Assessing the Contribution of Tricellular Junctions to Epithelial Mechanics – ACTRICE

This project was based on an integrated approach combining complementary biological models, advanced imaging, targeted genetic manipulations, quantitative mechanical measurements and theoretical modelling to study the role of tricellular junctions in the organisation of epithelia.

Two model systems were used for comparison: Drosophila epithelia, which allowed for genetic and dynamic analysis in vivo, and vertebrate models comprising polarised epithelial cell lines and intestinal organoids derived from stem cells. During the project, the Xenopus leavis embryo ectoderm model was implemented within Partner 1's team. This comparative strategy made it possible to identify conserved mechanisms while taking into account the specificities of each system.

The assembly and dynamics of tricellular and bicellular junctions were analysed using high- and super-resolution real-time imaging, including Airyscan, RIM, SIM and expansion microscopy, combined with 3D segmentation and morphometric quantification tools. Non-invasive genetic approaches (mosaic analysis (Drosophila), CRISPR/Cas9 for fluorescent tag insertion at the locus, RNAi) were used to disrupt or precisely track junctional components.

The contribution of mechanical forces was studied using laser nano-ablation, traction force microscopy and optogenetics to measure and manipulate tensions locally at tricellular and bicellular junctions. These experimental data were integrated into theoretical models to link force production, cell geometry and tissue organisation.

This combination of multi-scale and interdisciplinary methods has enabled us to move from a detailed description of tricellular junctions to a mechanistic understanding of their role in cell division, intercellular communication and homeostasis in proliferative epithelia.

Epithelial tissues line our organs and form an essential barrier to protect the body. Their strength depends on specialised junctions that connect cells to each other. Our project aimed to understand how these junctions are organised, adapt to mechanical stresses and contribute to maintaining tissue integrity, under both normal and pathological conditions.

Using Drosophila as a model, we have shown that tricellular junctions, located at the point of contact between three cells, form independently of the more conventional junctions connecting two cells. We have identified key proteins necessary for their assembly and demonstrated that their alteration disrupts the epithelial barrier, highlighting their central role in tissue cohesion.

We have also highlighted the remarkable ability of epithelia to compensate for junctional defects. When certain junctions are weakened, cells strengthen their contacts and adapt their internal mechanics. This process is accompanied by a reorganisation of membrane protein trafficking, favouring recycling rather than degradation, in order to preserve tissue stability.

In Xenopus embryo, a vertebrate model, we have identified a conserved protein in tricellular junctions that is essential to their functioning. This protein also plays a key role in the cellular movements necessary for the formation of certain tissues, demonstrating that tricellular junctions are involved in both the architecture and dynamics of epithelia.

In a cancer model in Drosophila, we have shown that disruption of junctions promotes the collective extrusion of tumour cells from the epithelium. Our work reveals that tissue organisation and cell adhesion preferences determine the direction of this extrusion, a key phenomenon in tumour progression.

Finally, in mammals, we discovered that tricellular junctions are highly dynamic during cell division. They reorganise themselves to allow division while maintaining the mechanical cohesion of the tissue. A protein characteristic of these junctions also intervenes at the very end of division, ensuring the correct separation of daughter cells.

Overall, this project highlights the central role of tricellular junctions in the stability, adaptation and plasticity of epithelial tissues, with important implications for development, regeneration and diseases such as cancer.

By combining cutting-edge imaging, genetic approaches, mechanical analyses and modelling, this project has begun to provide an integrated mechanistic view of the role of tricellular junctions in the homeostasis of proliferative epithelia. The results obtained have made it possible to initiate a new conceptual framework linking cellular architecture, mechanical forces and cellular decisions, with major implications for understanding development, tissue regeneration and epithelial pathologies.

In epithelia, at tricellular contacts where three cells meet, bicellular junctions are disjointed and specialized structures called tricellular junctions (TCJs) are assembled. In addition to ensure epithelial barrier functions, TCJs just emerged as hot spots for integrating epithelial tension. In this context, we aim to understand how TCJ components are assembled and regulate actomyosin cytoskeleton to control forces at cell-cell contacts. We will compare symmetric and asymmetric cell division models within Drosophila and mammalian epithelia that have a distinct organization of junction domains and molecular components, using approaches that include advanced imaging, genetic, mechanical perturbations and modelling. This multidisciplinary project will provide a comprehensive understanding on how TCJs control the geometry and force applied on cell-cell contacts, hence the strength of cell communication, to regulate the epithelium organization and homeostasis of progenitors and stem cells