Biology subjects

Martin-Lemaitre, C.

Publications and source records attributed to Martin-Lemaitre, C..

7 recordsLinked to original sources

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

Assembly of tight junction belts by surface condensation and actin elongation

Formation of biomolecular condensates via phase separation enables compartmentation of many cellular processes. However, how cells can control condensation at specific locations to create complex cellular structures remains poorly understood. Here, we investigated the mechanism of tight junction formation, which involves condensation of scaffold proteins at cell-cell contacts and elongation of the condensates into a belt around the cellular perimeter. Using cell biology, reconstitution, and thermodynamic theory, we discovered that cells use surface phase transitions to control local condensation at the membrane far below bulk saturation. Surface condensation of junctional ZO-scaffold proteins is mediated by receptor binding and regulated by the receptors oligomerization state. Functionally, ZO surface condensation is directly coupled to actin polymerization and bundling, which drives elongation of receptor-ZO-actin condensates similar to tight junction belt formation in cells. We conclude that surface phase transitions provide a robust mechanism to control the position and shape of protein condensates. One-Sentence SummaryLocal surface binding of cytosolic scaffold proteins provides spatial control of protein condensation to assemble adhesion junctions.

biophysics↗

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

Wetting of junctional condensates along the apical interface promotes tight junction belt formation

Biomolecular condensates enable cell compartmentalization by acting as membrane-less organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. Here, we studied formation of tight junctions, which initially assemble as condensates that over time elongate around the membrane cell perimeter to form a closed junctional barrier2. We discovered that the elongation of junctional condensates is driven by a physical wetting process around the apical membrane interface. Using temporal proximity proteomics in combination with live and super-resolution imaging, we found that wetting is mediated by the apical protein PATJ, which promotes adhesion of condensates to the apical membrane resulting in an interface formation and linear spreading into a closed belt. Using PATJ mutations we show that apical adhesion of junctional condensates is necessary and sufficient for stable tight junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates and membrane interfaces to shape mesoscale structures.

cell biology↗

bioRxiv2020

Optimization of mouse embryonic stem cell culture for organoid and chimeric mice production.

In vitro stem cell culture is demanding in terms of manpower and media supplements. In recent years, new protocols have been developed to expand pluripotent embryonic stem cells in suspension culture, which greatly simplifies cell handling and scalability. However, it is still unclear how suspension culture protocols with different supplements affect pluripotency, cell homogeneity and cell differentiation compared to established adherent culture methods. Here we tested four different culture conditions for mouse embryonic stem cells (mESC) and quantified chimerism and germ line transmission as well as in vitro differentiation into three-dimensional neuro-epithelia. We found that suspension culture supplemented with CHIR99021/LIF offers the best compromise between culturing effort, robust pluripotency and cell homogeneity. Our work provides a guideline for simplifying mESC culture and should encourage more cell biology labs to use stem cell-based organoids as model systems.

cell biology↗

bioRxiv2019

Phase separation of zonula occludens proteins drives formation of tight junctions

Tight junctions are cell adhesion complexes that seal tissues and are involved in cell polarity and signalling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands is dependent on the two membrane associated scaffolding proteins ZO1 and ZO2. To understand how ZO proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight junction proteins including adhesion receptors, cytoskeletal adapters and transcription factors. Our results suggest that an active phase transition of ZO proteins into a condensed membrane bound compartment drives claudin polymerization and coalescence of a continuous tight junction belt.

cell biology↗