High-Throughput Mechanomic Screening Reveals Novel Regulators of Single-Cell Mechanics
Laura von Selzam,
Katarzyna Plak,
Christine Schweitzer,
Cornelia Liebers,
Paul Müller,
Marta Urbanska,
Martin Kräter,
Buzz Baum,
Jona Kayser, et al.
Biophysical Journal
125
(12)
3060-3073
(2026)
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The mechanical properties of cells are dynamic, allowing them to adjust to different needs in different biological contexts. In recent years, advanced biophysical techniques have enabled the rapid, high-throughput assessment of single-cell mechanics, providing new insights into the regulation of the mechanical cell phenotype. However, the molecular mechanisms by which cells maintain and regulate their mechanical properties remain poorly understood. Here, we present a genome-scale RNA interference (RNAi) screen investigating the roles of kinase and phosphatase genes in regulating single-cell mechanics using Real-Time Fluorescence and Deformability Cytometry (RT-FDC). Our screen identified 82 known and novel mechanical regulators across diverse cellular functions from 214 targeted genes, leveraging RT-FDC’s unique capabilities for comprehensive, high-throughput mechanical phenotyping with single-cell and cell cycle resolution. These findings refine our understanding of how signaling pathways coordinate structural determinants of cell mechanical phenotypes and provide a starting point for uncovering new molecular targets involved in biomechanical regulation across diverse biological systems.
Automation and improvement of WBC mechanical profiling in deformability cytometry
Sara Kaliman,
Shada Abuhattum Hofemeier,
Benedikt Hartmann,
Jochen Guck
Biophysical Journal
125
(12)
3048-3059
(2026)
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Deformability cytometry (DC) is a powerful biophysical technique that enables cost-effective, high-throughput characterization of disease-associated changes in blood cell mechanics. Mechanical profiling of living white blood cells (WBCs) is particularly valuable due to their critical role in the immune response. However, reliably identifying and classifying WBC subtypes in a label-free manner remains a significant challenge. Until now, the analysis pipeline has relied on manual gating by trained experts, limiting scalability and reproducibility. In this study, we present a fully automated and generalizable framework for WBC classification in shear flow DC experiments, based on box filters and unsupervised clustering of cell populations. Both box filters and unsupervised clustering rely on cell shape features derived from high-accuracy segmentation and on cell texture features derived from bright-field images. This unsupervised approach not only improves reproducibility and reduces processing time but also overcomes key limitations of supervised models that require extensive training data and often suffer from reduced performance under varying imaging conditions. We validated our method by comparing cell features obtained through manual gating and automated classification across six experimental sets. These sets incorporated variations in blood donors, anticoagulants (EDTA and citrate), blood collection sources (capillary and venous), and device brightness settings. Each set included five repeated measurements. The results consistently confirmed the reliability and robustness of the method across all tested conditions and WBC types. Importantly, this automated pipeline enables the inclusion of WBCs with membrane protrusions—typically excluded from standard analyses—allowing for morphological characterization of potentially activated cells. Moreover, by using shape features derived from the original contour rather than the convex hull, we improve morphological accuracy and reduce measurement variability. This approach thus enhances the accuracy, consistency, and scalability of WBC mechanophenotyping and enables high-throughput analysis across large cohorts.
Stem Cell Differentiation Disperses Transcriptional Clusters via a Conserved Surface‐Condensate Trajectory
Tim Klingberg,
Irina Wachter,
Agnieszka Pancholi,
Matthias Akyel,
Yomna Gohar,
Priya Kumar,
Ana Miguel Fernandes,
Yuzhi Bao,
Alica Schmidt‐Heydt, et al.
Stem cells exhibit exceptionally prominent transcriptional clusters, which dissolve with progressing differentiation. Although these clusters are assigned central roles in embryonic gene regulation, their formation and loss during differentiation remain poorly understood. This study reveals that these prominent clusters disperse along a conserved trajectory in mouse embryonic stem cells, fruit fly testes, and zebrafish embryos. Imaging and lattice simulations show that these clusters form via surface condensation on H3K27ac-marked super-enhancer regions, which act as genomic scaffolds. Upon differentiation, partial loss of these active epigenetic marks and transcription-driven unfolding lead to dispersal of the prominent clusters. The block copolymer-based lattice simulations explain this process as a conserved trajectory through a three-dimensional state space, governed by surface condensation principles that extend beyond canonical liquid–liquid phase separation. This work marks surface condensation as a biophysical mechanism for the dynamic organization of stem cell-specific transcriptional hubs and demonstrates evolutionary conservation in several organisms. By uncovering a conserved biophysical mechanism for transcriptional organization in development, our work illustrates how polymer properties can contribute to the control of cell identity and fate.
Fibronectin matrix remodelling modulates the active nematic dynamics of cancer-associated fibroblasts
Cécile Jacques,
Louisiane Perrin,
Joseph Ackermann,
Samuel Bell,
Olivier Zajac,
Ambre Lapierre,
Lucas Anger,
Clément Hallopeau,
Carlos Pérez-González, et al.
Cancer-associated fibroblasts are major architects of the tumour stroma, where their aligned, elongated morphology forms a capsule that mechanically restrains tumour expansion. However, it is unclear how this supracellular organization emerges and persists. Here we show that fibroblasts generate a fibronectin matrix that progressively acquires the same nematic order as the cell layer, and that this matrix in turn feeds back to immobilize both cells and topological defects. Using long-term live imaging, traction force microscopy, matrix microfabrication and hydrodynamic modelling, we find that this reciprocal coupling induces an ageing process in which cellular flows and defect motion slow dramatically and ultimately freeze. Despite this arrest, the monolayer remains active, with defects concentrating contractile forces that may represent mechanical weak points. Disrupting fibronectin production fluidizes the capsule, reactivates defect dynamics and compromises its barrier-like function. These findings reveal a self-organizing mechanism by which fibroblasts and their matrix co-evolve to create a mechanically stable, yet active, stromal architecture with direct implications for tumour dissemination.
Fibrin hydrogels regulate human astrocyte state and neuronal reprogramming
Thomas Distler,
Katherina Konrad Daga,
Martina Bürkle,
Sebastián Vásquez-Sepúlveda,
Kristian Franze,
Magdalena Götz,
Giacomo Masserdotti
Astrocytes are key components in reactive gliosis after brain injury, yet defined in vitro models dissecting the influence of extracellular matrix components enriched after injury, such as fibrin, on human astrocyte behavior and function are still missing. Here, we use fibrinogen-derived fibrin and fibrin-alginate-RGD (FAR) 3D hydrogel substrates to examine their influence on human induced pluripotent stem cell (hiPSC)-derived astrocyte behavior and on their direct conversion into neurons. Astrocytes develop complex morphologies in 3D-FAR hydrogels, while they are more proliferative and migratory in 3D-fibrin hydrogels (3D-fibrin). Interestingly, gene expression analysis revealed different reactive states of astrocytes in 3D-fibrin and 3D-FAR, which persist over time. The highly inflammatory state and stiffness of 3D-FAR are largely incompatible with direct neuronal reprogramming, hampering the direct conversion even at early stages. Conversely, astrocytes in 3D-fibrin hydrogels can convert into neuronal cells, demonstrating a potent influence of how fibrin is presented on distinct astrocyte states, with great relevance for fate conversion.
Glycan atlassing enables functional tracing of cell state
Dijo Moonnukandathil Joseph,
Nazlican Yurekli,
Sarah Fritsche,
Reem Hashem,
Oana-Maria Thoma,
Imen Larafa,
Tina Boric,
Chloé Bielawski,
Karim Almahayni, et al.
The glycocalyx is a complex layer of glycosylated molecules that surrounds all cells in the human body. It is involved in regulating critical cellular processes, including immune response modulation, cell adhesion and host–pathogen interactions. Despite these insights, the functional relationship between the glycocalyx architecture and cellular state has remained elusive, largely due to the structural diversity of glycocalyx constituents and their nanoscale organization. Here we show that DNA-tagged lectin labelling and metabolic oligosaccharide engineering enable multiplexed super-resolution microscopy of the glycocalyx constituents, yielding an atlas of glycocalyx architecture with nanometre resolution. Quantitative analysis of the obtained nanoscale map of glycocalyx constituents facilitates the extraction of characteristic spatial relationships that accurately report on the cellular state. We demonstrate the capacity of our approach, which we term glycan atlassing, across cell and tissue types, ranging from cultured cell lines to primary immune cells, neurons and primary patient tissue. Glycan atlassing establishes a transformative strategy for investigating glycocalyx remodelling in development and disease, potentially enabling the development of glycocalyx-centred targets in diagnosis and therapy.
Hyaluronic acid and tissue mechanics orchestrate mammalian digit tip regeneration
Byron W. H. Mui,
Joseph J. Y. Wong,
Camille E. Dumas,
Jia Hua Wang,
Toni Bray,
Kentaro Hirose,
Lauren Connolly,
Alexander Winkel,
Sebastian Timmler, et al.
Although regenerating complex tissues is a feature of many lower vertebrates, most mammals have traded this capacity for rapid wound healing and fibrotic scarring. In adult humans, this “regenerative window” is almost entirely closed, with the distal digit tip remaining as one of few tissues capable of complete multitissue regeneration that restores its original structure; more proximal amputations scar. Historically, research into this phenomenon has prioritized molecular signaling pathways and cellular origins. However, the role of extrinsic cues—specifically, the physical and mechanical signals from the surrounding microenvironment—has remained poorly understood in the context of whole-tissue regeneration.
Cellular aggregate formation: Continuum modelling and computational aspects
Soheil Firooz,
B. Daya Reddy,
Vasily Zaburdaev,
Paul Steinmann
Computer Methods in Applied Mechanics and Engineering
451
118687
(2026)
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In this manuscript, we carry out a systematic study on the mechanics and rheology of cellular aggregate formations. We study how cell–cell and cell–matrix interactions influence cellular aggregation, which can be described as an active phase separation process. Furthermore, we investigate the mechanisms underlying the coalescence of aggregates. The problem is analyzed in both Eulerian and Lagrangian frameworks and the computational intricacies for each approach are highlighted. Using our recently developed micromorphic-based artificial diffusion method, we circumvent the numerical instabilities arising from the convective nature of the problem. Finally, via a comprehensive numerical study, we investigate the dynamics of the cellular aggregate formation under various conditions. A notable agreement between the numerical and experimental results is observed. Our work provides significant insights into the mechanics of cellular aggregates which paves the way for better understanding the role of active mechanical forces in biological systems.
Dynamic heterogeneity and hidden fluidity in dense epithelial tissues
Yuan Shen,
Wang Xi,
René-Marc Mège,
Walter Kob,
Benoît Ladoux
Science Advances
12
eaec3773
(2026)
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Epithelial tissues maintain organ integrity while continuously remodeling during morphogenesis, repair, and disease. At high cell densities, these tissues often appear mechanically arrested in a disordered, solid-like state, raising the question of how they retain the ability to reorganize. Here, we show that, unlike thermal glasses, dense epithelial tissues do not exhibit caging behavior but instead behave as a complex fluid. Cells display subdiffusive creep together with Fickian yet non-Gaussian dynamics and compressed exponential relaxation, hallmarks of stress-driven fluidity. This fluidity arises from the tissue’s structural and mechanical organization rather than from cell division or extrusion, which only transiently enhance local dynamics. Fast-moving cells organize into collective, anisotropic clusters whose spatial heterogeneity correlates with local structural entropy and soft vibrational modes. Together, these findings reveal a hidden fluidity in densely packed epithelia that supports mechanical stability while preserving the capacity for remodeling during development, wound healing, and early tumor invasion.
Diffractive Neural Networks for High‐Throughput Classification of Objects in Microfluidic Systems
Jingli Li,
Steffen Schoenhardt,
Jeffrey Harmon,
Jochen Guck,
Min Gu,
Elena Goi
Advanced Photonics Research
7
e202500272
(2026)
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The integration of optical imaging and machine learning on microfluidic platforms makes it possible to achieve high-content minimally invasive characterization of a population of samples on a single chip As the analysis of this high-content information is typically conducted in the electronic domain optoelectronic conversion speeds and the bandwidth available for data processing put a limitation on the throughput of these methods In this work we present an analysis system based on diffractive neural networks with the potential for integration in microfluidic systems for high-content classification of objects with high sampling rates We show that such a system can distinguish objects by size through passive optical inference with a numerical test accuracy of 98.2 and an experimental test accuracy of 83.4 in an environment compatible with a microfluidic chamber This development paves the way for novel approaches in high-speed phenotyping of large cell populations based on all-optical or hybrid optoelectronic neuromorphic information processing.
Lena Pollinger,
Johannes N. Greve,
Melanie Grosch,
Sara Kaliman,
Shada Abuhattum Hofemeier,
Martin Kräter,
Ina Brauer,
Jan T. Schaefer,
Francesca Pasutto, et al.
Kidney International Reports
11
106343
(2026)
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INTRODUCTION: Pathogenic variants in myosin heavy chain 9 (MYH9) encoding the heavy chain of nonmuscle myosin IIA (NMMIIA) cause autosomal-dominant MYH9-related disease that may include proteinuric kidney disease macrothrombocytopenia cataract sensorineural deafness and elevated liver enzymes. METHODS: Whole exome sequencing and segregation analysis were performed in a patient with end-stage renal disease Histology of kidney and liver biopsies was assessed and blood smears were examined for the presence of Döhle-like bodies Deformability cytometry and monocyte migration assays were performed Immortalized podocytes and primary skin fibroblasts of 1 patient were transfected with plasmids containing MYH9 wild type (WT) or the p.(Arg424Gly) variant Biochemical studies using recombinantly produced proteins were conducted to assess the variant’s impact on adenosine triphosphate (ATP) turnover and motor function. RESULTS: We identified the likely pathogenic heterozygous MYH9 variant c.1270C>G p.(Arg424Gly) in all affected members of a nonconsanguineous family Typical microscopic findings such as Döhle-like bodies or NMMIIA conglomerates were absent Nonetheless all patients presented with proteinuric kidney disease elevated liver enzymes and intermittent thrombocytopenia The altered protein showed increased ATP turnover in the presence of actin and enhanced motor activity under both unloaded and loaded conditions. CONCLUSION: We identified a novel fully segregating MYH9 variant causing MYH9-related disease Based on biochemical findings we report the first gain-of-function variant of MYH9 We propose that the enhanced intrinsic motor activity of the p.(Arg424Gly) variant is a key contributor to the disease mechanism Incorporation of the p.(Arg424Gly) variant into nonmuscle myosin IIA filaments and higher-order actomyosin assemblies may in principle affect actomyosin dynamics.
Cytoplasmic crowding acts as a porous medium reducing macromolecule diffusion
Olivier Destrian,
Nicolas Moisan,
René-Marc Mège,
Benoît Ladoux,
Benoit Goyeau,
Morgan Chabanon
Proceedings of the National Academy of Sciences of the United States of America
123
e2519599123
(2026)
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Intracellular transport of macromolecules is crucial for the proper functioning of most cellular processes. Although intracellular crowding is known to strongly alter macromolecule mobility, how cytoplasmic structures physically modulate diffusion remains largely unexplored. Here, we investigated the mechanisms by which cytoplasmic crowding controls diffusivity using live-cell experiments and porous media modeling approaches. Confocal microscopy combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy measurements revealed an anticorrelation between free green fluorescent protein diffusivity and the heterogeneous cytoplasmic structure abundance in live mammalian cells. This motivated the development of a multiscale model, where the cytoplasm is treated as a hierarchical porous medium with nanometric and micrometric obstacles. Numerically solving the model allowed us to predict the effective cytoplasmic diffusion coefficient for various obstacle volume fractions and to identify tortuous and porous hydrodynamic hindrances as key diffusion reduction mechanisms. Comparison with our experimental results highlighted the importance of hydrodynamic interactions between diffusing molecules and nanometric obstacles. Importantly, we found that the effective cytoplasmic diffusivity was not dependent on specific intracellular regions but rather on the local intracellular obstacle volume fraction. Finally, the model was extended to predict the diffusivity of larger macromolecules, showing excellent agreement with literature data for several macromolecules and cell lines. This study provides insights into the physical mechanisms impeding intracellular diffusion, demonstrating the potential of porous media modeling approaches to predict transport mechanisms in dynamic or heterogeneous intracellular structures, as in cell motility, blebbing, and apoptosis.
Long-range chemical signalling in vivo is regulated by mechanical signals
Eva K. Pillai,
Sudipta Mukherjee,
Niklas Gampl,
Ross J. McGinn,
Katrin A. Mooslehner,
Julia M. Becker,
Alexander K. Winkel,
Amelia J. Thompson,
Kristian Franze
Nature Materials
25
687-697
(2026)
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Biological processes are regulated by chemical and mechanical signals, yet how these signalling modalities interact remains poorly understood. Here we identify a crosstalk between tissue stiffness and long-range chemical signalling in the developing Xenopus laevis brain. Targeted knockdown of the mechanosensitive ion channel Piezo1 in retinal ganglion cells or in the brain tissue surrounding retinal ganglion cells causes pathfinding errors in vivo. In the brain parenchyma, Piezo1 downregulation decreases the expression of the diffusive long-range chemical guidance cues Semaphorin3A (Sema3A) and Slit1, which instruct turning responses in distant cells. Furthermore, Piezo1 knockdown results in tissue softening due to reduced expression of the adhesion proteins NCAM1 and N-cadherin. Targeted depletion of NCAM1 and N-cadherin similarly reduces tissue stiffness and Sema3A expression. Conversely, increasing environmental stiffness ex vivo enhances tissue-level force generation and Slit1 and Sema3A expression. Finally, in vivo stiffening of soft brain regions induces ectopic Sema3A production via a Piezo1-dependent mechanism. Overall, these findings demonstrate that tissue mechanics locally modulates the availability of diffusive, long-range chemical signals, thus influencing cell function at sites distant from the mechanical cue.
Cell viscosity influences haematogenous dissemination and metastatic extravasation of tumour cells
Valentin Gensbittel,
Zeynep Yesilata,
Louis Bochler,
Gautier Follain,
Laurie Nemoz-Billet,
Olivier Lefebvre,
Klemens Uhlmann,
Annabel Larnicol,
Giulia E. M. Ammirati, et al.
Metastases arise from a multistep process during which tumour cells face several microenvironmental mechanical challenges which influence metastatic success However how circulating tumour cells (CTCs) adapt their mechanics to such microenvironments is not fully understood Here we report that the deformability of CTCs affects their haematogenous dissemination and identify mechanical phenotypes that favour metastatic extravasation Combining intravital microscopy with CTC-mimicking elastic beads mechanical tuning in tumour lines and profiling of tumour-patient-derived cells we demonstrate that the inherent mechanical properties of circulating objects dictate their ability to enter constraining vessels We identify cellular viscosity as a rheostat of CTC circulation and arrest and show that cellular viscosity is crucial for efficient extravasation Moreover we find that mechanical properties that favour extravasation and subsequent metastatic outgrowth can be opposite Altogether our results establish CTC viscosity as a key biomechanical parameter that shapes several steps of metastasis.
Fine-tuning cell-mimicking polyacrylamide microgels: Sensitivity to microscale reaction conditions in droplet microfluidics
Ruchi Goswami,
Kyoohyun Kim,
Aldo R. Boccaccini,
Jochen Guck,
Salvatore Girardo
Materials and Design
262
115450
(2026)
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Shaping polyacrylamide (PAAm) hydrogels via droplet microfluidics enables production of microgels that mimic cellular physical properties, advancing mechanobiology research. Controlling microgel size and elasticity is essential but challenging, as multiple factors influence polymerization and network formation. Although chemical reactions in microdroplets are generally faster and more uniform than in bulk, these microreactors are highly sensitive: small changes in chemical or physical conditions can cause significant variations in microgel properties. Our study identifies flow conditions as a crucial factor affecting both microgel elasticity and size by modulating interfacial transport during gelation. Using a flow-focusing microfluidic chip, we generated pre-gel droplets with the same composition in an oil phase, systematically varying the PAAm-to-oil flow rate ratio while maintaining a constant total flow rate. This method produced droplets with minimal size variation (<1 µm), but beads exhibited distinct Young’s moduli despite identical monomer concentrations. Further analysis showed that catalyst transport across the oil–water interface strongly impacts polymerization efficiency and network structure. These findings demonstrate that while droplet polymerization offers advantages, reproducible microgel properties demand precise flow control. This work emphasizes the critical role of microfluidic parameter tuning in advancing PAAm microgel applications in biophysics.
Tissue stress measurements with Bayesian inversion stress microscopy
Lucas Anger,
Andreas Schoenit,
Fanny Wodrascka,
Carine Rossé,
René-Marc Mège,
Benoît Ladoux,
Philippe Marcq
The European Physical Journal E
49
2
(2026)
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Cells within biological tissue are constantly subjected to dynamic mechanical forces. Measuring the internal stress of tissues has proven crucial for our understanding of the role of mechanical forces in fundamental biological processes like morphogenesis, collective migration, cell division, or cell elimination and death. Previously, we have introduced Bayesian inversion stress microscopy (BISM), which relies on measuring cell-generated traction forces in vitro and has proven particularly useful to measure absolute stresses in confined cell monolayers. We further demonstrate the applicability and robustness of BISM across various experimental settings with different boundary conditions, ranging from confined tissues of arbitrary shape to monolayers composed of different cell types. Importantly, BISM does not require assumptions on cell rheology. Therefore, it can be applied to complex heterogeneous tissues consisting of different cell types, as long as they can be grown on a flat substrate. Finally, we compare BISM to other common stress measurement techniques using a coherent experimental setup, followed by a discussion on its limitations and further perspectives.
Red Blood Cell-derived Extracellular Vesicles as biomaterials: the opportunity of freezing-induced accelerated aging
Lucia Paolini,
Miriam Romano,
Valentina Mangolini,
Selene Tassoni,
Shuhan Jiang,
Elena Laura Mazzoldi,
Angelo Musicò,
Andrea Zendrini,
Anna Kashkanova, et al.
Biomaterials Science
14
122-139
(2026)
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Red blood cell-derived extracellular vesicles (RBC-EVs) are emerging as promising biomaterials for next-generation drug delivery, owing to their intrinsic biocompatibility, immune evasion properties, and minimal oncogenic risk. However, their broader application is currently limited by unresolved challenges related to heterogeneity, reproducibility, and long-term storage stability. By combining discontinuous sucrose density gradient separation with high-resolution interferometric nanoparticle tracking analysis, we identified a sharp bimodal size distribution of the vesicles in freshly prepared samples. We then tracked how long-term storage at −80 °C drove its conversion into a monomodal distribution. To reproduce these conditions in a shorter time frame, we developed an “accelerated-ageing” protocol based on freeze–thaw cycles that generates RBC-EV samples with homogeneous density, size distribution, and biological activity, effectively replicating the properties of preparations stored for six months at −80 °C. This new vesicle population results stable and retains membrane integrity and cellular internalization capacity, as confirmed by surface-associated enzymatic activity assays and uptake tests in cancer cell lines. These results suggest that freezing-induced “accelerated ageing” represents an effective method for the optimization and standardization of RBC-EVs as building blocks for biomaterial and bioengineering applications.
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