Optical characterization of molecular interaction strength in protein condensates.

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques - Brillouin microscopy and quantitative phase imaging (QPI) - to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

Brillouin microscopy.

The field of Brillouin microscopy and imaging was established approximately 20 years ago, thanks to the development of non-scanning high-resolution optical spectrometers. Since then, the field has experienced rapid expansion, incorporating technologies from telecommunications, astrophotonics, multiplexed microscopy, quantum optics and machine learning. Consequently, these advancements have led to much-needed improvements in imaging speed, spectral resolution and sensitivity. The progress in Brillouin microscopy is driven by a strong demand for label-free and contact-free methods to characterize the mechanical properties of biomaterials at the cellular and subcellular scales. Understanding the local biomechanics of cells and tissues has become crucial in predicting cellular fate and tissue pathogenesis. This Primer aims to provide a comprehensive overview of the methods and applications of Brillouin microscopy. It includes key demonstrations of Brillouin microscopy and imaging that can serve as a reference for the existing research community and new adopters of this technology. The article concludes with an outlook, presenting the authors' vision for future developments in this vibrant field. The Primer also highlights specific examples where Brillouin microscopy can have a transformative impact on biology and biomedicine.

Thermally Assisted Microfluidics to Produce Chemically Equivalent Microgels with Tunable Network Morphologies.

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.

Longitudinal associations between depressive symptoms and cell deformability: do glucocorticoids play a role?

Cell deformability of all major blood cell types is increased in depressive disorders (DD). Furthermore, impaired glucocorticoid secretion is associated with DD, as well as depressive symptoms in general and known to alter cell mechanical properties. Nevertheless, there are no longitudinal studies examining accumulated glucocorticoid output and depressive symptoms regarding cell deformability. The aim of the present study was to investigate, whether depressive symptoms predict cell deformability one year later and whether accumulated hair glucocorticoids mediate this relationship. In 136 individuals (nfemale = 100; Mage = 46.72, SD = 11.28; age range = 20-65), depressive symptoms (PHQ-9) and hair glucocorticoids (cortisol and cortisone) were measured at time point one (T1), while one year later (T2) both depressive symptoms and hair glucocorticoids were reassessed. Additionally, cell deformability of peripheral blood cells was assessed at T2. Depression severity at T1 predicted higher cell deformability in monocytes and lymphocytes at T2. Accumulated hair cortisol and cortisone concentrations from T1 and T2 were not associated with higher cell deformability and further did not mediate the relationship between depressive symptoms and cell deformability. Elevated depressive symptomatology in a population based sample is longitudinally associated with higher immune cell deformability, while long-term integrated glucocorticoid levels seem not to be implicated in the underlying mechanism.

Omega-3 supplementation changes the physical properties of leukocytes but not erythrocytes in healthy individuals: An exploratory trial.

n3-PUFA impact health in several ways, including cardiovascular protection and anti-inflammatory effects, but the underlying mechanisms are not fully understood. In this exploratory study involving 31 healthy subjects, we aimed to investigate the effects of 12 weeks of fish-oil supplementation (1500 mg EPA+DHA/day) on the physical properties of multiple blood cell types. We used deformability cytometry (DC) for all cell types and Laser-assisted Optical Rotational Red Cell Analysis (Lorrca) to assess red blood cell (RBC) deformability. We also investigated the correlation between changes in the physical properties of blood cells and changes in the Omega-3 Index (O3I), defined as the relative content of EPA+DHA in RBCs. Following supplementation, the mean±SD O3I increased from 5.3 %±1.5 % to 8.3 %±1.4 % (p < 0.001). No significant changes in RBC properties were found by both techniques. However, by DC we observed a consistent pattern of physical changes in lymphocytes, neutrophils and monocytes. Among these were significant increases in metrics correlated with the cells' deformability resulting in less stiff cells. The results suggest that leukocytes become softer and have an increased ability to deform under induced short-term physical stress such as hydrodynamic force in the circulation. These changes could impact immune function since softer leukocytes can potentially circulate more easily and could facilitate a more rapid response to systemic inflammation or infection. In conclusion, fish-oil supplementation modulates some physical properties of leukocyte-subfractions, potentially enhancing their biological function. Further studies are warranted to explore the impact of n3-PUFA on blood cell biology, particularly in disease states associated with leukocyte dysregulation.

Estimation of the mass density of biological matter from refractive index measurements.

The quantification of physical properties of biological matter gives rise to novel ways of understanding functional mechanisms. One of the basic biophysical properties is the mass density (MD). It affects the dynamics in sub-cellular compartments and plays a major role in defining the opto-acoustical properties of cells and tissues. As such, the MD can be connected to the refractive index (RI) via the well known Lorentz-Lorenz relation, which takes into account the polarizability of matter. However, computing the MD based on RI measurements poses a challenge, as it requires detailed knowledge of the biochemical composition of the sample. Here we propose a methodology on how to account for assumptions about the biochemical composition of the sample and respective RI measurements. To this aim, we employ the Biot mixing rule of RIs alongside the assumption of volume additivity to find an approximate relation of MD and RI. We use Monte-Carlo simulations and Gaussian propagation of uncertainty to obtain approximate analytical solutions for the respective uncertainties of MD and RI. We validate this approach by applying it to a set of well-characterized complex mixtures given by bovine milk and intralipid emulsion and employ it to estimate the MD of living zebrafish (Danio rerio) larvae trunk tissue. Our results illustrate the importance of implementing this methodology not only for MD estimations but for many other related biophysical problems, such as mechanical measurements using Brillouin microscopy and transient optical coherence elastography.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

High-throughput viscoelastic characterization of cells in hyperbolic microchannels.

Extensive research has demonstrated the potential of cell viscoelastic properties as intrinsic indicators of cell state, functionality, and disease. For this, several microfluidic techniques have been developed to measure cell viscoelasticity with high-throughput. However, current microchannel designs introduce complex stress distributions on cells, leading to inaccuracies in determining the stress-strain relationship and, consequently, the viscoelastic properties. Here, we introduce a novel approach using hyperbolic microchannels that enable precise measurements under a constant extensional stress and offer a straightforward stress-strain relationship, while operating at a measurement rate of up to 100 cells per second. We quantified the stresses acting in the channels using mechanical calibration particles made from polyacrylamide (PAAm) and found that the measurement buffer, a solution of methyl cellulose and phosphate buffered saline, shows strain-thickening following a power law up to 200 s-1. By measuring oil droplets with varying viscosities, we successfully detected changes in the relaxation times of the droplets and our approach could be used to get the interfacial tension and viscosity of liquid-liquid droplet systems from the same measurement. We further applied this methodology to PAAm microgel beads, demonstrating the accurate recovery of Young's moduli and the near-ideal elastic behavior of the beads. To explore the influence of altered cell viscoelasticity, we treated HL60 human leukemia cells with latrunculin B and nocodazole, resulting in clear changes in cell stiffness while relaxation times were only minimally affected. In conclusion, our approach offers a streamlined and time-efficient solution for assessing the viscoelastic properties of large cell populations and other microscale soft particles.

Single-Cell Mechanics: Structural Determinants and Functional Relevance.

The mechanical phenotype of a cell determines its ability to deform under force and is therefore relevant to cellular functions that require changes in cell shape, such as migration or circulation through the microvasculature. On the practical level, the mechanical phenotype can be used as a global readout of the cell's functional state, a marker for disease diagnostics, or an input for tissue modeling. We focus our review on the current knowledge of structural components that contribute to the determination of the cellular mechanical properties and highlight the physiological processes in which the mechanical phenotype of the cells is of critical relevance. The ongoing efforts to understand how to efficiently measure and control the mechanical properties of cells will define the progress in the field and drive mechanical phenotyping toward clinical applications.

AI-driven projection tomography with multicore fibre-optic cell rotation.

Optical tomography has emerged as a non-invasive imaging method, providing three-dimensional insights into subcellular structures and thereby enabling a deeper understanding of cellular functions, interactions, and processes. Conventional optical tomography methods are constrained by a limited illumination scanning range, leading to anisotropic resolution and incomplete imaging of cellular structures. To overcome this problem, we employ a compact multi-core fibre-optic cell rotator system that facilitates precise optical manipulation of cells within a microfluidic chip, achieving full-angle projection tomography with isotropic resolution. Moreover, we demonstrate an AI-driven tomographic reconstruction workflow, which can be a paradigm shift from conventional computational methods, often demanding manual processing, to a fully autonomous process. The performance of the proposed cell rotation tomography approach is validated through the three-dimensional reconstruction of cell phantoms and HL60 human cancer cells. The versatility of this learning-based tomographic reconstruction workflow paves the way for its broad application across diverse tomographic imaging modalities, including but not limited to flow cytometry tomography and acoustic rotation tomography. Therefore, this AI-driven approach can propel advancements in cell biology, aiding in the inception of pioneering therapeutics, and augmenting early-stage cancer diagnostics.

p21 Prevents the Exhaustion of CD4+ T Cells Within the Antitumor Immune Response Against Colorectal Cancer.

BACKGROUND & AIMS: T cells are crucial for the antitumor response against colorectal cancer (CRC). T-cell reactivity to CRC is nevertheless limited by T-cell exhaustion. However, molecular mechanisms regulating T-cell exhaustion are only poorly understood. METHODS: We investigated the functional role of cyclin-dependent kinase 1a (Cdkn1a or p21) in cluster of differentiation (CD) 4+ T cells using murine CRC models. Furthermore, we evaluated the expression of p21 in patients with stage I to IV CRC. In vitro coculture models were used to understand the effector function of p21-deficient CD4+ T cells. RESULTS: We observed that the activation of cell cycle regulator p21 is crucial for CD4+ T-cell cytotoxic function and that p21 deficiency in type 1 helper T cells (Th1) leads to increased tumor growth in murine CRC. Similarly, low p21 expression in CD4+ T cells infiltrated into tumors of CRC patients is associated with reduced cancer-related survival. In mouse models of CRC, p21-deficient Th1 cells show signs of exhaustion, where an accumulation of effector/effector memory T cells and CD27/CD28 loss are predominant. Immune reconstitution of tumor-bearing Rag1-/- mice using ex vivo-treated p21-deficient T cells with palbociclib, an inhibitor of cyclin-dependent kinase 4/6, restored cytotoxic function and prevented exhaustion of p21-deficient CD4+ T cells as a possible concept for future immunotherapy of human disease. CONCLUSIONS: Our data reveal the importance of p21 in controlling the cell cycle and preventing exhaustion of Th1 cells. Furthermore, we unveil the therapeutic potential of cyclin-dependent kinase inhibitors such as palbociclib to reduce T-cell exhaustion for future treatment of patients with colorectal cancer.

Bile Is a Selective Elevator for Mucosal Mechanics and Transport.

Mucus mechanically protects the intestinal epithelium and impacts the absorption of drugs, with a largely unknown role for bile. We explored the impacts of bile on mucosal biomechanics and drug transport within mucus. Bile diffused with square-root-of-time kinetics and interplayed with mucus, leading to transient stiffening captured in Brillouin images and a concentration-dependent change from subdiffusive to Brownian-like diffusion kinetics within the mucus demonstrated by differential dynamic microscopy. Bile-interacting drugs, Fluphenazine and Perphenazine, diffused faster through mucus in the presence of bile, while Metoprolol, a drug with no bile interaction, displayed consistent diffusion. Our findings were corroborated by rat studies, where co-dosing of a bile acid sequestrant substantially reduced the bioavailability of Perphenazine but not Metoprolol. We clustered over 50 drugs based on their interactions with bile and mucin. Drugs that interacted with bile also interacted with mucin but not vice versa. This study detailed the dynamics of mucus biomechanics under bile exposure and linked the ability of a drug to interact with bile to its abbility to interact with mucus.

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment.

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in humans and other mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a contributing factor to regeneration failure in mammals. We demonstrate that the SLRPs chondroadherin, fibromodulin, lumican, and prolargin are enriched in rodent and human but not zebrafish CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer mechano-structural properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as inhibitory ECM factors that impair axon regeneration by modifying tissue mechanics and structure, and identifies their enrichment as a feature of human brain and spinal cord lesions. These findings imply that SLRPs may be targets for therapeutic strategies to promote CNS regeneration.

Human T cells loaded with superparamagnetic iron oxide nanoparticles retain antigen-specific TCR functionality.

Background: Immunotherapy of cancer is an emerging field with the potential to improve long-term survival. Thus far, adoptive transfer of tumor-specific T cells represents an effective treatment option for tumors of the hematological system such as lymphoma, leukemia or myeloma. However, in solid tumors, treatment efficacy is low owing to the immunosuppressive microenvironment, on-target/off-tumor toxicity, limited extravasation out of the blood vessel, or ineffective trafficking of T cells into the tumor region. Superparamagnetic iron oxide nanoparticles (SPIONs) can make cells magnetically controllable for the site-specific enrichment. Methods: In this study, we investigated the influence of SPION-loading on primary human T cells for the magnetically targeted adoptive T cell therapy. For this, we analyzed cellular mechanics and the T cell response after stimulation via an exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65 and compared them to T cells that had not received SPIONs. Results: SPION-loading of human T cells showed no influence on cellular mechanics, therefore retaining their ability to deform to external pressure. Additionally, SPION-loading did not impair the T cell proliferation, expression of activation markers, cytokine secretion, and tumor cell killing after antigen-specific activation mediated by the TCR. Conclusion: In summary, we demonstrated that SPION-loading of T cells did not affect cellular mechanics or the functionality of the endogenous or an exogenous TCR, which allows future approaches using SPIONs for the magnetically enrichment of T cells in solid tumors.

Varying the Stiffness and Diffusivity of Rod-Shaped Microgels Independently through Their Molecular Building Blocks.

Microgels are water-swollen, crosslinked polymers that are widely used as colloidal building blocks in scaffold materials for tissue engineering and regenerative medicine. Microgels can be controlled in their stiffness, degree of swelling, and mesh size depending on their polymer architecture, crosslink density, and fabrication method-all of which influence their function and interaction with the environment. Currently, there is a lack of understanding of how the polymer composition influences the internal structure of soft microgels and how this morphology affects specific biomedical applications. In this report, we systematically vary the architecture and molar mass of polyethylene glycol-acrylate (PEG-Ac) precursors, as well as their concentration and combination, to gain insight in the different parameters that affect the internal structure of rod-shaped microgels. We characterize the mechanical properties and diffusivity, as well as the conversion of acrylate groups during photopolymerization, in both bulk hydrogels and microgels produced from the PEG-Ac precursors. Furthermore, we investigate cell-microgel interaction, and we observe improved cell spreading on microgels with more accessible RGD peptide and with a stiffness in a range of 20 kPa to 50 kPa lead to better cell growth.

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.

Alcohol-sourced acetate impairs T cell function by promoting cortactin acetylation.

Alcohol is among the most widely consumed dietary substances. Excessive alcohol consumption damages the liver, heart, and brain. Alcohol also has strong immunoregulatory properties. Here, we report how alcohol impairs T cell function via acetylation of cortactin, a protein that binds filamentous actin and facilitates branching. Upon alcohol consumption, acetate, the metabolite of alcohol, accumulates in lymphoid organs. T cells exposed to acetate, exhibit increased acetylation of cortactin. Acetylation of cortactin inhibits filamentous actin binding and hence reduces T cell migration, immune synapse formation and activation. While mutated, acetylation-resistant cortactin rescues the acetate-induced inhibition of T cell migration, primary mouse cortactin knockout T cells exhibited impaired migration. Acetate-induced cytoskeletal changes effectively inhibited activation, proliferation, and immune synapse formation in T cells in vitro and in vivo in an influenza infection model in mice. Together these findings reveal cortactin as a possible target for mitigation of T cell driven autoimmune diseases.

Dynamics of cell rounding during detachment.

Animal cells undergo repeated shape changes, for example by rounding up and respreading as they divide. Cell rounding can be also observed in interphase cells, for example when cancer cells switch from a mesenchymal to an ameboid mode of cell migration. Nevertheless, it remains unclear how interphase cells round up. In this article, we demonstrate that a partial loss of substrate adhesion triggers actomyosin-dependent cortical remodeling and ERM activation, which facilitates further adhesion loss causing cells to round. Although the path of rounding in this case superficially resembles mitotic rounding in involving ERM phosphorylation, retraction fiber formation, and cortical remodeling downstream of ROCK, it does not require Ect2. This work provides insights into the way partial loss of adhesion actives cortical remodeling to drive cell detachment from the substrate. This is important to consider when studying the mechanics of cells in suspension, for example using methods like real-time deformability cytometry (RT-DC).

Rapid single-cell physical phenotyping of mechanically dissociated tissue biopsies.

During surgery, rapid and accurate histopathological diagnosis is essential for clinical decision making. Yet the prevalent method of intra-operative consultation pathology is intensive in time, labour and costs, and requires the expertise of trained pathologists. Here we show that biopsy samples can be analysed within 30 min by sequentially assessing the physical phenotypes of singularized suspended cells dissociated from the tissues. The diagnostic method combines the enzyme-free mechanical dissociation of tissues, real-time deformability cytometry at rates of 100-1,000 cells s-1 and data analysis by unsupervised dimensionality reduction and logistic regression. Physical phenotype parameters extracted from brightfield images of single cells distinguished cell subpopulations in various tissues, enhancing or even substituting measurements of molecular markers. We used the method to quantify the degree of colon inflammation and to accurately discriminate healthy and tumorous tissue in biopsy samples of mouse and human colons. This fast and label-free approach may aid the intra-operative detection of pathological changes in solid biopsies.