Biology subjects

Kruse, K.

Publications and source records attributed to Kruse, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Tissue-scale mechanics controls differentiation strategy and dynamics of epithelial multilayering

Generating and maintaining multilayered epithelia requires coordinated cell division, differentiation, and tissue architecture, yet the precise mechanisms of multilayering remain unclear. Using the developing mouse epidermis, we show that basal stem cells adopt distinct multilayering strategies depending on tissue mechanics. Combining quantitative morphometry, embryo live imaging and physical modeling, we observe that early in development, the epidermis is fluid-like, allowing undifferentiated cells to move suprabasally through perpendicular divisions or basal detachment before differentiating. As the tissue matures and rigidifies, a mechanical barrier is established that only allows upward movement of basal cells that have committed to differentiation. The final step of this commitment is delamination that requires Notch signaling, triggered by increased tissue stiffness and jamming. This mechanical regulation orchestrates a feedback loop that induces cell upward motion precisely when the basal layer becomes crowded. Together, our findings identify tissue mechanics as the key determinant of how tissues drive multilayering and reveal mechanically regulated Notch signaling as a driver of epidermal delamination.

cell biology↗

bioRxiv2026

Modular gene tagging in C. elegans

Tagging a gene endogenously can identify when the gene is expressed and where the protein is localized. CRISPR is the primary tool for generating tags of endogenous genes, but it is error-prone and requires unique reagents for each gene and tag. Recombinases can insert DNA in an error-free and modular manner. Here, we tested eight recombinases for germline function in the nematode C. elegans, and introduce PhIT, a recombinase-based method for protein tagging. First, a short 39bp PhiC31 attB landing pad is inserted into the locus by CRISPR. This strain is a resource which can be used to insert a variety of modular tags. Second, tags are inserted by the integrase PhiC31, and in tandem, extraneous backbone sequences are removed by a tyrosine recombinase. Current modular tags include seven different fluorescent proteins, FLP-regulated cell-specific expression constructs, and degron tags. Importantly, tags can be inserted by genetic crosses instead of by microinjection.

genomics↗

bioRxiv2025

Colorectal cancer heterogeneity co-evolves with tumor architecture to determine disease outcome

Intratumoral heterogeneity, originating from genetic, epigenetic, and phenotypic cellular diversity, is pervasive in cancer. As these heterogeneous states employ diverse mechanisms to promote tumor progression, metastasis, and therapy resistance, emergence of cancer heterogeneity is one of the most significant barriers to curative treatment. Here we leverage deep learning approaches to develop a high-throughput image-analysis paradigm with subcellular resolution that quantifies and predicts colorectal cancer (CRC) patient outcome based on tissue architecture and nuclear morphology. We further combine this approach with spatial transcriptomics, multiplex immunohistochemistry, and patient-derived organoids to uncover a dynamic, co-evolutionary relationship between tumor architecture and cell states. We identify clinically relevant architectural interfaces in CRC tissue that diversify cellular identities by favoring distinct cancer stem cell states and thus promote evolution of tumor heterogeneity. Specifically, tissue fragmentation and associated compressive forces promote loss of classic stem cell signature and acquisition of fetal/regenerative stem cell states, which initially emerges as a hybrid state with features of epithelial-to-mesenchyme (EMT) transition. Additional tumor stroma communication then diversifies these states into distinct stem cell and EMT states at the invasive margins of tumors. Reciprocally, Wnt signaling state of tumor cells tunes their responsiveness to tissue architecture. Collectively, this work uncovers a feedback loop between cell states and tissue architecture that drives cancer heterogeneity and cell state diversification. Machine learning-based analyses harness this co-dependency, independently of mutational status or cancer stage, to predict patient outcome.

cancer biology↗

bioRxiv2025

Force patterning drives cell flows and 3D spatial order in auditory epithelia

During development, coordinated cell behavior drives the morphogenesis of epithelia into intricate structures essential for their physiological functions. How this coordination is achieved in epithelia composed of multiple cell types remains unclear. We study development of the avian auditory epithelium comprising sensory hair cells (HCs) and non-sensory supporting cells (SCs). Initially, HCs and SCs are arranged into mosaics by Notch-Delta signaling. During development, HCs partially extrude from the epithelium, establish a ten-fold gradient in apical surface area across the tissue, and intercalate with SCs to form near-hexagonal order. Using a combination of experiments and a 3D-vertex model, we show that an increase of contractility at the apical junctions between SCs compared to those between HCs and SCs drives spatial order within the epithelial plane and along the apical-basal axis. A faster increase of HC apical area at one end of the epithelium than the other leads to opposite fluxes of HCs and SCs and establishes the observed gradients in apical surface area and density of HCs along the auditory epithelium while maintaining a uniform degree of hexagonal order throughout. Our findings reveal that patterned junctional contractility can spatially coordinate cell behavior across both the plane and depth of a mixed epithelium, providing a general mechanism for generating complex three-dimensional tissue architectures during organogenesis.

developmental biology↗

bioRxiv2025

Interplay between Rac1/RhoA and actin waves in giant epithelial cells : experiment and theory

The acto-myosin cytoskeleton is a key driver of cellular shape changes in vivo and in vitro. Acto-myosin organization results from actin assembly and interactions between actin and myosin, which are both regulated by small Rho GTPases like Rac1 and RhoA. To uncover principles governing cytoskeletal organization, we analyzed actin patterns using live microscopy and theory. In giant Madin-Darby Canine Kidney (MDCK) epithelial cells and REF52 fibroblasts, we observed acto-myosin stress fibres and propagating waves. Stress fibres were stationary and correlated with homogeneous distributions of Rac and RhoA activity. Waves propagated at {approx} 1m/min and were associated with density variations of actin, Rac and active RhoA. Some waves transported cellular components or generated protrusions at the cell edge. Essential features of wave propagation are captured by a polar reaction-diffusion system for actin and Rac. Notably, two colliding waves annihilate each other. In cells, myosin activity was not required for the emergence of waves, but tended to suppress them. Consistently, local activation of RhoA slowed down or stopped and broke waves. These results highlight how the coupling between acto-myosin and Rho GTPase generates a variety of cytoskeletal structures and dynamics. O_TEXTBOXSIGNIFICANCEThe cytoskeleton shapes cells in vitro and in vivo. Its molecular mechanisms have been extensively documented, but the mesoscopic structures resulting from the interplay between the activator and the motor activity have been poorly characterized. Here we show that two structures are conserved in two systems, stress fibres with RhoA activity and diffusion actin waves regulated by Rac1. The annihilation of waves and the alteration of the wave dynamics are reproduced by a minimal model based on a polar reaction-diffusion model. This approach could pave the way to a generic description of regulated active gels in cells with potential biological functions, such as stress generation for fibres and probing of space and homogenization of the cortex by actin waves. C_TEXTBOX

biophysics↗

bioRxiv2025

Phase Separation and a Hydrodynamic Instability Localize Proteins at Growing Microtubule Ends

Regulating microtubule dynamics is essential for cellular function, and precise localization of regulatory proteins at microtubule ends is critical. End-binding protein EB3, a key regulator of microtubule growth, accumulates over an extended region at the growing end, forming a comet that gradually fragments into transient droplets along the microtubule shaft. Here, we combine in vitro reconstitution experiments with theoretical analysis to show that surface-mediated condensation of EB3 effectively localizes the protein at microtubule ends. Our results reveal that a Rayleigh-Plateau instability limits the condensates extent, producing a finite comet length and discrete droplets along the shaft. Remarkably, the comet size is independent of the GTP-cap size. This finding is supported by experiments on cells showing that modulation of microtubule growth velocity and hence GTP-cap size do not consistently alter EB3 comet length. Furthermore, our theory shows that rapid droplet evaporation requires a transition of EB3 to a non-phase-separating state. Overall, our work challenges the view that comet length directly reflects GTP-cap size and highlights a novel mechanism for regulating microtubule dynamics.

biophysics↗

bioRxiv2024

Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells

Acquisition of specific cell shapes and morphologies is a central component of cell fate transitions. Although signaling circuits and gene regulatory networks that regulate pluripotent stem cell differentiation have been intensely studied, how these networks are integrated in space and time with morphological transitions and mechanical deformations to control state transitions remains a fundamental open question. Here, we focus on two distinct models of pluripotency, primed pluripotent stem cells and pre-implantation inner cell mass cells of human embryos to discover that cell fate transitions associate with rapid changes in nuclear shape and volume which collectively alter the nuclear mechanophenotype. Mechanistic studies in human induced pluripotent stem cells further reveal that these phenotypical changes and the associated active fluctuations of the nuclear envelope arise from growth factor signaling-controlled changes in chromatin mechanics and cytoskeletal confinement. These collective mechano-osmotic changes trigger global transcriptional repression and a condensation-prone environment that primes chromatin for a cell fate transition by attenuating repression of differentiation genes. However, while this mechano-osmotic chromatin priming has the potential to accelerate fate transitions and differentiation, sustained biochemical signals are required for robust induction of specific lineages. Our findings uncover a critical mechanochemical feedback mechanism that integrates nuclear mechanics, shape and volume with biochemical signaling and chromatin state to control cell fate transition dynamics.

cell biology↗

bioRxiv2024

Topology changes of the regenerating Hydra define actin nematic defects as mechanical organizers of morphogenesis.

AbstractHydra is named after the mythological animal for its regenerative capabilities, but contrary to its mythological counterpart, it only regenerates one head when cut. Here we show that soft compression of head regenerating tissues induces the regeneration of viable, two headed animals. Topological defects in the supracellular nematic organization of actin were previously correlated with the new head regeneration site1. Soft compression creates new topological defects associated with additional heads. To test the necessity of topological defects in head regeneration, we changed the topology of the tissue. By compressing the head regenerating tissues along their body axis, topological defects of the foot and of the regenerating head fused together, forming a toroid with no defects. Perfectly ordered toroids did not regenerate over eight days and eventually disintegrated. Spheroids made from excised body column tissue partially lose their actin order during regeneration. Compression of spheroids generated toroids with actin defects. These tissues regenerated into toroidal animals with functional head and foot, and a bifurcated body. Our results show that topological defects in the actin order are necessary to shape the head of the regenerating Hydra, supporting the notion that actin topological defects are mechanical organizers of morphogenesis.

developmental biology↗

bioRxiv2024

Sphingolipid metabolism orchestrates the establishment of the adult hair follicle stem cell niche to control skin homeostasis

Bioactive sphingolipids serve as an essential building block of membranes, forming a selective barrier that ensures subcellular compartmentalization and facilitates cell type-specific intercellular communication through regulation of the plasma membrane receptor repertoire. How cell type-specific lipid compositions are achieved and what is their functional significance in tissue morphogenesis and maintenance has remained unclear. Here, we identify a stem-cell specific role for ceramide synthase 4 (CerS4) in orchestrating fate decisions in skin epidermis. Deletion of CerS4 in the epidermis prevents the effective development of the adult hair follicle bulge stem cell (HFSCs) niche due to altered differentiation trajectories of HFSC precursors towards upper hair follicle and inner bulge fates. Mechanistically, HFSC differentiation defects arise from an imbalance of key ceramides and their derivate sphingolipids in HFSCs associated with hyperactivity of canonical Wnt signaling. Impaired HFSC niche establishment leads to disruption of hair follicle architecture and hair follicle barrier function, ultimately triggering a T helper cell 2 - dominated immune infiltration closely resembling human atopic dermatitis. This work uncovers a fundamental role for a cell state-specific sphingolipid profile in epidermal stem cell homeostasis and the role of an intact stem cell niche in maintaining an intact skin barrier.

developmental biology↗

bioRxiv2022

Single cell transcriptomics reveals functionally specialized vascular endothelium in brain

The blood-brain barrier (BBB) limits the entry of leukocytes and potentially harmful substances from the circulation into the central nervous system (CNS). While BBB defects are a hallmark of many neurological disorders, the cellular heterogeneity at the neurovascular interface and the mechanisms governing neuroinflammation are not fully understood. Through single cell RNA sequencing of non-neuronal cell populations of the murine cerebral cortex during development, adulthood, ageing and neuroinflammation, we identify reactive endothelial venules (REVs), a compartment of specialised post-capillary endothelial cells (ECs) that are characterized by consistent expression of cell adhesion molecules, preferential leukocyte transmigration, association with perivascular macrophage populations, and endothelial activation initiating CNS immune responses. Our results provide novel insights into the heterogeneity of the cerebral vasculature and a useful resource for the molecular alterations associated with neuroinflammation and ageing.

physiology↗

bioRxiv2022

The effect of motor-induced shaft dynamics on microtubule stability and length

Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depends on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in presence of kinesin-1 molecular motors. Although the average microtubule dynamics can be captured by an effective rescue rate, the dynamics of individual microtubules differs dramatically when rescue occurs only at exchange sites. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network. SIGNIFICANCEThe microtubule network is essential for vital cellular processes like the organization of intracellular transport and division. Although microtubule assembly occurs at its tips, it has recently been reported that tubulin is exchanged along the microtubule shaft. Tubulin exchange plays an essential role in regulating microtubule dynamics and can be induced by molecular motors. Here, we provide the first systematic study of the impact of shaft dynamics on the regulation of rescue events, where a microtubule switches from shrinking to growing. Our results illustrate how the usage of microtubules as tracks for intracellular transport regulates the microtubule network and thus offers a novel perspective on intracellular organization.

biophysics↗

bioRxiv2021

Motor usage imprints microtubule stability in the shaft

Tubulin dimers assemble into dynamic microtubules which are used by molecular motors as tracks for intracellular transport. Organization and dynamics of the microtubule network is commonly thought to be regulated at the polymer ends, where tubulin-dimers can be added or removed. Here we show that molecular motors running on microtubules cause exchange of dimers along the shaft. These sites of dimer exchange act as rescue sites where depolymerising microtubules stop shrinking and start re-growing. Consequently, the average length of microtubules increases depending on how frequently they are used as motor tracks. An increase of motor activity densifies the cellular microtubule network and enhances cell polarity. Running motors leave marks in the shaft serving as traces of microtubule usage to organize the polarity landscape of the cell.

cell biology↗

bioRxiv2020

Pinching the cortex of live cells reveals thickness instabilities caused by Myosin II motors

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration and division. Because the cortical thickness is below optical resolution, it has been generally considered as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension due to actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape changing capacity of highly contractile cells that use amoeboid-like migration.

biophysics↗

bioRxiv2020

Integer topological defects organize stresses driving tissue morphogenesis

Tissues acquire their function and shape via differentiation and morphogenesis. Both processes are driven by coordinating cellular forces and shapes at the tissue scale, but general principles governing this interplay remain to be discovered. Here, we report that self-organization of myoblasts around integer topological defects, namely spirals and asters, triggers localized differentiation and, when differentiation is inhibited, drives the growth of cylindrical multicellular protrusions. Both localized differentiation and growth require specific stress patterns. By analyzing the experimental velocity and orientation profiles through active gel theory, we show that integer topological defects can concentrate compressive stresses, which we measure by using deformable pillars. Altogether, we envision topological defects as mechanical organizational centers that control differentiation and morphogenesis to establish tissue architecture.

biophysics↗

bioRxiv2020

FAN-C: A Feature-rich Framework for the Analysis and Visualisation of C data

Chromosome conformation capture data, particularly from high-throughput approaches such as Hi-C and its derivatives, are typically very complex to analyse. Existing analysis tools are often single-purpose, or limited in compatibility to a small number of data formats, frequently making Hi-C analyses tedious and time-consuming. Here, we present FAN-C, an easy-to-use command-line tool and powerful Python API with a broad feature set covering matrix generation, analysis, and visualisation for C-like data (https://github.com/vaquerizaslab/fanc). Due to its comprehensiveness and compatibility with the most prevalent Hi-C storage formats, FAN-C can be used in combination with a large number of existing analysis tools, thus greatly simplifying Hi-C matrix analysis.

genomics↗

bioRxiv2019

A new twist on bacterial motility - Two distinct type IV pili revealed by cryoEM

Many bacteria express flexible protein filaments on their surface that enable a variety of important cellular functions. Type IV pili are examples of such filaments and are comprised of a helical assembly of repeating pilin subunits. Type IV pili are involved in motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). They are therefore powerful structures that enable bacterial proliferation and genetic adaptation, potentially leading to the development of pathogenicity and antibiotic resistance. They are also targets for drug development.\n\nBy a complement of experimental approaches, we show that the bacterium Thermus thermophilus produces two different forms of type IV pilus. We have determined the structures of both and built atomic models. The structures answer key unresolved questions regarding the molecular architecture of type IV pili and identify a new type of pilin. We also delineate the roles of the two filaments in promoting twitching and natural transformation.

biophysics↗

bioRxiv2019

Cohesin disrupts polycomb-dependent chromosome interactions

How chromosome organisation is related to genome function remains poorly understood. Cohesin, loop-extrusion, and CTCF have been proposed to create structures called topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find as in other cell types that cohesin is required to create TADs and regulate A/B compartmentalisation. However, in the absence of cohesin we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system, depend on PRC1, and we discover that cohesin counteracts these interactions. This disruptive activity is independent of CTCF and TADs, and regulates gene repression by the polycomb system. Therefore, in contrast to the proposal that cohesin creates structure in chromosomes, we discover a new role for cohesin in disrupting polycomb-dependent chromosome interactions to regulate gene expression.

genetics↗

bioRxiv2019

Transposable elements drive reorganisation of 3D chromatin during early embryogenesis

Transposable elements are abundant genetic components of eukaryotic genomes with important regulatory features affecting transcription, splicing, and recombination, among others. Here we demonstrate that the Murine Endogenous Retroviral Element (MuERV-L/MERVL) family of transposable elements drives the 3D reorganisation of the genome in the early mouse embryo. By generating Hi-C data in 2-cell-like cells, we show that MERLV elements promote the formation of insulating domain boundaries through-out the genome in vivo and in vitro. The formation of these boundaries is coupled to the upregulation of directional transcription from MERVL, which results in the activation of a subset of the gene expression programme of the 2-cell stage embryo. Domain boundaries in the 2-cell stage embryo are transient and can be remodelled without undergoing cell division. Remarkably, we find extensive inter-strain MERVL variation, suggesting multiple non-overlapping rounds of recent genome invasion and a high regulatory plasticity of genome organisation. Our results demonstrate that MERVL drive chromatin organisation during early embryonic development shedding light into how nuclear organisation emerges during zygotic genome activation in mammals.

genomics↗