2 spike protein can alter blood-brain barrier integrity. Surface vimentin also acts as a co-receptor between the SARS-CoV-2 spike protein and the cell-surface angiotensin-converting enzyme 2 receptor. Therefore, we also investigated the permeability of MCF-10a and MCF-7 monolayers upon treatment with extracellular recombinant vimentin, and its modulation of the SARS-CoV-2 receptor binding domain. These findings show that binding of extracellular recombinant vimentin to the cell surface enhances the permeability of both MCF-10a and MCF-7 monolayers. However, with SARS-CoV-2 receptor binding domain addition, this effect is lost with MCF-7 monolayers, as the extracellular vimentin binds directly to the viral domain. This defines an influence of extracellular vimentin in SARS-CoV-2 infections.

Persistence-Speed Coupling Enhances the Search Efficiency of Migrating Immune Cells.

Migration of immune cells within the human body allows them to fulfill their main function of detecting pathogens. We present experimental evidence showing the optimality of the search strategy of these cells, which is of crucial importance to achieve an efficient immune response. We find that the speed and directional persistence of migrating dendritic cells in our in vitro experiments are highly correlated, which enables them to reduce their search time. We introduce theoretically a new class of random search optimization problems by minimizing the mean first-passage time (MFPT) with respect to the strength of the coupling between influential parameters. We derive an analytical expression for the MFPT in a confined geometry and verify that the correlated motion enhances the search efficiency if the mean persistence length is sufficiently shorter than the confinement size. Our correlated search optimization approach provides an efficient searching recipe and predictive power in a broad range of correlated stochastic processes.

Prestress and Area Compressibility of Actin Cortices Determine the Viscoelastic Response of Living Cells.

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.

Farnesylated Glycol Chitosan as a Platform for Drug Delivery: Synthesis, Characterization, and Investigation of Mucus-Particle Interactions.

Amphiphilic polymer-based drug delivery systems hold potential in enhancing pharmacokinetics and therapeutic efficacy due to their ability to simultaneously codeliver different drugs in a controlled manner. We propose here a facile method for synthesizing a new amphiphilic polymer, farnesylated glycol chitosan (FGC), which self-assembles into nanoparticles upon being dispersed in aqueous media. The characteristics of FGC nanoparticles, in particular the size, could be tuned in a range from 200 to 500 nm by modulating the degree of farnesylation and the pH and polymer concentration during particle preparation. Carrier capacity, release kinetics, and surface modification of the established system were investigated using different model compounds. The colloids were biocompatible and stable at biologically relevant pH values. The interactions between the carriers and human mucus were examined by multiple particle tracking, which revealed that ∼80% of the particles remain immobilized within the mucus matrix. These results postulate FGC as a versatile drug delivery platform.

Myosin II Activity Softens Cells in Suspension.

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.

Extracellular vimentin: Battle between the devil and the angel.

Vimentin, an intracellular cytoskeletal protein, can be secreted by various cells in response to conditions such as injury, stress, senescence, and cancer. Once vimentin is secreted outside of the cell, it is called extracellular vimentin. This extracellular vimentin is significantly involved in pathological conditions, particularly in the areas of viral infection, cancer, immune response, and wound healing. The effects of extracellular vimentin can be either positive or negative, for example it can enhance axonal repair but also mediates SARS-CoV-2 infection. In this review, we categorize the functional implications of extracellular vimentin based on its localization outside the cell. Specifically, we classify extracellular vimentin into two distinct forms: surface vimentin, which remains bound to the cell surface, and secreted vimentin, which refers to the free form that is completely released outside the cell. Overall, extracellular vimentin has a dual nature that encompasses both beneficial and detrimental effects on the functionality of cells, organs and whole organisms. Here, we summarize its effects in viral infection, cancer, immune response and wound healing. We find that surface vimentin is often associated with negative consequences, whereas secreted vimentin manifests predominantly with positive influences. We found that the observed effects of extracellular vimentin strongly depend on the specific circumstances under which its expression occurs in cells.

A low-cost alternative method of generating fibronectin micropatterned lines for cellular applications.

The cellular microenvironment contributes to the architecture, differentiation, polarity, mechanics and functions of the cell [1]. Spatial confinement of cells using micropatterning techniques allows to alter and regulate the cellular microenvironment for a better understanding of cellular mechanisms [2]. However, commercially available micropatterned consumables such as coverslips, dishes, plates etc. are expensive. These methods are complex and based on deep UV patterning [3,4]. In this study, we establish a low-cost method for effective micropatterning using Polydimethylsiloxane (PDMS) chips.•We demonstrate this method by generating fibronectin-coated micropatterned lines (width, 5 µm) on a glass bottom dish.•As a proof of concept, we culture macrophages on these lines. We additionally show that this method allows to determine the cellular polarity by measuring the position of the nucleus within a cell on a micropatterned line.

Chromatin decondensation and nuclear softening accompany Nanog downregulation in embryonic stem cells.

The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself-even prior to differentiation-is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development.

The regulatory role of cell mechanics for migration of differentiating myeloid cells.

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytoskeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.