Cryogenic light microscopy with Ångstrom precision deciphers structural conformations of PIEZO1
Hisham Mazal,
Alexandra Schambony,
Vahid Sandoghdar
bioRxiv 10.1101/2024.12.22.629944
(2025)
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Despite the impressive progress in molecular biochemistry and biophysics, many questions regarding the conformational states of large (transmembrane) protein complexes persist. In the case of the PIEZO protein, investigations by cryogenic electron microscopy (Cryo-EM) and atomic force microscopy (AFM) have established a symmetric trimer structure with three long-bladed domains in a propeller-like configuration. A transition of PIEZO protein from curved to flat conformation is hypothesized to actuate closed and open channels for the flow of ions. However, conclusive high-resolution data on the molecular organization of PIEZO in its native form are lacking. To address this shortcoming, we exploit single-particle cryogenic light microscopy (spCryo-LM) to decipher the conformational states of the mouse PIEZO1 protein (mPIEZO1) in the cell membrane. Here, we implement a high-vacuum cryogenic shuttle to transfer shock-frozen unroofed cell membranes in and out of a cryostat for super-resolution microscopy at liquid helium temperature. By localizing fluorescent labels placed at the extremities of the three blades with Ångstrom precision, we ascertain three configurations of the protein with radii of 6, 12, and 20 nm as projected onto the membrane plane. Our data suggest that in the smallest configuration, the blades form a nano-dome structure that is more strongly curved than previously observed and predicted by AlphaFold-3. In the largest conformation, we believe the structure must fully unbend in an anticlockwise manner to form a flat extended state. We attribute the 12 nm conformation, the most frequently occupied state, to an intermediate state and discuss our results in the context of the findings from other groups. Combination of spCryo-LM and Cryo-EM measurements together with in situ photothermal stimulation promises to provide quantitative insight into the interplay between structure and function of PIEZO and other biomolecular complexes in their native environments.
Science Advances, to appear
Consensus statement on Brillouin light scattering microscopy of biological materials
Pierre Bouvet,
Carlo Bevilacqua,
Yogeshwari Ambekar,
Giuseppe Antonacci,
Joshua Au,
Silvia Caponi,
Sophie Chagnon-Lessard,
Juergen Czarske,
Thomas Dehoux, et al.
Nature Photonics
19
681-691
(2025)
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Brillouin light scattering (BLS) spectroscopy is a non-invasive, non-contact, label-free optical technique that can provide information on the mechanical properties of a material on the submicrometre scale. Over the past decade, BLS has found increasing microscopy applications in the life sciences, driven by the observed importance of mechanical properties in biological processes, the realization of more sensitive BLS spectrometers and the extension of BLS to an imaging modality. As with other spectroscopic techniques, BLS measurements detect not only signals that are characteristic of the investigated sample, but also those of the experimental apparatus, and can be substantially affected by measurement conditions. Here we report a consensus between researchers in the field. We aim to improve the comparability of BLS studies by providing reporting recommendations for the measured parameters and detailing common artefacts. Given that most BLS studies of biological matter are still at proof-of-concept stages and use different, often self-built, spectrometers, a consensus statement is particularly timely to ensure unified advancement.
Small U-Net for Fast and Reliable Segmentation in Imaging Flow Cytometry
Sara Kaliman,
Raghava Alajangi,
Nadia Sbaa,
Paul Müller,
Nadine Ströhlein,
Jeffrey Harmon,
Martin Kräter,
Jochen Guck,
Shada Abuhattum Hofemeier
Imaging flow cytometry requires rapid and accurate segmentation methods to ensure high-quality cellular morphology analysis and cell counting. In deformability cytometry (DC), a specific type of imaging flow cytometry, accurately detecting cell contours is critical for evaluating mechanical properties that serve as disease markers. Traditional thresholding methods, commonly used for their speed in high-throughput applications, often struggle with low-contrast images, leading to inaccuracies in detecting the object contour. Conversely, standard neural network approaches like U-Net, though effective in medical imaging, are less suitable for high-speed imaging applications due to long inference times. To address these issues, we present a solution that enables both fast and accurate segmentation, designed for imaging flow cytometry. Our method employs a small U-Net model trained on high-quality, curated, and annotated data. This optimized model outperforms traditional thresholding methods and other neural networks, delivering a 35× speed improvement on CPU over the standard U-Net. The enhanced performance is demonstrated by a significant reduction in systematic measurement errors in blood samples analyzed using DC. The tools developed in this study are adaptable for various imaging flow cytometry applications. This approach improves segmentation quality while maintaining the rapid processing necessary for high-throughput environments.
Cell state-specific cytoplasmic density controls spindle architecture and scaling
Nature Cell Biology
27
959-971
(2025)
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Mitotic spindles are dynamically intertwined with the cytoplasm they assemble in. How the physicochemical properties of the cytoplasm affect spindle architecture and size remains largely unknown. Using quantitative biochemistry in combination with adaptive feedback microscopy, we investigated mitotic cell and spindle morphology during neural differentiation of embryonic stem cells. While tubulin biochemistry and microtubule dynamics remained unchanged, spindles changed their scaling behaviour; in differentiating cells, spindles were considerably smaller than those in equally sized undifferentiated stem cells. Integrating quantitative phase imaging, biophysical perturbations and theory, we found that as cells differentiated, their cytoplasm became more dilute. The concomitant decrease in free tubulin activated CPAP (centrosomal P4.1-associated protein) to enhance the centrosomal nucleation capacity. As a consequence, in differentiating cells, microtubule mass shifted towards spindle poles at the expense of the spindle bulk, explaining the differentiation-associated switch in spindle architecture. This study shows that cell state-specific cytoplasmic density tunes mitotic spindle architecture. Thus, we reveal physical properties of the cytoplasm as a major determinant in organelle size control.
Cellular morphodynamics as quantifiers for functional states of resident tissue macrophages in vivo
Miriam Schnitzerlein,
Eric Greto,
Anja Wegner,
Anna Möller,
Oliver Aust,
Oumaima Ben Brahim,
David B. Blumenthal,
Vasily Zaburdaev,
Stefan Uderhardt, et al.
PLOS Computational Biology
21
e1011859
(2025)
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Resident tissue macrophages (RTMs) are essential for tissue homeostasis. Their diverse functions, from monitoring interstitial fluids to clearing cellular debris, are accompanied by characteristic morphological changes that reflect their functional status. While current knowledge of macrophage behaviour comes primarily from in vitro studies, their dynamic behavior in vivo is fundamentally different, necessitating a more physiologically relevant approach to their understanding. In this study, we employed intravital imaging to generate dynamic data from peritoneal RTMs in mice under various conditions and developed a comprehensive image processing pipeline to quantify RTM morphodynamics over time, defining human-interpretable cell size and shape features. These features allowed for the quantitative and qualitative differentiation of cell populations in various functional states, including pro- and anti-inflammatory activation and endosomal dysfunction. The study revealed that under steady-state conditions, RTMs exhibit a wide range of morphodynamical phenotypes, constituting a naïve morphospace of behavioral motifs. Upon challenge, morphodynamic patterns changed uniformly at the population level but predominantly within the constraints of this naïve morphospace. Notably, aged animals displayed a markedly shifted naïve morphospace, indicating drastically different behavioral patterns compared to their young counterparts. The developed method also proved valuable in optimizing explanted tissue setups, bringing RTM behavior closer to the physiological native state. Our versatile approach thus provides novel insights into the dynamic behavior of bona fide macrophages in vivo, enabling the distinction between physiological and pathological cell states and the assessment of functional tissue age on a population level.
Cryogenic light microscopy of vitrified samples with Ångstrom precision
Hisham Mazal,
Franz Wieser,
Daniel Bollschweiler,
Vahid Sandoghdar
bioRxiv 10.1101/2025.05.27.656160
(2025)
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High-resolution studies in structural biology are commonly based on diffraction methods and on electron microscopy. However, these approaches are limited by the difficulty in crystallization of biomolecules or by a low contrast that makes high-resolution measurements very challenging in crowded samples such as a cell membrane. The exquisite labeling specificity of fluorescence microscopy gets around these issues. Indeed, several recent reports have reached resolutions down to the Ångstrom level in super-resolution microscopy, but to date, these works used fixed samples. To establish light microscopy as a workhorse in structural biology, two main requirements must be fulfilled: near-native sample preservation and near-atomic optical resolution. Here, we demonstrate a technique that satisfies these key criteria with particular promise for conformational studies on membrane proteins and their complexes. To prepare cell membranes in their near-native state, we adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing and transfer of samples. We developed a high-vacuum cryogenic shuttle system that allows us to transfer vitrified samples in and out of a liquid-helium cryostat that houses a super-resolution fluorescence microscope. Sample temperatures below 10 K help dissipate the heat from laser illumination, thus maintaining intact vitreous ice. We utilize the photoblinking of organic dye molecules attached to well-defined positions of a protein to localize one label fluorophore at a time. We present various characterization studies of the vitreous ice, photoblinking behavior, and the effects of the laser intensity. Moreover, we benchmark our method by demonstrating Ångstrom precision in resolving the full assembled configuration of the heptameric membrane protein alpha-hemolysin (αHL) in a synthetic lipid membrane as a model system. Additionally, we report on the technique’s capability to resolve membrane proteins in their native cellular membrane environment. Our method, which we term single-particle cryogenic light microscopy (spCryo-LM), enables structural studies of membrane protein tertiary and quaternary conformations without the need for chemical fixation or protein isolation. The approach can also integrate other super-resolution or spectroscopic techniques with particular promise in correlative microscopy with images from Cryo-EM and related techniques.
Differential tissue deformability underlies fluid pressure-driven shape divergence of the avian embryonic brain and spinal cord
Susannah B.P. McLaren,
Shi-Lei Xue,
Siyuan Ding,
Alexander K. Winkel,
Oscar Baldwin,
Shreya Dwarakacherla,
Kristian Franze,
Edouard Hannezo,
Fengzhu Xiong
An enlarged brain underlies the complex central nervous system of vertebrates. The dramatic expansion of the brain that diverges its shape from the spinal cord follows neural tube closure during embryonic development. Here, we show that this differential deformation is encoded by a pre-pattern of tissue material properties in chicken embryos. Using magnetic droplets and atomic force microscopy, we demonstrate that the dorsal hindbrain is more fluid than the dorsal spinal cord, resulting in a thinning versus a resisting response to increasing lumen pressure, respectively. The dorsal hindbrain exhibits reduced apical actin and a disorganized laminin matrix consistent with tissue fluidization. Blocking the activity of neural-crest-associated matrix metalloproteinases inhibits hindbrain expansion. Transplanting dorsal hindbrain cells to the spinal cord can locally create an expanded brain-like morphology in some cases. Our findings raise questions in vertebrate head evolution and suggest a general role of mechanical pre-patterning in sculpting epithelial tubes.
Hierarchical Heterogeneities in Spatio-Temporal Dynamics of the Cytoplasm
Understanding of the dynamics inherent to biological matter is crucial for illuminating the physical mechanisms underlying cellular processes. In this study, we employ bright-field differential dynamic microscopy (DDM) to investigate density fluctuations inherent in a cell-free model of eukaryotic cytoplasm. Our measurements reveal subdiffusive fractional Brownian motion and non-Gaussian displacement distributions, highlighting cytoplasmic heterogeneity. We introduce an empirical model that combines fractional Brownian motion with an inverse Gaussian distribution of diffusivities to describe the observed non-Gaussianity. Validated through Monte Carlo simulations, this model allows us to estimate the fractional diffusivity and exponent effectively. By altering macromolecular composition, the addition of energy, and assembly of a cytoskeleton, we identify three independent mechanisms that result in similar fractional exponents yet distinct diffusivities. We find that energy addition leads to non-stationary dynamics, in contrast to the stationary behavior observed under passive conditions. Presence of microtubules introduces a secondary dynamical timescale, which we describe using a two-state fractional Brownian motion model to differentiate between cytosolic and microtubule network associated contributions. Our findings demonstrate the effectiveness of DDM as a label-free tool for quantifying viscoelastic and heterogeneous properties of the cytoplasm and provide insights into how physical and biochemical factors, including cytoskeletal organization, govern subcellular dynamics.
Viscoelastic characterization of cells in microfluidic channels with 3D hydrodynamic focusing
Benedikt Hartmann,
Felix Reichel,
Conrad Möckel,
Jochen Guck
The viscoelastic nature of biological cells has emerged as an increasingly important research subject due to its relevance for cellular functions under physiological and pathological conditions. Advancements in microfluidics have made this technology a promising tool to study the viscoelasticity of cells. However, significant challenges remain, including the complex distribution of stresses acting on cells depending on the channel geometry, and the difficulty of keeping cells in the focal plane for imaging. Here, we report a new approach using hyperbolic channels for measuring cell viscoelasticity. A channel height much larger than the typical cell size minimized shear stresses so that normal stresses in the hyperbolic region dominated the stress distribution. Reducing the complexity of the stress-strain relationship allowed us to use polyacrylamide microgel beads to calibrate the stress curve. Additionally, we introduced 3D hydrodynamic focusing which enabled us to focus cells and microgel beads in the center of the channel. Finally, Kelvin-Voigt and power-law rheology models were employed to extract the mechanical properties of microgel beads and human leukemia HL60 cells. The measurement technique described here will help establish the viscoelastic properties of cells as an important readout in biophysical research in health and disease.
Flocking and giant fluctuations in epithelial active solids
Proceedings of the National Academy of Sciences of the United States of America
122
e2421327122
(2025)
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The collective motion of epithelial cells is a fundamental biological process which plays a significant role in embryogenesis, wound healing, and tumor metastasis. While it has been broadly investigated for over a decade both in vivo and in vitro, large-scale coherent flocking phases remain underexplored and have so far been mostly described as fluid. In this work, we report an additional mode of large-scale collective motion for different epithelial cell types in vitro with distinctive features. By tracking individual cells, we show that cells move over long time scales coherently not as a fluid, but as a polar elastic solid with negligible cell rearrangements. Our analysis reveals that this solid flocking phase exhibits signatures of long-range polar order, accompanying with scale-free correlations of the transverse component of velocity fluctuations, anomalously large density fluctuations, and shear waves. Based on a general theory of active polar solids, we argue that these features result from massless orientational Goldstone mode, which, in contrast to polar fluids where they are generic, require the decoupling of global rotations of the polarity and in-plane elastic deformations in polar solids. We theoretically show and consistently observe in experiments that the fluctuations of elastic deformations diverge for large system sizes in such polar active solid phases, leading eventually to rupture and thus potentially loss of tissue integrity at large scales.
Interferometric scattering microscopy
Naomi S. Ginsberg,
Chia-Lung Hsieh,
Philipp Kukura,
Marek Piliarik,
Vahid Sandoghdar
Over the past two decades, interferometric scattering (iSCAT) microscopy has become a powerful label-free imaging method with a range of applications in fundamental science and technology. iSCAT detects the scattering of subwavelength entities through interference with a reference beam of light. Performed in a variety of illumination and detection schemes, iSCAT has exploited both amplitude and phase information to reach single-molecule detection sensitivity; to determine the size, mass and refractive index of nanoparticles; to achieve high spatiotemporal precision in 3D tracking of nanoparticles; to image subcellular nanostructures; and to quantify ultrafast diffusion and transport of energy in solids. In this Primer, we describe the basic principles of iSCAT detection and imaging from theoretical and practical points of view. We discuss various factors that affect the attainable signal-to-noise ratio, which in turn determines crucial performance features such as sensitivity and speed. We survey selected applications in which iSCAT has been instrumental in providing new insights. Finally, we discuss some of the current challenges and potential avenues for advancing the technique further.
Selective plane illumination microscopy (SPIM), also known as light sheet fluorescence microscopy, provides high specificity through fluorescence labeling. However, it lacks complementary structural information from the surrounding context, which is essential for the comprehensive analysis of biological samples. Here, we present a high-resolution, multimodal imaging system that integrates SPIM with full-field optical coherence tomography (FF-OCT), without requiring modifications to the existing SPIM setup. Both SPIM and FF-OCT offer low phototoxicity and intrinsic optical sectioning, making them well-suited for in vivo imaging. Their shared detection path enables seamless and efficient co-registration of fluorescence and structural data. We demonstrate the functionality of this combined system by performing in vivo imaging of zebrafish larvae.
Resolving spatiotemporal dynamics in bacterial multicellular populations: approaches and challenges
Suyen Solange Espinoza Miranda,
Gorkhmaz Abbaszade,
Wolfgang R. Hess,
Knut Drescher,
Antoine-Emmanuel Saliba,
Vasily Zaburdaev,
Liraz Chai,
Klaus Dreisewerd,
Alexander Grünberger, et al.
Microbiology and Molecular Biology Reviews
89
e00138-24
(2025)
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The development of multicellularity represents a key evolutionary transition that is crucial for the emergence of complex life forms. Although multicellularity has traditionally been studied in eukaryotes, it originates in prokaryotes. Coordinated aggregation of individual cells within the confines of a colony results in emerging, higher-level functions that benefit the population as a whole. During colony differentiation, an almost infinite number of ecological and physiological population-forming forces are at work, creating complex, intricate colony structures with divergent functions. Understanding the assembly and dynamics of such populations requires resolving individual cells or cell groups within such macroscopic structures. Addressing how each cell contributes to the collective action requires pushing the resolution boundaries of key technologies that will be presented in this review. In particular, single-cell techniques provide powerful tools for studying bacterial multicellularity with unprecedented spatial and temporal resolution. These advancements include novel microscopic techniques, mass spectrometry imaging, flow cytometry, spatial transcriptomics, single-bacteria RNA sequencing, and the integration of spatiotemporal transcriptomics with microscopy, alongside advanced microfluidic cultivation systems. This review encourages exploring the synergistic potential of the new technologies in the study of bacterial multicellularity, with a particular focus on individuals in differentiated bacterial biofilms (colonies). It highlights how resolving population structures at the single-cell level and understanding their respective functions can elucidate the overarching functions of bacterial multicellular populations.
Measurement force, speed, and postmortem time affect the ratio of CNS gray-to-white-matter elasticity
Julia Monika Becker,
Alexander Kevin Winkel,
Eva Kreysing,
Kristian Franze
For several decades, many attempts have been made to characterize the mechanical properties of gray and white matter, which constitute the two main compartments of the central nervous system, with various methods and contradictory results. In particular, the ratio of gray-to-white-matter elasticity is sometimes larger than 1 and sometimes smaller; the reason for this apparent discrepancy is currently unknown. Here, we exploited atomic force microscopy-based indentation measurements to systematically investigate how the measurement force, measurement speed, postmortem interval, and temperature affect the measured elasticity of spinal cord tissue and, in particular, the ratio of gray-to-white-matter elasticity (Kg/Kw). Within the explored parameter space, increasing measurement force and speed increased the measured elasticity of both gray and white matter. However, Kg/Kw declined from values as high as ∼5 at low forces and speeds to ∼1 for high forces and speeds. Kg/Kw also strongly depended on the anatomical plane in which the measurements were conducted and was considerably higher in transverse sections compared with longitudinal sections. Furthermore, the postmortem interval impacted both the absolute measured tissue elasticity and Kg/Kw. Gray matter elasticity started decreasing ∼3 h postmortem until reaching a plateau after ∼6 h. In contrast, white matter elasticity started declining from the beginning of the measurements until ∼6 h postmortem, when it also leveled off. As a result, Kg/Kw increased until ∼6 h postmortem before stabilizing. Between 20 and 38°C, both gray and white matter elasticity decreased at a similar rate without affecting Kg/Kw. We have thus identified differences in the response of gray and white matter to varying strains and strain rates, and the postmortem interval, and excluded temperature as a factor affecting Kg/Kw. These differential responses likely contribute to the contradictory results obtained with different methods working in different strain regimes.
in Press
Force transmission is a master regulator of mechanical cell competition
Andreas Schoenit,
Siavash Monfared,
Lucas Anger,
Carine Rosse,
Varun Venkatesh,
Lakshmi Balasubramaniam,
Elisabetta Marangoni,
Philippe Chavrier,
René-Marc Mège, et al.
Nature Materials
24
966-976
(2025)
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Cell competition is a tissue surveillance mechanism for eliminating unwanted cells, being indispensable in development, infection and tumourigenesis. Although studies have established the role of biochemical mechanisms in this process, due to challenges in measuring forces in these systems, how mechanical forces determine the competition outcome remains unclear. Here we report a form of cell competition that is regulated by differences in force transmission capabilities, selecting for cell types with stronger intercellular adhesion. Direct force measurements in ex vivo tissues and different cell lines reveal that there is an increased mechanical activity at the interface between two competing cell types, which can lead to large stress fluctuations resulting in upward forces and cell elimination. We show how a winning cell type endowed with a stronger intercellular adhesion exhibits higher resistance to elimination and benefiting from efficient force transmission to the neighbouring cells. This cell elimination mechanism could have broad implications for keeping the strong force transmission ability for maintaining tissue boundaries and cell invasion pathology.
Femtosecond Fieldoscopy for super-resolution label-free microscopy
Soyeon Jun,
Andreas Herbst,
Kilian Scheffter,
Daniel Wehner,
Anchit Srivastava,
Hanieh Fattahi
Microscopy and Analysis
39
42-44
(2025)
Accessing complete electric field information of a laser pulse interacting with a medium at visible to near-infrared (near-petahertz) frequencies has traditionally required complex laboratory systems operating in vacuum conditions. Recent advancements, however, have enabled the measurement of electric fields at near-petahertz frequencies in ambient air. This capability is critical for understanding ultrafast phenomena and for achieving quantitative detection of molecular species in various samples. This article introduces Femtosecond Fieldoscopy, a field-resolved detection technique for label-free spectroscopy and microscopy. This approach delivers exceptional detection sensitivity and dynamic range at petahertz bandwidths by combining attosecond temporal resolution with temporal isolation of target molecular responses from environmental and excitation pulse effects. Furthermore, Femtosecond Fieldoscopy holds promise for achieving sub-diffraction spatial resolution, opening new horizons for high-precision label-free spectro-microscopy.
Electrostatic All-Passive Force Clamping of Charged Nanoparticles
Yazgan Tuna,
Amer Al-Hiyasat,
Anna D. Kashkanova,
Andreas Dechant,
Eric Lutz,
Vahid Sandoghdar
In the past decades, many techniques have been explored for trapping microscopic and nanoscopic objects, but the investigation of nano-objects under arbitrary forces and conditions remains nontrivial. One fundamental case concerns the motion of a particle under a constant force, known as force clamping. Here, we employ metallic nanoribbons embedded in a glass substrate in a capacitor configuration to generate a constant electric field on a charged nanoparticle in a water-filled glass nanochannel. We estimate the force fields from Brownian trajectories over several micrometers and confirm the constant behavior of the forces both numerically and experimentally. Furthermore, we manipulate the diffusion and relaxation times of the nanoparticles by tuning the charge density on the electrode. Our highly compact and controllable setting allows for the trapping and force-clamping of charged nanoparticles in a solution, providing a platform for investigating nanoscopic diffusion phenomena.
De novo identification of universal cell mechanics gene signatures
Marta Urbanska,
Yan Ge,
Maria Winzi,
Shada Abuhattum Hofemeier,
Syed Shafat Ali,
Maik Herbig,
Martin Kräter,
Nicole Toepfner,
Joanne Durgan, et al.
Cell mechanical properties determine many physiological functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors that govern the mechanical properties is therefore a subject of great interest. Here, we present a mechanomics approach for establishing links between single-cell mechanical phenotype changes and the genes involved in driving them. We combine mechanical characterization of cells across a variety of mouse and human systems with machine learning-based discriminative network analysis of associated transcriptomic profiles to infer a conserved network module of five genes with putative roles in cell mechanics regulation. We validate in silico that the identified gene markers are universal, trustworthy, and specific to the mechanical phenotype across the studied mouse and human systems, and demonstrate experimentally that a selected target, CAV1, changes the mechanical phenotype of cells accordingly when silenced or overexpressed. Our data-driven approach paves the way toward engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions.
Biphasic inflammation control by dedifferentiated fibroblasts enables axon regeneration after spinal cord injury in zebrafish
Nora John,
Thomas Fleming,
Julia Kolb,
Olga Lyraki,
Sebastián Vásquez-Sepúlveda,
Asha Parmer,
Kyoohyun Kim,
Maria Tarczewska,
Kanwarpal Singh, et al.
Fibrosis and persistent inflammation are interconnected processes that inhibit axon regeneration in the mammalian central nervous system (CNS). In zebrafish, by contrast, fibroblast-derived extracellular matrix deposition and inflammation are tightly regulated to facilitate regeneration. However, the regulatory cross-talk between fibroblasts and the innate immune system in the regenerating CNS remains poorly understood. Here, we show that zebrafish fibroblasts possess a dual role in inducing and resolving inflammation, which are both essential for regeneration. We identify a transient, injury-specific cthrc1a+ fibroblast state with an inflammation-associated, less differentiated, and non-fibrotic profile. Induction of this fibroblast state precedes and contributes to the initiation of the inflammatory response. At the peak of neutrophil influx, cthrc1a+ fibroblasts coordinate the resolution of inflammation. Disruption of these inflammation dynamics alters the mechano-structural properties of the lesion environment and inhibits axon regeneration. This establishes the biphasic inflammation control by dedifferentiated fibroblasts as a pivotal mechanism for CNS regeneration.
Dynamic forces shape the survival fate of eliminated cells
Lakshmi Balasubramaniam,
Siavash Monfared,
Aleksandra Ardaševa,
Carine Rosse,
Andreas Schoenit,
Tien Dang,
Chrystelle Maric,
Mathieu Hautefeuille,
Leyla Kocgozlu, et al.
Tissues eliminate unfit, unwanted or unnecessary cells through cell extrusion, and this can lead to the elimination of both apoptotic and live cells. However, the mechanical signatures that influence the fate of extruding cells remain unknown. Here we show that modified force transmission across adherens junctions inhibits apoptotic cell eliminations. By combining cell experiments with varying levels of E-cadherin junctions and three-dimensional modelling of cell monolayers, we find that these changes not only affect the fate of the extruded cells but also shift extrusion from the apical to the basal side, leading to cell invasion into soft collagen gels. We generalize our findings using xenografts and cysts cultured in matrigel, derived from patients with breast cancer. Our results link intercellular force transmission regulated by cell–cell communication to cell extrusion mechanisms, with potential implications during morphogenesis and invasion of cancer cells.
Cytoskeleton-functionalized synthetic cells with life-like mechanical features and regulated membrane dynamicity
Sebastian Novosedlik,
Felix Reichel,
Thijs van Veldenhuisen,
Yudong Li,
Hanglong Wu,
Henk Janssen,
Jochen Guck,
Jan van Hest
Nature Chemistry
17
356-364
(2025)
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The cytoskeleton is a crucial determinant of mammalian cell structure and function, providing mechanical resilience, supporting the cell membrane and orchestrating essential processes such as cell division and motility. Because of its fundamental role in living cells, developing a reconstituted or artificial cytoskeleton is of major interest. Here we present an approach to construct an artificial cytoskeleton that imparts mechanical support and regulates membrane dynamics. Our system involves amylose-based coacervates stabilized by a terpolymer membrane, with a cytoskeleton formed from polydiacetylene fibrils. The fibrils bundle due to interactions with the positively charged amylose derivative, forming micrometre-sized structures mimicking a cytoskeleton. Given the intricate interplay between cellular structure and function, the design and integration of this artificial cytoskeleton represent a crucial advancement, paving the way for the development of artificial cell platforms exhibiting enhanced life-like behaviour.
Thermally Assisted Microfluidics to Produce Chemically Equivalent Microgels with Tunable Network Morphologies
Dirk Rommel,
Bernhard Häßel,
Philip Pietryszek,
Matthias Mork,
Oliver Jung,
Meike Emondts,
Nikita Norkin,
Iris Christine Doolaar,
Yonka Kittel, et al.
Angewandte Chemie, International Edition in English
64
(1)
(2025)
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Although micron-sized microgels have become important building blocks in regenerative materials, offering decisive interactions with living matter, their chemical composition mostly significantly varies when their network morphology is tuned. Since cell behavior is simultaneously affected by the physical, chemical, and structural properties of the gel network, microgels with variable morphology but chemical equivalence are of interest. This work describes a new method to produce thermoresponsive microgels with defined mechanical properties, surface morphologies, and volume phase transition temperatures. A wide variety of microgels is synthesized by crosslinking monomers or star polymers at different temperatures using thermally assisted microfluidics. The diversification of microgels with different network structures and morphologies but of chemical equivalence offers a new platform of microgel building blocks with the ability to undergo phase transition at physiological temperatures. The method holds high potential to create soft and dynamic materials while maintaining the chemical composition for a wide variety of applications in biomedicine.
How intercellular forces regulate cell competition
Andreas Schoenit,
Siavash Monfared,
Lucas Anger,
Carine Rosse,
Varun Venkatesh,
Lakshmi Balasubramaniam,
Elisabetta Marangoni,
Philippe Chavrier,
René‐Marc Mège, et al.
