Biology subjects

Tans, S. J.

Publications and source records attributed to Tans, S. J..

17 recordsLinked to original sources

bioRxiv2026

Low CAR affinity enhances T-cell synapse dynamics and activation

Low affinity chimeric antigen receptors (CARs) can clinically outperform high-affinity variants, yet the molecular mechanisms underlying affinity-dependent activity remain unclear. Here, we show that low CAR affinity enables faster CAR cluster motion, which is central to T-cell activation. We compared low-affinity CAT and high-affinity FMC63 CARs signaling at different stages of activation on supported lipid bilayers functionalized with defined CD19 densities. At low CAR expression on dense CD19 bilayers, low-affinity CAT CARs formed more mature and dynamic synapses, recruited ZAP70 more efficiently, and showed faster microcluster motion. These proximal signaling changes were accompanied by stronger calcium flux and increased NFAT nuclear translocation. Our data support a model in which weaker, lower-affinity CAR-antigen interactions promote receptor turnover, microcluster mobility, and signaling-adaptor recruitment, thereby enhancing downstream signal propagation. Our work provides a mechanistic basis for the superior functional performance reported for affinity-tuned CD19 CAR-T cells.

biophysics↗

bioRxiv2026

NuclearIDTracker resolves intestinal cell identity and lineage dynamics through nuclear phenotypic signatures

Organoid models have transformed our understanding of intestinal renewal. Fluorescent imaging has been extensively used to identify key cell types and their differentiation pathways, but immunofluorescence provides only static readouts, whereas live imaging requires fluorescent-reporter engineering and is constrained by limited multiplexing and spectral overlap. Here, we introduce NuclearIDTracker, an explainable machine-learning framework that infers cell identity directly from 3D nuclear segmentations. Using a single nuclear marker, NuclearIDTracker accurately classifies intestinal cell types and integrates with single-cell tracking to resolve lineages and reconstruct dynamic state transitions during organoid development. We show that TA-like cells, rather than stem cells, drive early crypt formation and generate enterocyte and Paneth lineages, as well as the stem-cell population, which emerges only later and subsequently replenishes the TA-like compartment. Following stem-cell ablation, crypt regeneration was not driven by a single discrete cell type. Instead, multiple epithelial populations converged on a proliferative regenerative state with a nuclear phenotypic signature that resembled, but remained distinct from, that of homeostatic TA-like cells, and a YAP/TAZ-associated fetal-like transcriptional signature. Thus, nuclear phenotypic signatures resolve cell identity and reveal coordinated epithelial plasticity during crypt regeneration. NuclearIDTracker establishes a non-perturbative tool to quantify cell identity and state dynamics at single-cell resolution, revealing previously inaccessible biological dynamics and expanding the toolkit for studying epithelial homeostasis, regeneration, and disease.

cell biology↗

bioRxiv2026

Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

Immune cell receptor - ligand interactions are key to cancer immunotherapy. However, receptor-ligand affinities often fail to predict T-cell mediated cancer killing, while immune-target cell binding strength measurements are limited by low precision and high non-specific binding. Here we present bilayer acoustic force spectroscopy (BAFS), a method to quantify the binding strength of receptors in immune synapses that virtually eliminates non-specific binding and increases the resolving power by up to 50-fold. By replacing target cells with a supported lipid bilayer functionalized with antigens, BAFS avoids antigen-independent interactions and target cell heterogeneity, while maintaining the spatial self-organization of receptors that typifies active immune synapses. We demonstrate the high sensitivity and control by showing how CAR T-cell synapse strength depends on CD19 antigen density, and by revealing that CD8 synergistically strengthens {beta}TCR-pMHC synapses independently of Lck recruitment to CD8. BAFS is a general method that can be used broadly in immunotherapy screening and to dissect the complex molecular interactions that underpin immune synapse activation.

biophysics↗

bioRxiv2025

NAC promotes co-translational folding at the ribosomal tunnel exit

The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While NACs function as a dynamic hub for other factors is well-established, its direct role in co-translational folding is unclear. By proteome-wide profiling NAC co-translational interactions in human cells, we found that NAC recognizes emerging segments enriched in hydrophobicity and -helical propensity, within folded domains of cytonuclear proteins. Single-molecule and structural analyses reveal that NAC, via its {beta}-barrel domain, dynamically interacts with nascent chains at the ribosomal tunnel exit and is capable of promoting on-pathway folding. Compartment-specific nascent chain interactions of NAC further elucidate its role in targeting to the endoplasmic reticulum and mitochondrial membrane protein biogenesis. Together, these findings show that NAC acts as a bona fide co-translational chaperone that facilitates early protein folding at the ribosomal tunnel exit, expanding its functional repertoire in protein biogenesis.

biochemistry↗

bioRxiv2024

Mature tuft cell phenotypes are sequentially expressed along the intestinal crypt-villus axis following cytokine-induced tuft cell hyperplasia

Intestinal tuft cells are epithelial sentinels that trigger host defense upon detection of parasite-derived compounds. While representing interesting targets for immunomodulatory therapies in inflammation-driven intestinal diseases, their detailed functioning is poorly understood. Although two distinct intestinal tuft cell types have been described, we reveal common intermediary transcriptomes among tuft cells in mouse and human. Tuft cell-specific reporter knock-ins in organoids show that the two tuft types are sequentially expressed transcriptomic states that represent different maturation stages. Moreover, cytokines interleukin-4 and interleukin-13 only induce lineage specification to Nrep+ tuft-1 cells, while BMP and cholinergic signalling advance differentiation towards immune-related ChAT+ tuft-2 phenotypes. Functionally, both tuft cell states have chemosensory capacity and respond to stimuli like succinate, but reaction probability increases during tuft cell maturation. Our tuft type-specific reporters and optimized differentiation strategy in organoids provide an experimental platform to study the functioning of tuft cells and their unique chemosensory properties.

cell biology↗

bioRxiv2023

Structural basis of membrane targeting and coatomer assembly by human GBP1

Guanylate-Binding Proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) that mediate immune effector functions against intracellular pathogens. A key step for the antimicrobial activity of GBPs is the formation of homo- and heterooligomeric complexes on the membrane of pathogen-associated compartments or cytosolinvasive bacteria. Similar to other large GTPases of the dynamin family, oligomerisation and membrane association of GBPs depend on their GTPase activity. How nucleotide binding and hydrolysis prime GBPs for membrane targeting and coatomer formation remains unclear. Here, we report the cryo-EM structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and resolve the molecular ultrastructure of GBP1 coatomer assemblies on liposomes and bacterial lipopolysaccharide membranes. We show how nucleotide binding promotes large-scale conformational changes of the middle and GTPase effector domains that expose the isoprenylated carboxyl-terminus for association with lipid membranes. Our structure reveals how the -helical stalks of the middle domain form a parallel arrangement firmly held in a unique cross-over arrangement by intermolecular contacts between adjacent monomers. This conformation is critical for GBP1 dimers to assemble into densely packed coatomers on target membranes. The extended -helix of the effector domain is flexible and permits intercalation into the dense lipopolysaccharide layer on the outer membrane of gram-negative bacterial pathogens. We show that nucleotide-dependent oligomerisation and GTP hydrolysis yield GBP1 membrane scaffolds with contractile abilities that promote the formation of tubular membrane protrusions and membrane fragmentation. Collectively, our data provide a structural and mechanistic framework for interrogating the molecular basis for GBP1 effector functions in intracellular immunity.

biophysics↗

bioRxiv2022

The interplay between metabolic stochasticity and regulation in single E. coli cells

Metabolism is inherently stochastic at the cellular level. Whether cells actively regulate processes in response to these random internal variations is a fundamental problem that remains unaddressed, yet critical to understanding biological homeostasis. Here, we show that in E. coli cells, expression of the main catabolic enzymes is continuously adjusted in response to metabolic fluctuations under constant external conditions. This noise feedback is performed by the cAMP-CRP system, which controls transcription of the catabolic enzymes by modulating concentrations of the second messenger cAMP upon changes in metabolite abundance. Using time-lapse microscopy, genetic constructs that selectively disable cAMP-CRP noise feedback, and mathematical modelling, we show how fluctuations circulate through this hybrid metabolic-genetic network at sub cell-cycle timescales. This circulation of stochastic fluctuations is explained by four distinct noise propagation modes, one of which describes the continuous cAMP-CRP regulation. The model successfully predicts how noise circulation is impacted by C-sector under and over-expression. The results raise the question whether the cAMP-CRP system, as well as other metabolic regulation mechanisms, have evolved to manage internal metabolic fluctuations in addition to external growth conditions. We conjecture that second messengers may broadly function to control metabolic stochasticity and achieve cellular homeostasis.

systems biology↗

bioRxiv2022

Mother cells control daughter cell proliferation in intestinal organoids to minimize proliferation fluctuations

During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias towards proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry, but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations, that may be broadly relevant for other tissues.

cell biology↗

bioRxiv2022

Minimizing cell number fluctuations in self-renewing tissues with a stem cell niche

Self-renewing tissues require that a constant number of proliferating cells is maintained over time. This maintenance can be ensured at the single-cell level or the population level. Maintenance at the population level leads to fluctuations in the number of proliferating cells over time. Often, it is assumed that those fluctuations can be reduced by increasing the number of asymmetric divisions, i.e. divisions where only one of the daughter cells remains proliferative. Here, we study a model of cell proliferation that incorporates a stem cell niche of fixed size, and explicitly model the cells inside and outside the niche. We find that in this model fluctuations are minimized when the difference in growth rate between the niche and the rest of the tissue is maximized and all divisions are symmetric divisions, producing either two proliferating or two non-proliferating daughters. We show that this optimal state leaves visible signatures in clone size distributions and could thus be detected experimentally.

cell biology↗

bioRxiv2021

Direct observation of Hsp90-induced compaction in a protein chain

The chaperone Hsp90 is well known to undergo important conformational changes, which depend on nucleotide, co-chaperones, substrate interactions and post-translational modifications. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally, yet central to its function. Here, using optical tweezers and luciferase and glucocorticoid receptor substrates, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable kinases and receptor clients, regulates their activity, and how Hsp90 cooperates with Hsp70.

biophysics↗

bioRxiv2021

Single Cell Variability of CRISPR-Cas Interference and Adaptation

CRISPR-Cas defence is a combination of adaptation to new invaders by spacer acquisition, and interference by targeted nuclease activity. While these processes have been studied on a population level, the individual cellular variability has remained unknown. Here, using a microfluidic device combined with time-lapse microscopy, we monitor invader clearance in a population of Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we show large cell-to-cell variability of clearance times, which originates from primed CRISPR adaptation. Faster growth and cell division, as well as higher levels of Cascade, increase the chance of clearance by interference. In contrast, faster growth is associated with decreased chances of clearance by priming. A mathematical model explains the experimental findings, and identifies Cascade binding to the mutated invader DNA, rather than spacer integration, as the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond to invading threats in a timely manner. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

microbiology↗

bioRxiv2020

Weak catch bonds make strong networks

Molecular catch bonds are ubiquitous in biology and well-studied in the context of leukocyte extravasion1, cellular mechanosensing2,3, and urinary tract infection4. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this remarkable ability enables cells to increase their adhesion in fast fluid flows1,4, and hence provides strength-on-demand. Recently, cytoskeletal crosslinkers have been discovered that also display catch bonding5-8. It has been suggested that they strengthen cells, following the strength-on-demand paradigm9,10. However, catch bonds tend to be weaker compared to regular (slip) bonds because they have cryptic binding sites that are often inactive11-13. Therefore, the role of catch bonding in the cytoskeleton remains unclear. Here we reconstitute cytoskeletal actin networks to show that catch bonds render them both stronger and more deformable than slip bonds, even though the bonds themselves are weaker. We develop a model to show that weak binding allows the catch bonds to mitigate crack initiation by moving from low- to high-tension areas in response to mechanical loading. By contrast, slip bonds remain trapped in stress-free areas. We therefore propose that the mechanism of catch bonding is typified by dissociation-on-demand rather than strength-on-demand. Dissociation-on-demand can explain how both cytolinkers5-8,10,14,15 and adhesins1,2,4,12,16-20 exploit continuous redistribution to combine mechanical strength with the adaptability required for movement and proliferation21. Our findings provide a new perspective on diseases where catch bonding is compromised11,12 such as kidney focal segmental glomerulosclerosis22,23, caused by the -actinin-4 mutant studied here. Moreover, catch bonds provide a route towards creating life-like materials that combine strength with deformability24.

biophysics↗

bioRxiv2020

The ribosome modulates folding inside the ribosomal exit tunnel

Proteins commonly fold cotranslationally on the ribosome, while the nascent chain emerges from the ribosomal tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is still not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not reproduce the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Polypeptide collapse modulation and folding stimulation by GroEL-ES

Unfolded proteins ubiquitously collapse into a compact yet dynamic state1,2. While this compaction is pivotal to protein folding3, aggregation4,5, intrinsic disorder6, and phase separation7, its role in protein quality control mechanisms remains obscure8. Collapse has been characterized mainly for polypeptides that are free in solution, in terms of kinetics, chain expansion, and effect on folding9,10. Yet, theory suggests that the solvent-mediated forces driving collapse can be altered near hydrophobic and charged surfaces, which are observed for many proteins including GroEL-ES11,12. Notably, while GroEL-ES is the archetypal protein-folding chaperone, its folding mechanism remains unresolved13,14. GroEL-ES is proposed to sterically confine polypeptides within its closed chamber15, unfold misfolded states16,17, or promote folding indirectly by suppressing aggregation18,19. Here, using integrated protein manipulation and imaging, we show that GroEL-ES can strengthen the collapse of polypeptide substrates, and hence stimulate folding directly. Strikingly, attractive forces pull substrate chains into the open GroEL cavity -unclosed by GroES-, and hence trigger a gradual compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL, and is aided by GroES binding to the cavity rim, and by the amphiphilic C-terminal tails at the cavity bottom. Peptides corresponding to these C-termini alone are sufficient to strengthen the collapse. The results show a mechanism that allows folding to be stimulated: by strengthening the collapse, residues are brought together that must contact to fold. The notion that one protein can modulate the collapse of another may be generally important in protein conformation and coacervation control, for systems ranging from the GroEL-ES homologue TRiC/CCT20, to the oncogenic c-Myc/Max complex21, and the nuclear pore transporter transportin22.

biophysics↗

bioRxiv2020

OrganoidTracker: efficient cell tracking using machine learning and manual error correction

Time-lapse microscopy is routinely used to follow cells within organoids, allowing direct study of division and differentiation patterns. There is an increasing interest in cell tracking in organoids, which makes it possible to study their growth and homeostasis at the single-cell level. As tracking these cells by hand is prohibitively time consuming, automation using a computer program is required. Unfortunately, organoids have a high cell density and fast cell movement, which makes automated cell tracking difficult. In this work, a semi-automated cell tracker has been developed. To detect the nuclei, we use a machine learning approach based on a convolutional neural network. To form cell trajectories, we link detections at different time points together using a min-cost flow solver. The tracker raises warnings for situations with likely errors. Rapid changes in nucleus volume and position are reported for manual review, as well as cases where nuclei divide, appear and disappear. When the warning system is adjusted such that virtually error-free lineage trees can be obtained, still less than 2% of all detected nuclei positions are marked for manual analysis. This provides an enormous speed boost over manual cell tracking, while still providing tracking data of the same quality as manual tracking.

bioinformatics↗

bioRxiv2019

Evolutionary constraints in regulatory networks defined by partial order between phenotypes

Gene regulation networks allow organisms to adapt to diverse environmental niches. However, the constraints underlying the evolution of regulatory phenotypes remain ill-defined both theoretically and experimentally. Here, we show that the concept of partial order identifies such constraints, and test the predictions by experimentally evolving an engineered signal-integrating network in multiple environments. We find that populations: 1) expand in fitness space along the Pareto-optimal front predicted by conflicts in regulatory demands, by fine-tuning binding affinities within the network, 2) expand beyond this constraint by changes in the network structure, thus allowing access to new fitness domains. Strikingly, the constraint predictions are based on whether the network output increases or decreases in response to the different signals, and do not require information on the network architecture or underlying genetics. Overall, our findings show that limited knowledge on current regulatory phenotypes can provide predictions on future evolutionary constraints.

evolutionary biology↗

bioRxiv2018

Long-term expanding human airway organoids for disease modelling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar biopsies or lavage material. The pseudostratified airway organoid epithelium consists of basal cells, functional multi-ciliated cells, mucus-producing goblet cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoid culture conditions also allow gene editing as well as the derivation of various types of lung cancer organoids. Respiratory syncytial virus (RSV) infection recapitulated central disease features and dramatically increases organoid cell motility, found to be driven by the non-structural viral NS2 protein. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

cell biology↗