ABSTRACT Fibroblast transcriptomic states reflect physiological and pathological tissue contexts, yet the upstream determinants that stabilize these states remain poorly defined. Integrins mediate extracellular matrix (ECM) adhesion and biochemical signaling, but whether they encode mechanical constraints into stable transcriptomic programs is unclear. Using engineered mouse fibroblasts, bulk and single-cell transcriptomics, and controlled micromechanical confinement, we show that integrins can shape the transcriptomic landscape. The bulk transcriptome of fibroblasts expressing αV- and β1-class indicates a shared mechanosensitive baseline, except when these integrin classes are expressed individually. We also found integrin-specific gene clusters, including β1-class integrin-dependent enrichment of Areg, Epha7, Lhpp and Igf2r , which regulate development, regeneration and disease, and altered YAP1 targeted gene expression. At single-cell resolution under confinement, β1-class integrins sustain a progenitor-associated program, whereas their loss or αV-class enrichment promotes a constitutively activated state linked to injury repair and wound healing. Mechanical confinement and confinement duration further reshapes these states in an integrin-identity-dependent manner. Our findings establish integrin-identity as a determinant of how fibroblasts transduce mechanotemporal inputs from the cell surface to the nucleus.

The biochemical and mechanical properties of extracellular matrix proteins govern cell adhesion, mechanics, and migration. How cells use integrins to discriminate between the arginine-glycine-aspartic acid motifs presented by different extracellular matrix proteins, a process central to tissue homeostasis and disease, has remained unclear. Here we show that mammalian cells mount a distinct “biphasic” mechanical response through αV-class integrins to the arginine-glycine-aspartic acid motif of vitronectin compared with fibronectin, osteopontin, and cyclic arginine-glycine-aspartic acid. Within seconds of contact with vitronectin, we find that αV-class integrins strengthen cell adhesion through two load-dependent mechanotransduction pathways in which αVβ3 and αVβ5 integrins take complementary roles. Under low load, we demonstrate that the first phase requires both integrins together with an intact, pre-tensed actomyosin cortex, talin, paxillin, and focal adhesion kinase activity, with αVβ5 integrin additionally engaging clathrin-mediated endocytosis. Under higher load, we show that the second phase is dominated by αVβ3 integrin–directed actin-related protein 2/3, cellular Src kinase, and phosphatidyl inositol-3-kinase signaling, which organizes the consensus adhesome, while αVβ5 integrin concurrently drives cellular stiffening. Taken together, we find that αV-class integrins rapidly deploy arginine-glycine-aspartic acid -motif- and β-subunit-specific programs that cooperatively tune cell adhesion and mechanics according to the extracellular matrix composition. Here the authors show that αV-class integrins distinguish the RGD motif of vitronectin from other ligands and, within seconds, deploy β-subunit-specific, load-dependent programs in which αVβ3 and αVβ5 cooperate to tune fibroblast adhesion and mechanics to ECM composition.

Abstract In recent year, it became clear that the cell nucleus can undergo large deformations, during immune cell migration and tumor growth. These deformations generate signals that allow cells to sense their environment and adapt to it. How cells cope with and respond to large deformations thus strongly depends on the nuclear mechanics, but our understanding of the physical properties of the nucleus remains incomplete. In particular, it is not clear how the nuclear volume responds to deformation. Here we combine controlled confinement assays, high-resolution imaging and atomic force microscopy with theoretical modelling to propose a physical model of the cell nucleus that accounts for its surface and bulk properties and addresses both steady-state and transient regimes. Our results establish the nucleus as a poroelastic body in which mechanics are dominated by the envelope and dynamics by the chromatin, and suggest that regulating water permeability may be as important as softening the envelope for cells migrating rapidly through dense tissues.

β-arrestins, pivotal regulators of G protein-coupled receptor (GPCR) signaling, assemble with hundreds of GPCRs. How this assembly rises to functionally distinct complexes in which β-arrestin engages the GPCR tail, core or both, remains a central question. Here employing single-molecule force spectroscopy and molecular dynamics simulations, we monitor assembly of β2-adrenergic receptor (β2AR)-β-arrestin2 (βarr2) tail, core and tail-core complexes in phospholipid membranes and dissect their mechanical and kinetic stabilities. We show that βarr2 engages the phosphorylated β2AR carboxy-terminus (C-tail) within milliseconds, much faster than the active receptor core. In addition, the phospholipid membrane contributes substantially to complex stability, with phosphatidylinositol 4,5-bisphosphate (PIP2) modulating stability and conformation. While PIP2 stabilizes the β2AR-βarr2 core, it precludes βarr2 from concomitantly binding the phosphorylated β2AR C-tail. βarr2 activation and PIP2 strengthen βarr2-membrane association through insertion of the C-edge and finger loop. These findings establish PIP2, alongside ligand binding and receptor phosphorylation, as a central determinant of β2AR-βarr2 complex assembly, offering mechanistic insight into the regulation of GPCR signaling.

Cells operate as networks of proteins, membranes, condensates and compartments, each sensing and displaying distinct intracellular mechanical properties. However, cells also sense, adapt and respond to manifold mechanical properties of the environment, including adhesion, tension, stiffness, shear, viscoelasticity, plasticity, pressure and confinement. By gauging these properties at various timescales and across nano to macro length scales, cellular systems alter their collective responses. The field of mechanobiology aims to elucidate how cellular systems such as tissues, organoids or organs perceive, respond to and influence mechanical cues, and how these impact physiological processes including homeostasis, growth, division, differentiation, movement, development, adaptation and apoptosis. This Perspective highlights challenges within mechanobiology that must be systematically tackled to advance exploration and deepen our understanding of the mechanical attributes of intricate multicellular organisms. Such understanding necessitates the engineering of multicellular models as reference systems, the development of new tools to rigorously quantify and manipulate mechanical properties from the nanoscale to macroscale and theoretical frameworks to decode mechanobiological complexities. Ultimately, addressing these challenges will improve the analysis, monitoring and prediction of mechanobiological processes across molecular, multicellular and organismal scales, thus advancing mechanodiagnostics and mechanomedicine.

With the passing of Andreas Engel on 1 April 2026, the structural biology and biophysics communities have lost one of their most visionary and influential scientists. Over a career spanning more than five decades, Andreas fundamentally shaped how we visualize and understand biological macromolecules at the nanoscale. His pioneering contributions to scanning transmission electron microscopy (STEM), atomic force microscopy (AFM) and electron crystallography opened entirely new avenues for studying the architecture and function of membrane proteins and supramolecular protein complexes.

Crown rot caused by Fusarium graminearum is a major wheat disease, but only a few soils are known to suppress this disease. Here, 49 French soils were screened in greenhouse plant tests to determine their capacity to limit wheat crown rot upon seed inoculation with F. graminearum. As many as 14 of the soils (29%) showed disease suppressiveness, whereas 15 soils were disease-conducive and 20 soils of intermediate status. The suppressive soils originated from different regions, but overall higher disease symptoms were found with North-West soils. The suppressive soils represented various soil types, i.e. Cambisols, Fluvisols, Leptosols and Luvisols. Chemical properties did not differ markedly in suppressive vs conducive soils, but organic carbon content was lower in suppressive soils than in conducive soils. Overall, soil suppressiveness to F. graminearum-mediated crown rot disease was rather widespread in French regions and present across contrasted farm soils.

In this review we discuss how atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) approaches can be applied to monitor the unfolding and folding pathways of individual membrane proteins. Particularly, we focus on the insertion and folding of prokaryotic α-helical and β-barrel membrane proteins and compare their unassisted insertion and folding pathways with those assisted by insertases, translocases, and chaperones. We highlight examples in which SMFS is applied to detect the misfolding of membrane proteins such as induced by the lipid composition of the membrane or resulting from unassisted folding. While SMFS can monitor how soluble and transmembrane chaperones reduce misfolding of structural segments, it can also monitor how insertases and translocases guide their stepwise insertion and folding into membranes until the membrane protein has completed folding. Examples show that the inner membrane insertase YidC inserts structural segments in a random order, whereas the SecYEG translocon inserts transmembrane α-helices sequentially. However, when acting together, SecYEG dominates over YidC, consistent with the role of the translocon in directing membrane protein insertion and folding. Finally, we discuss β-barrel membrane protein folding in the bacterial outer membrane, including the β-barrel assembly machinery (BAM) complex, and how SMFS applied to native outer membrane vesicles provides access to monitor the insertion and folding of membrane proteins in the native-like membrane environment.

BACKGROUND:Plant microbiota has received increasing attention in recent years. In particular, the microbiota associated with cereals is being extensively studied to identify bacterial strains that can promote plant health and growth. Barley is the fourth most important cereal worldwide in terms of agricultural production. Intensive barley agriculture requires the use of chemical fertilizers to compensate for nutrient deficiencies in soils and limit pathogen development. The isolation and use of bacteria that can enhance the bioavailability of soil nutrients and inhibit the development of plant pathogens could ultimately limit the use of these chemicals. In this study, we have isolated from a barley microbiota three bacterial strains belonging to the genus Streptomyces. These strains were characterized and named GPA1, GPAT2, and GPN2. RESULTS:These three closely related isolates were from the same bacterial genus Streptomyces. Based on a phylogenetic analysis, the strains GPAT2 and GPN2 were classified as Streptomyces murinus, while GPA1 was identified as a new species. All strains showed antagonistic activity against two microorganisms that inhibit barley germination: Pseudomonas sp. MRN1 and Fusarium sp. CK. In addition, these strains exhibited different effects on the growth of barley cultivated under hydroponic and axenic conditions. In fact, GPN2 appeared to have no effect whereas the inoculation of barley seedlings with GPAT2 and GPA1 resulted in a reduction and an increase in root length after two weeks of growth, respectively. GPA1 had various Plant Growth-Promoting (PGP) abilities, including phosphate and zinc solubilization and siderophore production. A metabolite profiling of the GPA1 bacterial culture also showed its production and excretion of indole-3-acetic acid (IAA). CONCLUSION:In this study, we have characterized three closely related bacteria, which display different effects on barley seedlings growth. These results revealed that the type of interactions of Streptomyces with barley is strain-dependent, suggesting that these interactions may arise from specific molecular mechanisms acquired through coevolutionary processes.

Malignancies of epithelial tissues, called carcinomas, account for the majority of cancer cases. Much cancer research has focused on genetic alterations and their relation to different carcinoma phenotypes. Besides a rewiring in the signalling networks, carcinoma progression is accompanied by mechanical changes in the epithelial cells and the extracellular matrix. Here, we reveal intricate morphologies in the basement membrane at the onset of bladder cancer, and propose that they emerge from a mechanical buckling instability upon epithelial overgrowth. Using a combination of microscopy imaging of the mouse and human bladder tissue, elasticity theory, and numerical simulations of differential growth in the bladder mucosa, we find that aberrant tissue morphologies can emerge through stiffness changes in the different mucosa layers. The resulting thickening, wrinkles and folds exhibit qualitative and quantitative similarity with imaged early papillary tumors and carcinomas in situ . Atomic force microscopy indeed reveals local stiffness changes in the pathological basement membrane. Our findings suggest a mechanical origin of the different carcinoma subtypes in the bladder, which have vastly different clinical prognosis. They might provide the basis for a new line of attack in medical carcinoma treatment and prophylaxis.

Exposed to mechanical confinement, mammalian cells can establish remarkable unspecific adhesion, which is independent of integrins. How cells facilitate such adhesion remains unclear. Here, it is investigated how mammalian cells exposed to compression initiate unspecific and integrin-mediated adhesion. It is observed that with increasing compression, cells increase adhesion to collagen I or fibronectin and strengthen adhesion faster. Under low and medium compression, cells minimally increase unspecific adhesion to substrates that lack specific binding sites for cell surface receptors, such as integrins. However, under high compression, mammalian cells switch to a strong unspecific adhesion state, which significantly contributes to cell-extracellular matrix (ECM) adhesion. Thereby cells use the glycocalyx to directly facilitate strong unspecific adhesion and to enhance early integrin-mediated adhesion. The mechanistic insight of how cells unspecifically adhere to substrates under confinement opens avenues to better understand cell adhesion in development, homeostasis, disease, and in a wide range of biotechnological and medical applications in which cells are exposed to mechanical confinement.

Mammalian cells adjust integrin-mediated adhesion based on the composition and structure of the extracellular matrix (ECM). However, how spatially confined ECM ligands regulate cell adhesion initiation remains unclear. Here, we investigate how cells adapt early adhesion to different ECM protein areas. Through combining microcontact printing with single-cell force spectroscopy we measure cell adhesion initiation and strengthening to defined areas of ECM proteins. HeLa cells and mouse embryonic fibroblasts gradually increase adhesion with collagen I or fibronectin area, while reaching maximum adhesion force to ECM patterns having areas above certain thresholds. On much smaller patterns, both cell types switch to a different state and considerably increase the adhesion force per ECM protein area, which they strengthen much faster. This spatially enhanced adhesion state does not require talin or kindlin, indicating a fundamentally different adhesion mechanism. Mechanotransduction seems to play integrin and cell type-specific roles in the spatially enhanced adhesion state.

The fabrication of wedge-shaped cantilevers for Atomic Force Microscopy (AFM) remains a critical yet challenging task, particularly when precision and efficiency are required. In this study, we present a streamlined protocol for producing these wedges using NOA63 UV-curing polymer, which simplifies the process and eliminates the need for dedicated equipment. Our method reduces preparation time while maintaining the mechanical properties of the cantilevers, in line with the manufacturer's specifications. We demonstrate the effectiveness of our wedged cantilevers in stress-relaxation experiments performed by means of AFM and confocal microscopy on primary Chronic Lymphocytic Leukemia cells and the MEC1 cell line. These experiments highlight the effectiveness of using modified cantilevers to consistently apply precise uniaxial loading to soft, spherical cells. This technique offers a marked improvement in fabrication speed and operational ease compared to traditional methods, without compromising the accuracy or performance of the measurements. This protocol is not only time-saving, but also adaptable for use in a wide range of biological applications, making it a valuable tool for AFM-based research in cellular mechanics.

Spontaneous locomotion is a common feature of most metazoan cells, generally attributed to the properties of actomyosin networks. This force-producing machinery has been studied down to the most minute molecular details, especially in lamellipodium-driven migration. Nevertheless, how actomyosin networks work inside contraction-driven amoeboid cells still lacks unifying principles. Here, using stable motile blebs from HeLa cells as a model amoeboid motile system, we imaged the dynamics of the actin cortex at the single filament level and revealed the co-existence of three distinct rheological phases. We introduce “advected percolation,” a process where rigidity percolation and active advection synergize, spatially organizing the actin network’s mechanical properties into a minimal and generic locomotion mechanism. Expanding from our observations on simplified systems, we speculate that this model could explain, down to the single actin filament level, how amoeboid cells, such as cancer or immune cells, can propel efficiently through complex 3D environments.

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control.

Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light-driven proton pumps bacteriorhodopsin and blue light-absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer-controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH-sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color-controlled pH-adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications.

Plasma membrane rupture (PMR) and the release of cytosolic content are hallmarks of necrotic cell death. The plasma membrane protein ninjurin-1 (NINJ1) actively promotes cell lysis by polymerizing into membrane embedded filaments that induce the formation of large plasma membrane lesions. Yet the signals controlling NINJ1 polymerization and the opening of membrane lesions remain unknown. Here we combine cell biology and biophysical measurements to characterize the steps preceding NINJ1-induced PMR in cells undergoing ferroptosis. Our results show that NINJ1 lesions form through a 2-step mechanism requiring oligomerization, which is triggered by signal 1, and subsequent lesion opening driven by signal 2, which we determine to be cell swelling without a concomitant increase in intracellular pressure. The close homologue NINJ2 polymerizes with similar kinetics to NINJ1 but fails to form lesions during cell swelling, further highlighting that oligomerization and PMR are distinct events. Chimeras between NINJ1 and NINJ2 show that the unstructured N-terminal region of NINJ1 controls lesion opening in response to cell swelling. In summary, our data establish a new 2-step model for NINJ1-driven necrosis in which initial oligomerization causes the formation of NINJ1 filaments that serve as weak links in the plasma membrane and can break once cell swelling causes a rise in membrane tension and mechanical stress. ### Competing Interest Statement The authors have declared no competing interest.

Certain soils promote crop health because they are pathogen-suppressive (i.e., fungistatic) or disease-suppressive, but the effect of soil management on these properties is not fully understood. Here, we tested the hypothesis that manure could favor fungistasis by screening 26 manured or non-manured wheat fields from Serbia for their ability to control survival/growth of the fungal plant pathogen Fusarium graminearum Fg1. Quantitative PCR showed that the pathogen grew after inoculation in all 26 autoclaved soils. In absence of autoclaving, the pathogen was stable or grew in 16 soils (37 % manured) but declined in the 10 others (70 % manured). For most soils, there was no significant link between soil chemistry and fungistasis, except with Mionica in western/central Serbia. Mionica soils MI2 and MI3, which had received manure, exhibited higher levels of organic matter and potassium compared with soils MI4 and MI5, which had not received manure and were non-fungistatic. Using Mionica soils, we then tested the hypothesis that fungistatic (manured) soils rather than non-fungistatic (nonmanured) soils would protect wheat from F. graminearum disease. Indeed, fungistatic soils were suppressive to wheat damping-off. Non-fungistatic soil MI4 was conducive, as expected, but non-fungistatic soil MI5 turned out to be suppressive. Metabarcoding showed that the structure of prokaryotic and fungal rhizosphere communities depended mostly on field location, with a significant effect of F. graminearum inoculation. In conclusion, our findings show that certain farming practices (here, manure amendments) may promote soil fungistasis towards F. graminearum. However, both fungistatic and non-fungistatic soils can be suppressive to F. graminearum disease in wheat, and their differences in rhizosphere microbiota suggest different phytoprotection mechanisms.

Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.