Biology subjects

Riveline, D.

Publications and source records attributed to Riveline, D..

At least 19 recordsLinked to original sources

bioRxiv2025

Interaction dynamics between epithelial cysts captured by tissue rheology

Epithelial cysts are minimal structures involved in morphogenesis. They are fluid-filled cavities surrounded by an epithelial monolayer. Cysts grow during development and their interactions shape organs. While their growth dynamics as single structures are well characterised, the physical mechanisms underlying their interaction remain poorly understood. Here we design a minimal assay of interacting cyst doublets based on microfabrication, quantitative biology, and theory to show that Madin-Darby Canine Kidney (MDCK) cyst interactions are essentially determined by the rheological properties of their epithelial monolayers. We report two phases of interaction between epithelial cysts: coalescence of cellular monolayers and lumen fusion, with similar speeds of 0.3 {micro}m/h. We modulate the distribution of interaction phenotypes by reducing cell-cell adhesion using E-cadherin knock-out MDCK cells and we report that E-cadherin depletion promotes lumen fusion. Remarkably, the dynamics of coalescence and fusion are conserved between both cell lines. To understand the conserved speeds and the effect of cell-cell adhesion, we model the mechanical behavior of cyst doublets as a complex fluid to predict a speed determined by viscosity, a stretch-dependent monolayer tension, and the adhesion energy between cells. We measure these parameters through rheological experiments using micropipette aspiration and lumen drainage, which span the full range of stretch. A key insight from this analysis is that accounting for the tensions dependence on stretch is is essential to capture the different dynamics observed during cyst interaction. Using these rheological measurements, we successfully recapitulate the conserved speed. Altogether, our results open new perspectives to understand tissue dynamics during organogenesis through simple physical arguments. Significance StatementCysts are fluid-filled cavities sur-rounded by a cellular monolayer. Interactions between cysts are important during morphogenesis, yet their governing rules are poorly understood. Here we designed a doublet assay using microfabrication to control cyst geometry and characterized their interaction dynamics. We report that speeds of coalescence and fusion between epithelial cysts are conserved even for cell lines depleted in E-cadherin, a central adhesion protein. We measured their rheological parameters by two independent methods, micropipette aspiration and laser cutting. Using a simple model and scaling arguments, we demonstrate that these parameters are sufficient to explain interaction speeds. This study sheds light on the interplay between tissue dynamics and rheology and its implication in orchestrating morphogenetic events in vitro and in vivo.

biophysics↗

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

Teach your microscope how to print: Low-cost and rapid-iteration microfabrication for biology

The application of traditional microfabrication techniques to biological research is hindered by their reliance on clean rooms, expensive or toxic materials, and slow iteration cycles. We present an accessible microfabrication workflow that addresses these challenges by integrating consumer 3D printing techniques and repurposing standard fluorescence microscopes equipped with DMDs for maskless photolithography. Our method achieves micrometer-scale precision across centimeter-sized areas without clean room infrastructure, using affordable and readily available consumables. We demonstrate the versatility of this approach through four biological applications: inducing cytoskeletal protrusions via 1 m-resolution surface topographies; micropatterning to standardize cell and tissue morphology; fabricating multilayer microfluidic devices for confined cell migration studies; imprinting agar chambers for long-time tracking of C. elegans. Our protocol drastically reduces material costs compared to conventional methods and enables design-to-device turnaround within a day. By leveraging open-source microscope control software and existing lab equipment, our workflow lowers the entry barrier to micro-fabrication, enabling labs to prototype custom solutions for diverse experimental needs while maintaining compatibility with soft lithography and downstream biological assays.

cell biology↗

bioRxiv2024

Myosin cluster dynamics determines epithelial wound ring constriction

Collection of myosin motors and actin filaments can self-assemble into submicrometric clusters under the regulation of RhoA. Emergent dynamics of these clusters have been reported in a variety of morphogenetic systems, ranging from Drosophila to acto-myosin assays in vitro. In single cell cytokinetic rings, acto-myosin clusters are associated with stress generation when radial and transport when tangential with respect to the ring closure. Here, we show that these phenomena hold true for acto-myosin multi-cellular rings during wound closure in epithelial monolayers. We assessed the activity of RhoA using FRET sensors, and we report that cluster dynamics does not correlate with RhoA activity. Nevertheless, we show that bursts of RhoA activation precede recruitment of myosin. Altogether myosin clusters dynamics is conserved between single and multi-cellular systems and this suggests that they could be used as generic read-outs for mapping and predicting stress generation and shape changes in morphogenesis.

biophysics↗

bioRxiv2024

Asymmetry of acto-myosin cortices as active fluids shape cells in organoids

Cell cortex is a thin sheet of actin cytoskeleton spanning cell boundaries with rich out-of-equilibrium dynamics. A theoretical description of the cortex as an active fluid enables to capture cell shapes dynamics in 3D epithelial tissues. However models integrated with calibration of parameters and quantitative experiments are lacking so far. Here we report that cells in organoids and in cysts have conserved apico-basal-lateral asymmetric compositions in actin and in myosin, and we quantify their densities and mechanical properties. This allows to calibrate a new model coupling active fluids with a phase field which reproduces the main features of cell shapes. To test our approach, we successfully predict changes in cell shapes by modulating actin polymerisation, myosin activity, and adhesion in experiments and in simulations. Our study shows how active fluid theory integrated with experiments can determine cell shapes in epithelial tissues.

biophysics↗

bioRxiv2024

Control of lumen geometry and topology by cell proliferation rate and pressure

Many internal organs in multicellular organisms comprise epithelia which enclose fluid-filled cavities. These are referred to as lumens and their formation is regulated by a wide range of processes, including epithelial polarization, secretion, exocytosis and actomyosin contractility [1, 2]. While these mechanisms have shed light on lumen growth, what controls lumen morphology remains enigmatic. Here we use pancreas organoids to explore how lumens acquire either a spherical shape or a branched topology [3]. Combining computational simulations based on a phase field model with experimental measurements we reveal that lumen morphology arises from the balance between the cell cycle duration and lumen pressure, with more complex lumen at low pressure and fast proliferation rates. Moreover, the manipulation of proliferation and lumen pressure in silico and in vitro is sufficient to alter and reverse the morphological trajectories of the lumens. Increasing epithelial permeability of spherical lumens lead to lower lumen pressure and converts their morphology to complex lumen shapes, highlighting its crucial role. In summary, the study underscores the importance of balancing cell proliferation, lumen pressure, and epithelial permeability in determining lumen morphology, providing insights relevant to other organs, for tissue engineering and cystic disease understanding and treatment [4].

developmental biology↗

bioRxiv2024

Predicting Organoid Morphology Through a Phase Field Model: Insights into Cell Division and Lumenal Pressure

Organoids are ideal systems to predict the phenotypes of organs. However, there is currently a lack of understanding regarding the generalized rules that enable use of simple cellular principles to make morphological predictions of entire organoids. Therefore, we employed a phase field model with the following basic components: the minimum conditions for the timing and volume of cell division, lumen nucleation rules, and lumenal pressure. Through our model, we could compute and generate a myriad of organoid phenotypes observed till date. We propose morphological indices necessary to characterize the shapes and construct phase diagrams and show their dependencies on proliferation time and lumen pressure. Additionally, we introduced the lumen-index parameter, which helped in examining the criteria to maintain organoids as spherical structures comprising a single layer of cells and enclosing an intact lumen. Finally, we predict a star-like organoid phenotype that did not undergo differentiation, suggesting that the volume constraint during cell division may determine the final phenotype. In summary, our approach provides researchers with guidelines to test the mechanisms of self-organization and predict the shape of organoid. Author summaryIn nature, a wide variety of organ morphologies are observed. Owing to the complexity of the process underlying the acquisition of organs morphology, it is challenging to investigate the mechanisms that lead to such variations. A promising approach to study these variations is the use of "computational organoid" study, which is the computational-based study of self-organizing shapes in multicellular assemblies and fluid-filled cavities called lumens that develop from a few proliferating cells. This study explores general mechanisms that dictate how various mechanical factors affect the growing self-organized multicellular assembly. We relied on computer simulations of the mathematical model called multicellular phase-field model with lumens and explored the mechanical factor effects, such as the lumen pressure while considering the time and volume conditions required for cell division. These simulations generated and categorized a wide range of organoid phenotypes based on the varying lumen pressure and cell division conditions. These phenotypes were characterized into seven distinct classes, based on the morphological index sets, including a cellular monolayer/multilayer surrounding single or multiple lumens and branch formation. These phenotypes were obtained without the assumption of differentiation. Our study elucidates the mechanisms underlying the organoid and organ formation with different shapes, thereby highlighting the significance of mechanical forces in shaping these complex biological structures.

cell biology↗

bioRxiv2024

Generic rules of lumen nucleation and fusion in epithelial organoids

Many internal organs in the body harbor a fluid-filled lumen. The mechanisms of lumens initiation and fusion have been reported as dependent on organ-type during organogenesis. In contrast, the physics of lumen suggests that force balance between luminal pressure and cell mechanics could lead to conserved rules which may unify their self-organisation. However, this hypothesis lacks experimental evidence. Here we compare lumen dynamics for three different systems (MDCK cysts, pancreatic spheres, and epiblast cysts) by using quantitative cell biology, microfabrication and theory. We report that initial cell number determines the maximum number of lumens but does not impact the steady state which is a final single lumen. In addition, lumen numbers exhibit two phases over time, a nucleation phase followed by a fusion phase. In the nucleation phase, lumens form between two cells in pancreatic and MDCK cysts whereas they form at the rosette stage between ten cells in epiblasts. In the second phase, lumens fuse by an increase in lumen volume for pancreatic spheres and MDCK cysts, whereas cell convergent directional motion leads to lumens fusion in epiblasts. We show that these phenomena are associated to the luminal hydrostatic pressure. We support these results with theoretical arguments and numerical simulations. We finally use MDCK cysts to manipulate cell adhesion and lumen volume and we successfully reproduce the fusion dynamics of pancreatic spheres and epiblasts. Our results reveal self-organisation rules of lumens across systems with relevance for morphogenesis during development and for the design of synthetic organs.

biophysics↗

bioRxiv2023

UBAP2L drives scaffold assembly of nuclear pore complexes at the intact nuclear envelope

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a direct role of ubiquitin-associated protein 2-like (UBAP2L) in the biogenesis of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and drives the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L facilitates the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact nuclear envelope. TeaserLiao et al. show how UBAP2L drives the assembly of the scaffold elements into symmetrical and functional NPCs at the nuclear envelope in human cells.

cell biology↗

bioRxiv2023

The interplay between lumen pressure and cell proliferation determines organoid morphology in a multicellular phase field model

systems biology↗

bioRxiv2023

Tight junctions regulate lumen morphology via hydrostatic pressure and junctional tension

Formation of fluid filled lumen by epithelial tissues is a fundamental process for organ development. How epithelial cells regulate the hydraulic and cortical forces to control lumen morphology is not completely understood. Here, we quantified the mechanical role of tight junctions in lumen formation using genetically modified MDCKII cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for hydraulic inflation of lumen. However, depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements and perturbations of hydrostatic lumen pressure and junctional tension with modelling, we were able to predict lumen morphologies from the pressure-tension force balance. We found that in MDCK tissue the tight junction promotes formation of spherical lumen by decreasing cortical tension via inhibition of myosin. In addition, we found that the apical surface area of cells is largely uncoupled from lumen volume changes, suggesting that excess apical area contributes to lumen opening in the low-pressure regime. Overall, our findings provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

cell biology↗

bioRxiv2022

Polarity-driven three-dimensional spontaneous rotation of a cell doublet

Cell mechanical interactions play a fundamental role in the self-organisation of organisms. How these interactions drive coordinated cell movement in three-dimensions remains unclear. Here we report that cell doublets embedded in a 3D extracellular matrix undergo spontaneous rotations and we investigate the rotation mechanism using live cell imaging, quantitative measurements, mechanical perturbations, and theory. We find that rotation is driven by a polarized distribution of myosin within cell cortices. The mismatched orientation of this polarized distribution breaks the doublet mirror symmetry. In addition, cells adhere at their interface through adherens junctions and with the extracellular matrix through focal contacts near myosin clusters. Using a physical theory describing the doublet as two interacting active surfaces, we find that rotation is driven by myosin-generated gradients of active tension, whose profiles are dictated by interacting cell polarity axes. We show that interface three-dimensional shapes can be understood from the Curie principle: shapes symmetries are related to broken symmetries of myosin distribution in cortices. To test for the rotation mechanism, we suppress myosin clusters using laser ablation and we generate new myosin clusters by optogenetics. Our work clarifies how polarity-oriented active mechanical forces drive collective cell motion in three dimensions.

biophysics↗

bioRxiv2022

Interplay between cell height variations and planar pulsations in epithelial monolayers

Biological tissues change their shapes through collective interactions of cells. This coordination sets length and time scales for dynamics where precision is essential, in particular during morphogenetic events. However, how these scales emerge remains unclear. Here we address this question using the pulsatile domains observed in confluent epithelial MDCK monolayers where cells exhibit synchronous contraction and extension cycles of {approx}5 hours duration and {approx}200 m length scale. We report that the monolayer thickness changes gradually in space and time by more than two folds in order to counterbalance the contraction and extension of the incompressible cytoplasm. We recapitulate these pulsatile dynamics using a continuum model and show that incorporation of cell stiffness dependent height variations is critical both for generating temporal pulsations and establishing the domain size. We propose that this feedback between height and mechanics could be important in coordinating the length scales of tissue dynamics.

biophysics↗

bioRxiv2021

Spontaneous rotations in epithelia as an interplay between cell polarity and RhoA activity at boundaries.

Directed flows of cells in vivo are essential in morphogenesis. They shape living matter in phenomena involving cell mechanics and regulations of the acto-myosin cytoskeleton. However the onset of coherent motion is still poorly understood. Here we show that coherence is associated with spontaneous alignments of cell polarity by designing cellular rings of controlled dimensions. A tug-of-war between polarities dictates the onset of coherence, as assessed by tracking live cellular shapes and motions in various experimental conditions. In addition, we identify an internally driven constraint set by cellular acto-myosin cables at boundaries as essential to ensure coherence, and active force is generated as evaluated by the high RhoA activity. The cables are required to trigger coherence as shown by our numerical simulations based on a novel Vicsek-type model including free active boundaries. We quantitatively reproduce in silico coherence onsets and we predict criteria leading to coherence. Altogether, spontaneous coherent motion results from basic competitions between cell orientations and active cables at boundaries.

biophysics↗

bioRxiv2020

Control and mechanisms of pulsatile flows in epithelial monolayers

Collective motions of epithelial cells in vivo are essential for morphogenesis in developmental biology. Tissues elongate, contract, flow, and oscillate, thus sculpting embryos. These tissue level dynamics are known, but the physical mechanisms at the cellular level are unclear, with various behaviors depending on the tissues and species. Moreover, investigations on in vitro tissue behavior usually focus on only one type of cell dynamics and use diverse theoretical approaches, making systematic comparisons between studies challenging. Here, we show that a single epithelial monolayer of Madin Darby Canine Kidney (MDCK) cells can exhibit two types of local tissue kinematics, pulsations and long range coherent flows. We analyzed these distinct motions by using quantitative live imaging. We also report that these motions can be controlled with internal and external cues such as specific inhibitors, and friction modulation of the substrate by microcontact printing method. We further demonstrate with a unified vertex model that both behaviors depend on the competition between velocity alignment and random diffusion of cell polarization. When alignment and diffusion are comparable, a pulsatile flow emerges, whereas the tissue undergoes long-range flows when velocity alignment dominates. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics are important in regulating the dynamics of tissue morphogenesis.

biophysics↗

bioRxiv2020

Temporal correlation between oscillating force dipoles drives single cell migration in 3D.

Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In some cells, symmetry breaking manifests itself in a directional flow of actin from the front to the rear of the cell. Many cells, especially in physiological 3D matrices do not show such coherent actin dynamics and present seemingly competing protrusion/retraction dynamics at their front and back. How symmetry breaking manifests itself for such cells is therefore elusive. We take inspiration from the scallop theorem proposed by Purcell for micro-swimmers in Newtonian fluids: self-propelled objects undergoing persistent motion at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. We report similar observations for cells crawling in 3D. We quantified cell motion using a combination of 3D live cell imaging, visualization of the matrix displacement and a minimal model with multipolar expansion. We show that our cells embedded in a 3D matrix form myosin-driven force dipoles at both sides of the nucleus, that locally and periodically pinch the matrix. The existence of a phase shift between the two dipoles is required for directed cell motion which manifests itself as cycles with finite area in the dipole-quadrupole diagram, a formal equivalence to the Purcell cycle. We confirm this mechanism by triggering local dipolar contractions with a laser. This leads to directed motion. Our study reveals that these cells control their motility by synchronizing dipolar forces distributed at front and back. This result opens new strategies to externally control cell motion as well as for the design of micro-crawlers.

biophysics↗

bioRxiv2020

Ratchetaxis in channels: cells move directionally by pushing walls asymmetrically.

Cell motility is essential in a variety of biological phenomena ranging from early development to organ homeostasis and diseases. This phenomenon was so far mainly studied and characterized on flat surfaces in vitro whereas this situation is rarely seen in vivo. Recently, cell motion in 3D microfabricated channels was reported to be possible, and it was shown that confined cells push on walls. However, rules setting cell directions in this context were not characterized yet. Here, we show by using assays that ratchetaxis operates in 3D ratchets on fibroblasts and on epithelial cancerous cells. Open ratchets rectify cell motion, whereas closed ratchets impose a direct cell migration along channels set by the cell orientation at the channel entry point. We also show that nuclei are pressed at constrictions zones through mechanisms involving dynamic asymmetries of focal contacts, stress fibers, and intermediate filaments. Interestingly, cells do not pass these constricting zones when defective in the keratin fusion implicated in squamous cancer. By combining ratchetaxis with chemical gradients, we finally report that cells are sensitive to local asymmetries in confinement and that topological and chemical cues may be encoded differently by cells. Altogether our ratchet channels could mimic small blood vessels where cells are confined: cells would probe local asymmetries which would determine their entry into tissues and direction. Our results could shed light on invasions mechanisms in cancer.

biophysics↗

bioRxiv2019

Elongations of epithelial colony in vitro: symmetry breaking through collective effects

Epithelial tissues of the developing embryos elongate by different mechanisms, such as neighbor exchange, cell elongation, and oriented cell division. Since autonomous tissue self-organization is influenced by external cues such as morphogen gradients or neighboring tissues, it is difficult to distinguish intrinsic from directed tissue behavior. The mesoscopic processes leading to the different mechanisms remain elusive. Here, we study the spontaneous elongation behavior of spreading circular epithelial colonies in vitro. By quantifying deformation kinematics at multiple scales, we report that global elongation happens primarily due to cell elongations, and its direction correlates with the anisotropy of the average cell elongation. By imposing an external time-periodic stretch, the axis of this global symmetry breaking can be modified and elongation occurs primarily due to orientated neighbor exchange. These different behaviors are confirmed using a vertex model for collective cell behavior, providing a framework for understanding autonomous tissue elongation and its origins.

biophysics↗