Biology subjects

Sengupta, K.

Publications and source records attributed to Sengupta, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Remodelling of chromatin architecture and super-enhancer landscape in lamin A/C depleted HGSOC

Lamins are nuclear intermediate filament proteins that maintain nuclear architecture through interactions with the chromatin. The A- and B-type lamins tether the genome at the peripheral lamina underlying the inner nuclear membrane in the form of heterochromatic lamina-associated domains (LADs). However, LADs associated with A-type lamins are confined not only to lamina but also to the nuclear core, thereby pointing to their distinct and multifarious roles in chromatin organisation and regulation. Previous studies involving lamin B1 depletion depicted the detachment of LADs from the nuclear periphery, accompanied by alteration of chromatin distribution while preserving the topologically associating domains (TADs) in structurally intact form. In this piece of work, we have shown, for the first time in HGSOC, the effects of lamin A/C knockdown on chromatin organisation, which was characterised by significant detethering of gene-poor chromatin from the periphery along with active (A) to inactive (B) compartment switching. This was associated with a rewiring of oncogenic super-enhancer elements corroborated by the differential gene expression profile. Overall analysis tipped the scale in favour of reduced cellular proliferation upon lamin A/C knockdown. This finding was strengthened by the observed proliferative potential of spheroids ex vivo and tumours in a mouse xenograft model.

cancer biology↗

bioRxiv2026

T cell degranulation and payload delivery are modulated in space and time

Release of destructive payloads through degranulation is a hallmark of T cell cytotoxicity. Upon stimulation, tens to hundreds of granules are rapidly delivered to the interface with a target cell but only a small number of degranulation events occur. Two forms of degranulation have been observed and differ in the physical form of the payload released. The deposition of insoluble particles encapsulated in a proteinaceous shell may be more potent than release of purely soluble factors. The selection mechanisms and triggers for degranulation are still poorly understood, partly due to the lack of tools to dynamically map and distinguish each mode. Here we use high spatiotemporal resolution in vitro assays, coupled to a novel label-free strategy to characterize and track released particles, to show that degranulation is regulated not only by chemical but also physical aspects of the activation inputs. Furthermore, the released, insoluble particles are transported within the intermembrane junction with a preferred directionality. Immobilized stimulatory ligands favored the soluble degranulation mode suggesting that the particulate release is pertinent to mobile, physiological immune interfaces. Engineered chimeric antigen receptor (CAR) T cells have a defect in centralizing released particles, which may explain dampened toxicity and off-target tissue damage. Our results demonstrate how degranulation events are likely regulated by a combination of physical, chemical, and signaling mechanisms. We anticipate these findings will generate new strategies to bias degranulation outcomes and optimize the destructive capacity of single cytotoxic T cells. In the case of engineered T cells, efforts to restore the normal transport of insoluble particles could contribute to stronger therapeutics for cellular immunotherapy.

immunology↗

bioRxiv2026

Tuning T-cell immunological synapse by modular DNA-Nanobody engagers for precision immunotherapy

Bispecific T-cell engagers (TCEs) are a promising class of cancer immunotherapies, but their clinical use is limited by toxicity and insufficient specificity. Tuning the T cell- tumor interface through engager architecture may address these drawbacks. To this end, we engineered hybrid constructs composed of two nanobodies targeting CD3 and the model tumor antigen HER2, respectively, connected by rigid DNA linkers of variable length. Using cytotoxicity assays and hybrid biophysical platforms, we demonstrate a linker-length dependence of cell spreading on antigen, target killing and cytokine release, revealing a functional decoupling between killing and cytokine secretion, and implicating the glycocalyx as a key player. Through the addition of EGFR targeting, we also generate trispecific constructs implementing an "OR-gate" logic to address tumor heterogeneity and reduce resistance due to antigen loss. Overall, these versatile constructs show great therapeutic promise, and at the same time serve as platforms to test hypotheses on biophysical mechanisms.

synthetic biology↗

bioRxiv2026

An Unprecedented Cluster in a Methanotroph Acetol Dehydrogenase

Biocatalytic metal-containing clusters are nanometer-sized chemical reactors that enable enzymes to perform chemistry far beyond what would be possible with amino acids alone 1,2. These clusters display a remarkable variability3 and elucidating their exact mechanisms is often difficult to determine, requiring input from a multitude of techniques ranging from spectroscopic to structural and theoretical methods. Thus, for unknown clusters, it is essential to accumulate, collate, and interpret as much information as possible from every relevant technique available. Here we report the discovery and in-depth, interdisciplinary characterization of an entirely novel, [Cu-4Fe-4S] cluster in the protein acetol dehydrogenase (AceDH). AceDH, isolated directly from Methylacidiphilum fumariolicum SolV cells, catalyzed the oxidation of acetol to methylglyoxal, proving its role in the 2-propanol/acetone metabolism of methanotrophs4-6. Structural, spectroscopic and electrochemical analyses reveal the [Cu-4Fe-4S] cluster has a unique three-dimensional- and electronic structure involving electronic coupling between the copper and one of the iron atoms, likely contributing to its high, +275 mV, redox potential. The binding site for the novel cluster is composed of two protein subunits and involves a novel motif. These findings expand the known repertoire of biological metal cofactors and provide insight into how heterometallic clusters are adapted for biological processes.

biochemistry↗

bioRxiv2026

Chronic short sleep in early life accelerates cognitive decline via disrupted proteostasis

Chronic short sleep (CSS) is an emerging public health issue that frequently begins in adolescence and is common among healthcare professionals and others engaged in shift work. Epidemiological studies associate CSS and sleep disruption with metabolic disorders, cardiovascular disease, cognitive decline, and heightened Alzheimers disease risk. Building on our prior findings that sleep deprivation perturbs proteostasis and activates endoplasmic reticulum (ER) stress pathways, we investigated the long-term consequences of CSS in young adult wild-type mice over the course of one year. Mice exposed to CSS displayed impaired cognition in hippocampal dependent tasks by 28 weeks of age, indicating emerging memory deficits. At the molecular level, CSS disrupted hippocampal proteostasis--particularly protein folding processes--and triggered ER stress and activation of the unfolded protein response (UPR). Importantly, disrupted proteostasis preceded the behavioral decline, with diminution of the key chaperone and UPR regulator BiP occurring at 20-22 weeks of age. CSS also increased markers of cellular stress and neuroinflammation while reducing the expression of proteins associated with memory function. Age also seemed to be a cellular stressor, causing a longitudinal increase in UPR, ISR, and neuroinflammation markers. Together, these results indicate that both chronic short sleep and age compromise proteostasis and promote neuroinflammation, contributing to progressive cognitive dysfunction.

neuroscience↗

bioRxiv2025

Defects in lamin A-Prohibitin crosstalk leads to ROS elevation and OxPhos imbalance in laminocardiomyopathy

Lamins are critical for maintaining nuclear homeostasis, chromosome positioning, and cellular mechanotransduction, which involves the transfer of mechanical signals from the cellular microenvironment to the nucleus. Recent studies have also highlighted the involvement of lamin A in mitochondrial homeostasis and the regulation of reactive oxygen species production. Missense mutations in lamin A are linked to a spectrum of diseases known as laminopathies, which include conditions such as dilated cardiomyopathy (DCM), muscular dystrophy, and progeria. One such mutation, K97E, is associated with DCM, causing severe cardiac complications that can lead to myocardial infarction in extreme cases. Our study reveals a detailed pathogenic cascade in K97E-transfected cells involving disrupted interaction with Prohibitin-2, a key mitochondrial protein. Mitochondria exhibit increased fission, reduced fusion, and fragmentation, due to OPA1 downregulation and DRP1 recruitment driven by actin cytoskeletal remodelling. Impaired Rho-ERK- FAK signalling reduces F-actin assembly, elevating G-actin, which further promotes mitochondrial fission. This feedback loop leads to mitochondrial depolarisation, ATP deficiency, and global metabolic catastrophe, in particular cholesterol metabolism, accompanied by elevated ROS. In cardiomyocytes, such dysfunction may underlie contractile deficits and arrhythmias. Our findings establish PHB2 as a critical node linking nuclear integrity, cytoskeletal architecture, and mitochondrial homeostasis, offering new insights into DCM pathogenesis and therapeutic targets. Our findings elucidate the pivotal role of lamin A in cellular energetics and mechanotransduction, offering novel insights into DCM pathophysiology, which in turn opens avenues for developing targeted therapeutic strategies. TeaserLamin A K97E mutation alters cellular metabolome through disturbed mitochondrial and actin homeostasis in a feedback loop with PHB2 at its hub and causes gross pathogenesis of DCM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/665267v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@176d89aorg.highwire.dtl.DTLVardef@1868703org.highwire.dtl.DTLVardef@109dff8org.highwire.dtl.DTLVardef@14f469a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5C_FLOATNO Gross mitochondrial defects arising from PHB2 and actin perturbations leading to severe metabolic and bioenergetic effects during K97E mutation of lamin A C_FIG

cell biology↗

bioRxiv2025

Substrate viscoelasticity regulates fibroblast adhesion and migration

Mechanical properties of the extracellular matrix (ECM) modulate cell-substrate interactions and influence cellular behaviors such as contractility, migrations, and proliferation. Although the effects of substrate stiffness on mechanobiology have been well studied, the role of ECM viscoelasticity in fibrotic progression remains less understood. To examine how viscoelasticity affects the biophysical properties and regulates the signaling of human mammary fibroblasts, we engineered elastic (E) and viscoelastic (VE) polyacrylamide hydrogels with comparable storage moduli ([~]14.52 {+/-} 1.03 kPa) but distinctly different loss moduli. Fibroblasts cultured on E hydrogels spread extensively (2428.93 {+/-} 864.71 m{superscript 2}), developed prominent stress fibers with higher zyxin intensity, and generated higher traction stresses (2931.57 {+/-} 1732.61 Pa). In contrast, fibroblasts on VE substrates formed smaller focal adhesion areas (54.2% reduction), exhibited lower critical adhesion strengths (51.8%), and generated 21% lower traction stresses (p < 0.001), indicating weaker adhesions. These substrates also promoted migrations and showed enhanced proliferation accompanied by reduced YAP activity, suggesting a mechanotransduction shift that may involve alternative signaling pathways. In contrast, E substrates showed YAP nuclear translocation, consistent with greater cytoskeletal tension and contractility. These findings highlight the importance of energy dissipation mechanisms in regulating fibroblast function on substrates mimicking the fibrotic milieu. Our results demonstrate that tuning the ECM viscoelasticity is a useful strategy to regulate cell behaviors in tissue engineered scaffolds, and develop better disease modeling for regenerative medicine.

bioengineering↗

bioRxiv2024

Unveiling epigenetic regulatory elements associated with breast cancer development

Breast cancer is the most common cancer in women and the 2nd most common cancer worldwide, yearly impacting over 2 million females and causing 650 thousand deaths. It has been widely studied, but its epigenetic variation is not entirely unveiled. We aimed to identify epigenetic mechanisms impacting the expression of breast cancer related genes to detect new potential biomarkers and therapeutic targets. We considered The Cancer Genome Atlas database with over 800 samples and several omics datasets such as mRNA, miRNA, DNA methylation, which we used to select 2701 features that were statistically significant to differ between cancer and control samples using the Monte Carlo Feature Selection and Interdependency Discovery algorithm, from an initial total of 417,486. Their biological impact on cancerogenesis was confirmed using: statistical analysis, natural language processing, linear and machine learning models as well as: transcription factors identification, drugs and 3D chromatin structure analyses. Classification of cancer vs control samples on the selected features returned high classification weighted Accuracy from 0.91 to 0.98 depending on feature-type: mRNA, miRNA, DNA methylation, and classification algorithm. In general, cancer samples showed lower expression of differentially expressed genes and increased {beta}-values of differentially methylated sites. We identified mRNAs whose expression is well explained by miRNA expression and differentially methylated sites {beta}-values. We recognized differentially methylated sites possibly affecting NRF1 and MXI1 transcription factors binding, causing a disturbance in NKAPL and PITX1 expression, respectively. Our 3D models showed more loosely packed chromatin in cancer. This study successfully points out numerous possible regulatory dependencies.

cancer biology↗

bioRxiv2024

Cardiomyopathic mutations of lamin A perturb mutual interactions of lamin, nuclear membrane, and chromatin leading to LLPS

Lamins are nuclear intermediate filaments constituting the nuclear lamina which maintains the structural integrity of the nucleus and play a key role in the spatial organisation of the genome. Mutations in the lamin protein have been associated with diverse diseases collectively known as laminopathies. In this study, we focused on two lamin A mutants - E161K and K97E - associated with dilated cardiomyopathy (DCM). Through confocal imaging, we established that these mutations cause large scale disruption of the peripheral lamin and consequent heterochromatin organisation, along with the formation of lamin aggregates inside the nucleoplasm. Using coarse-grained polymer simulations, we uncovered the role of lamin-lamin, lamin-membrane and lamin-chromatin interactions in maintaining wild-type lamin and chromatin organisation and showed that disruptions in these interactions can reproduce the experimental observations in the lamin mutants. These predictions were verified using 3D-FISH experiments to quantify the reorganisation of chromosome territories in these mutants. Using advanced imaging methods, we characterised the dynamical properties of the lamin aggregates in the mutants to show for the first time a liquid-like state of the lamin aggregates through a liquid-liquid phase separation. The altered lamin and chromatin interactions in the mutants thus manifest as liquid-like aggregates in the nucleoplasm leading to disruption of the spatial organisation of the genome in these laminopathy-associated mutants.

biophysics↗

bioRxiv2024

Celldetective: an AI-enhanced image analysis toolfor unraveling dynamic cell interactions

Analysis of multimodal and multidimensional data capturing dynamic interactions between diverse cell populations is a current challenge in bioimaging, especially in the context of immunology and immunotherapy research. Here, we introduce Celldetective, an open-source Python-based software tool designed for high-performance, end-to-end analysis of image-based in vitro immune and immunotherapy assays. Celldetective is purpose-built for multicondition, 2D multi-channel time-lapse microscopy of mixed cell populations. Although it is optimised for the needs of immunology assays, it is nevertheless broadly applicable to any biological system involving interacting cell populations. The software seamlessly integrates AI-based segmentation, tracking, and automated single-cell event detection, all within an intuitive graphical interface that supports interactive visualisation, annotation, and training options. We showcase its capabilities with original datasets of single immune effector cell interactions with an activating surface mediated by bispecific antibodies, and pairwise interactions in antibody-dependent cell cytotoxicity events.

bioinformatics↗

bioRxiv2023

A "through-DNA" mechanism for metal uptake-vs.-efflux regulation

Transition metals like Zn are essential for all organisms including bacteria, but fluctuations of their concentrations in the cell can be lethal. Organisms have thus evolved complex mechanisms for cellular metal homeostasis. One mechanistic paradigm involves pairs of transcription regulators sensing intracellular metal concentrations to regulate metal uptake and efflux. Here we report that Zur and ZntR, a prototypical pair of regulators for Zn uptake and efflux in E. coli, respectively, can coordinate their regulation through DNA, besides sensing cellular Zn2+ concentrations. Using a combination of live-cell single-molecule tracking and in vitro single-molecule FRET measurements, we show that unmetallated ZntR can enhance the unbinding kinetics of Zur from DNA by directly acting on Zur-DNA complexes, possibly through forming heteromeric ternary and quaternary complexes that involve both protein-DNA and protein-protein interactions. This through-DNA mechanism may functionally facilitate the switching in Zn uptake regulation when bacteria encounter changing Zn environments; it could also be relevant for regulating the uptake-vs.-efflux of various metals across different bacterial species and yeast.

biophysics↗

bioRxiv2023

Chromatin network rearrangements in differentiation and cancer reveal connections between evolutionary processes and gene regulation

Cancer cells are highly plastic, allowing them to adapt to changing conditions. Genes related to basic cellular processes evolved in ancient species, while more specialized genes appeared later with multicellularity (metazoan genes) or even after mammals evolved. Transcriptomic analyses have shown that ancient genes are up-regulated in cancer, while metazoan-origin genes are inactivated. Despite the importance of these observations, the underlying mechanisms remain unexplored. Here, we study local and global epigenomic mechanisms that may regulate genes from specific evolutionary periods. Using evolutionary gene age data, we characterize the epigenomic landscape, gene expression regulation, and chromatin organization in three cell types: human embryonic stem cells, normal B-cells, and primary cells from Chronic Lymphocytic Leukemia, a B-cell malignancy. We identify topological changes in chromatin organization during differentiation observing patterns in Polycomb repression and RNA Polymerase II pausing, which are reversed during oncogenesis. Beyond the non-random organization of genes and chromatin features in the 3D epigenome, we suggest that these patterns lead to preferential interactions among ancient, intermediate, and recent genes, mediated by RNA Polymerase II, Polycomb, and the lamina, respectively. Our findings shed light on gene regulation according to evolutionary age and suggest this organization changes across differentiation and oncogenesis.

cancer biology↗

bioRxiv2023

Nucleo-cytoplasmic environment modulates spatio-temporal p53 phase separation

Phase separation of various transcription factors and nucleic acids into biomolecular condensates is known to play an essential role in the regulation of gene expression. Here, we show that p53, a tumor suppressor and transcription factor, phase separates and forms biomolecular condensates in the nucleus of cancer cells as well as when overexpressed in the various cell lines. Although the nuclear condensates of wild-type (WT) p53 maintain their liquid state and are able to bind DNA, cancer-associated mutations not only promote misfolding but also partially rigidify the p53 condensates, which are unable to bind the DNA. Irrespective of WT or mutant form, the cytoplasmic partitioning of p53 with time also results in biomolecular condensate formation, which eventually undergoes rigidification. In vitro, WT p53 core domain (p53C) forms biomolecular condensates, which rigidify with time and the process is further promoted by cancer-associated mutations. Both RNA and non-specific DNA promote LLPS of p53C, but specific DNA promotes the dissolution of p53C condensates. The result suggests that the cellular microenvironment regulates p53 LLPS, material property and its functions.

biophysics↗

bioRxiv2022

Talin and kindlin cooperate to control the density of integrin clusters.

Focal adhesions are important mechanosensitive structures, composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Cellular adhesion to the extracellular matrix depends on the activation of integrins by intracellular mechanisms. Talin and kindlin are major activators of integrins that are recruited to the inner membrane and bind to {beta}-integrin cytoplasmic tails. Many studies showed the importance of integrin activation and clustering and how the organization of extracellular ligands guides the nanoscale organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we performed experiments using a biomimetic in vitro system, made of Giant Unilamellar Vesicles, containing transmembrane integrins, on which purified talin, kindlin, and actomyosin assemble. Here we first show that talin and kindlin individually have the ability to cluster integrins. When added together, talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density in the talin-integrin, kindlin-integrin, and talin-kindlin-integrin clusters reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin significantly enhances the segregation of talin-integrin clusters induced by actomyosin contractility, suggesting that it increases the coupling of these clusters to the actin cytoskeleton. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion.

biochemistry↗

bioRxiv2022

Probing mechanical interaction of immune receptors and cytoskeleton by membrane nanotube extraction

The role of force application in immune cell recognition is now well established, the force being transmitted between the actin cytoskeleton to the anchoring ligands through receptors such as integrins. In this chain, the mechanics of the cytoskeleton to receptor link, though clearly crucial, remains poorly understood. To probe this link, we combine mechanical extraction of membrane tubes from T cells using optical tweezers, and fitting of the resulting force curves with a viscoelastic model taking into account the cell and relevant molecules. We solicit this link using four different antibodies against various membrane bound receptors: antiCD3 to target the T Cell Receptor (TCR) complex, antiCD45 for the long sugar CD45, and two clones of antiCD11 targeting open or closed conformation of LFA1 integrins. Upon disruption of the cytoskeleton, the stiffness of the link changes for two of the receptors, exposing the existence of a receptor to cytoskeleton link - namely TCR-complex and open LFA1, and does not change for the other two where no such a link was expected. Our integrated approach allows us to probe, for the first time, the mechanics of the intracellular receptor-cytoskeleton link in immune cells.

biophysics↗

bioRxiv2022

Intrinsic linking of chromatin fiber in human cells

MotivationWe propose a practical algorithm based on graph theory, with the purpose of identifying CTCF-mediated chromatin loops that are linked in 3D space. Our method is based finding clique minors in graphs constructed from pairwise chromatin interaction data obtained from the ChIA-PET experiments. We show that such a graph structure, representing a particular arrangement of loops, mathematically necessitates linking, if co-occurring in an individual cell. The presence of these linked structures can advance our understanding of the principles of spatial organization of the genome. ResultsWe apply our method to graphs created from in situ ChIA-PET data for GM12878, H1ESC, HFFC6 and WTC11 cell lines, and from long-read ChIA-PET data. We look at these datasets as divided into CCDs - closely interconnected regions defined based on CTCF loops. We find numerous candidate regions with minors, indicating the presence of links. The graph-theoretic characteristics of these linked regions, including betweenness and closeness centrality, differ from regions without, in which no minors were found, which supports their non-random nature. We also look at the position of the linked regions with respect to chromatin compartments. AvailabilityThe implementation of the algorithm is available at https://github.com/SFGLab/cKNOTs ContactDariusz.Plewczynski@pw.edu.pl

genomics↗

bioRxiv2022

Multi-scale phase separation by explosive percolation with single chromatin loop resolution

The 2m-long human DNA is tightly intertwined into the cell nucleus of the size of 10m. The DNA packing is explained by folding of chromatin fiber. This folding leads to the formation of such hierarchical structures as: chromosomal territories, compartments; densely packed genomic regions known as Chromatin Contact Domains (CCDs), and loops. We propose models of dynamical genome folding into hierarchical components in human lymphoblastoid, stem cell, and fibroblast cell lines. Our models are based on explosive percolation theory. The chromosomes are modeled as graphs where CTCF chromatin loops are represented as edges. The folding trajectory is simulated by gradually introducing loops to the graph following various edge addition strategies that are based on topological network properties, chromatin loop frequencies, compartmentalization, or epigenomic features. Finally, we propose the genome folding model - a biophysical pseudo-time process guided by a single scalar order parameter. The parameter is calculated by Linear Discriminant Analysis. We simulate the loop formation by using Loop Extrusion Model (LEM) while adding them to the system. The chromatin phase separation, where fiber folds into topological domains and compartments, is observed when the critical number of contacts is reached. We also observe that 80% of the loops are needed for chromatin fiber to condense in 3D space, and this is constant through various cell lines. Overall, our in-silico model integrates the high-throughput 3D genome interaction experimental data with the novel theoretical concept of phase separation, which allows us to model event-based time dynamics of chromatin loop formation and folding trajectories.

genomics↗

bioRxiv2022

Cellular forces during early spreading of T lymphocytes on ultra-soft substrates

Mechanical forces are increasingly recognized as critical regulators of T cell activation, yet their earliest dynamics remain poorly resolved. Here, we use traction force microscopy on ultra-soft, antigen-presenting-cell-like polyacrylamide substrates to quantify the first 15 minutes of force generation by Jurkat and primary human CD4 T cells under controlled activating conditions. By combining time-resolved stress mapping with spatial tensor analysis, we uncover previously unrecognized heterogeneity in early T cell mechanosensing. Rather than producing a single stereotyped mechanical response, T cells exhibit three distinct temporal force regimes: low-amplitude active fluctuations, intermittent force bursts, and sustained sigmoidal buildups of stress. These temporal programs tightly couple to spatial organization: fluctuating and intermittent behaviors associate with disordered stress distributions, whereas sustained sigmoidal responses predominantly accompany polarized, dipolar, and unexpectedly extensile stress patterns. Substrate stiffness strongly reshapes this distribution, with stiffer gels suppressing sustained high-energy responses and biasing cells toward fragmented mechanical engagement. Primary T cell subsets likewise display distinct force phenotypes: naive cells exhibit weak, fluctuating behaviors, whereas memory cells more readily enter sustained high-force states. Together, these findings support a two-stage model of early T cell mechanosensing in which filopodia-mediated probing generates low, intermittent forces that, upon sustained engagement, transition to a lamellipodia-driven spreading phase producing larger, persistent, and predominantly outward-directed stresses. This framework provides a mechanistic explanation for how substrate mechanics, receptor context, and immune cell state shape force generation during the earliest stages of activation. More broadly, our results identify force as a dynamic and structured component of antigen recognition rather than a passive consequence of T cell signaling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/480084v4_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@29e24dorg.highwire.dtl.DTLVardef@1c8399forg.highwire.dtl.DTLVardef@3f29edorg.highwire.dtl.DTLVardef@d9a581_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗