Biology subjects

Tans, S.

Publications and source records attributed to Tans, S..

11 recordsLinked to original sources

bioRxiv2026

Direct observation of ATP-driven ubiquitin chain handling by Cdc48

Cdc48 (p97 or VCP in metazoans) targets polyubiquitin to selectively disassemble and degrade proteins. Cdc48 is believed to move along the ubiquitin chain towards linked proteins, but this has not been directly observed. By following single molecules in time, we find that the polypeptide branch points of ubiquitin chains are repeatedly inserted and rejected from the Cdc48 pore in ATP-driven manner, in bursts lasting up to seconds. This non-processive mode either ends by terminal substrate rejection, or advances to processive action, which drives ubiquitin unfolding in two steps, branch point translocation, and the extrusion of ubiquitin and linked protein as polypeptide loops in trans. Final retrograde movement can bring polypeptide segments back to cis. Our results establish the dynamics of ubiquitin chain handling by Cdc48, reveal key hallmarks of kinetic proofreading. We speculate that Cdc48 translocation may play a role in ubiquitin chain selection.

biophysics↗

bioRxiv2025

BEST4/CA7+ and goblet cells are interdependent regulators of intestinal mucus homeostasis

The intestinal mucus layer is essential for the integrity of the intestinal barrier. It is produced by goblet cells, whose depletion is common in colonic inflammation but remains poorly understood. Here, we show that goblet cell survival relies on a reciprocal dependence with newly discovered BEST4/CA7+ cells. We developed a method to follow BEST4/CA7+ and goblet cells in time from birth to death in human colon organoids. Notably, goblet cells induce BEST4/CA7+ fates in sister cells and other neighbors, using DLL1-mediated lateral activation of Notch-signaling. BEST4/CA7+ cells in turn promote goblet survival, with the latter depleting rapidly after differentiation in absence of BEST4/CA7+ cells. This apoptosis inhibition does not require direct cell-cell contact and instead depends on their shared lumen. Such differentiation and survival interdependencies may be relevant beyond the maintenance of mucosal homeostasis.

cell biology↗

bioRxiv2025

Translation-driven temporal control for intertwined protein assembly

Protein complexes are essential to cells. However, how structurally intertwined protein subunits can assemble faithfully is poorly understood. Here, we reveal a "temporal control" mechanism driven by coupled ribosomes to form intertwined dimers. Using Disome Selective Profiling and optical tweezers, we show that the BTB domains of KEAP1, KLHL12, and PATZ1 form stable closed states as monomers, thus impeding proposed domain-swapping assembly routes. By contrast, the timed emergence of nascent chain segments during translation enables alternative folding-assembly pathways that bypass the closed monomeric state. Analysis indicates that this mechanism works in concert with dimerization quality control by the E3 ligase SCF-FBXL17, and is relevant across the BTB domain family. This study shows that ribosome cooperation expands the range of possible protein architectures.

molecular biology↗

bioRxiv2025

Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.

biophysics↗

bioRxiv2024

Cell tracking with accurate error prediction

Cell tracking is an indispensable tool for studying development by time-lapse imaging. However, existing cell trackers cannot assign confidence to predicted tracks, which prohibits fully automated analysis without manual curation. We present a fundamental advance: an algorithm that combines neural networks with statistical physics to determine cell tracks with error probabilities for each step in the track. From these we can obtain error probabilities for any tracking feature, from cell cycles to lineage trees, that function like p-values in data interpretation. Our method greatly speeds up tracking analysis by limiting manual curation to rare low-confidence tracking steps. Importantly, it also enables fully-automated analysis by retaining only high-confidence track segments, which we demonstrate by analyzing cell cycles and differentiation events at scale, for thousands of cells in multiple intestinal organoids. Our approach brings cell dynamics-based organoid screening within reach, and enables transparent reporting of cell tracking results and associated scientific claims.

bioinformatics↗

bioRxiv2023

Adaptation to a phenotypic trade-off requires an interplay between local and global regulators

Organisms depend on their ability to balance multiple phenotypes by evolutionary adaptation. For instance, cellular growth and movement jointly enable critical processes including microbial colonization and cancer invasiveness. Growth and movement are known to be controlled by local regulators that target single operons, as well as by global regulators that impact hundreds of genes. However, how these different levels of regulation interplay during evolution is unclear. Using Escherichia coli growth and motility as a model system, we show that global regulators enable the adaptation of two phenotypes bound by a trade-off, where improvement in one causes deterioration in the other. We measured how CRISPR-mediated knockdowns of global and local transcription factors impact growth and motility in different environments. We find that local regulators mostly modulate motility, while global regulators jointly modulate growth and motility. Genetic perturbations display complex high order interactions between genes and environments. Nevertheless, gene perturbations display consistent patterns in the growth-motility space when grouped by their position in the regulatory hierarchy. These patterns constrain evolutionary scenarios, where local regulators are typically mutated first to optimize motility, then global regulators allow cells to adjust the trade-off between growth and motility. These findings overall highlight the role of pleiotropic regulators for coordinating phenotypic responses in complex environments.

systems biology↗

bioRxiv2023

Co-translational ribosome pairing enables native assembly of misfolding-prone subunits

Protein complexes are pivotal to most cellular processes. Emerging evidence indicates that pairs of ribosomes ubiquitously drive the synchronized synthesis and assembly of two protein subunits into homodimeric complexes1-5. These observations suggest protein folding mechanisms of general importance enabled by contacts between nascent chains6,7 - which have thus far rather been considered detrimental8,9. However, owing to their dynamic and heterogeneous nature, the folding of interacting nascent chains remains unexplored. Here, we show that co-translational ribosome pairing allows their nascent chains to chaperone each other, thus enabling the formation of coiled-coil homodimers from subunits that misfold individually. We developed an integrated single-molecule fluorescence and force spectroscopy approach to probe the folding and assembly of two nascent chains extending from nearby ribosomes, using the intermediate filament lamin as a model system. Ribosome proximity in early translation stages was found to be critical: when interactions between nascent chains are inhibited or delayed, they become trapped in stable misfolded states that are no longer assembly competent. Conversely, early interactions allow the two nascent chains to nucleate native-like quaternary structures that grow in size and stability as translation advances. We conjecture that protein folding mechanisms enabled by ribosome cooperation are more broadly relevant to intermediate filaments and other protein classes.

molecular biology↗

bioRxiv2022

Following cell type transitions in space and time by combining live-cell tracking and endpoint cell identity in intestinal organoids

Elucidating the dynamics of cellular differentiation in space and time is key to advancing organoid biology and technology. Direct visualization of differentiation patterns is challenging, however, in part because of the difficulty of simultaneously detecting all relevant cell types by fluorescence imaging. Here we present TypeTracker, which determines the differentiation trajectories of all cells within a region of interest, including their type transitions, growth, divisions, and changes in spatial organization. We show how the lineage tree topology, as determined by automated cell tracking, allows for the retrospective identification of cell types across multiple generations. The data revealed various surprising aspects of intestinal organoid differentiation, including symmetric fate adoption by sister cells, cell type abundance regulated by division, and type commitment of cells occurring prior to their spatial reorganization. Our method can be applied broadly to study other organoid systems and to screen for compounds that affect differentiation programs.

biophysics↗

bioRxiv2020

ppGpp is a bacterial cell size regulator

Growth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the "adder" principle, by unknown mechanisms [1-4]. Growth is well known to be regulated by ppGpp, which controls anything from ribosome production to metabolic enzyme activity and replication initiation, and whose absence or excess can induce the stress response, filamentation, and yield growth-arrested miniature cells [5-8]. These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth. Here, to untangle effects of ppGpp and nutrients, we gained control of cellular ppGpp by inducing the synthesis and hydrolysis enzymes RelA and Mesh1. We found that ppGpp not only exerts control over the growth rate, but also over cell division and hence the steady state cell size. The added size responds rapidly to changes in the ppGpp level, aided by transiently accelerated or delayed divisions, and establishes its new constant value while the growth rate still adjusts. Moreover, the magnitude of the added size and resulting steady-state birth size correlate consistently with the ppGpp level, rather than with the growth rate, which results in cells of different size that grow equally fast. Our findings suggest that ppGpp serves as a critical regulator that coordinates cell size and growth control.

microbiology↗

bioRxiv2020

Early neurulation recapitulated in assemblies of embryonic and extraembryonic cells

Stem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo biology and engineering living systems with novel functions. To unlock that potential, we need new ways to elicit higher-level organization. Here we show that adding extraembryonic endoderm (XEN) cells to mouse gastruloids leads to the formation of neural epithelia. By single-cell RNA-seq, imaging and differentiation experiments, we demonstrate the neural characteristics and spatial patterning of the epithelial tissue. We further show that the XEN cells differentiate reciprocally to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish "XEN Enhanced Gastruloids" (XEGs) to explore heterotypic cellular interactions as a means to achieve complex, tissue-level organization in vitro.

developmental biology↗

bioRxiv2020

Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

CRISPR/Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences in human organoids awaits robust knock-in approaches. Here, we describe CRISPR/Cas9-mediated Homology-independent Organoid Transgenesis (CRISPR-HOT), which allows efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences at desired loci, without the necessity to inactivate TP53 in untransformed cells, previously used to increase HDR-mediated knock-in. CRISPR-HOT was employed to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter labelling the mitotic spindle by tagged tubulin and the cell membrane by tagged E-cadherin uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed TP53 involvement in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

cell biology↗