Gouirand, N. | Search bioRxiv

bioRxiv2026

A non-invasive approach for understanding localized force generation in 3D tissues

The development, maintenance and repair of epithelial tissues critically rely on adhesion complexes that ensure structural integrity while enabling dynamic remodeling. Such tissue remodeling underpins both physiological morphogenesis and pathological transformation. Central to these processes are mechanical forces, which tightly couple cytoskeletal organization to adhesion dynamics. Despite extensive investigations in two-dimensional (2D) systems, how these interactions are orchestrated within polarized three-dimensional (3D) epithelia remains largely unresolved. Here, we introduce a new, non-invasive strategy to probe localized force generation within 3D epithelial tissues. We engineered elastic polyacrylamide (PAAm) microbeads with cell-mimetic size and mechanical properties, enabling their seamless integration. In contrast to conventional bead injection approaches, these PAAm microbeads were spontaneously engulfed by the tissue, thereby establishing an intrinsic interface through which bead deformation can be directly correlated with local cytoskeletal architecture and adhesion organization, as visualized through high-resolution imaging combined with quantitative 3D computational reconstruction. Using this approach, we demonstrated that localized mechanical perturbations trigger pronounced cytoskeletal remodelling while preserving global tissue polarity. We further identified the extracellular matrix composition as key determinant of bead-tissue interactions, with collagen-I coating promoting robust adhesion and efficient incorporation. At the bead-cell interface, cells assembled tension-bearing focal adhesions and organized actin stress fibers, revealing the emergence of active cortical stress. Strikingly, quantitative analysis of bead deformation revealed a previously unrecognized mechanical duality: spatially segregated regions of pulling and pushing forces coexisted at the microscale, directly correlated with local cytoskeleton dynamics. This finding challenges the prevailing view of homogenous force application and instead supports a model in which cells deploy highly coordinated and spatially patterned force-generating strategies. Altogether, this integrative and non-invasive strategy offers a comprehensive pipeline for dissecting the dynamic interplay between cellular processes and tissue mechanics during morphogenesis in 3D model systems.

cell biology↗

bioRxiv2023

CRB3 and ARP2/3 regulate cell biomechanical properties to set epithelial monolayers for collective movement

Several cellular processes during morphogenesis, tissue healing or cancer progression involve epithelial to mesenchymal plasticity that leads to collective motion (plasticity?). Even though a rich variety of EMP programs exist, a major hallmark unifying them is the initial breaking of symmetry that modifies the epithelial phenotype and axis of polarity. During this process, the actin cytoskeleton and cellular junctions are extensively remodelled correlating with the build-up of mechanical forces. As the collective migration proceeds, mechanical forces generated by the actin cytoskeleton align with the direction of migration ensuring an organized and efficient collective cell behaviour, but how forces are regulated during the breaking of symmetry at the onset of EMP remains an unaddressed question. It is known that the polarity complex CRB3/PALS1/PATJ, and in particular, CRB3 regulates the organization of the actin cytoskeleton associated to the apical domain thus pointing at a potential role of CRB3 in controlling mechanical forces. Whether and how CRB3 influences epithelial biomechanics during the epithelial-mesenchymal plasticity remains, however, largely unexplored. Here, we systematically combine mechanical and molecular analyses to show that CRB3 regulates the biomechanical properties of collective epithelial cells during the initial breaking of symmetry of the EMP. CRB3 interacts with ARP2/3 and controls the remodelling of actin throughout the monolayer via the modulation of the Rho-/Rac-GTPase balance. Taken together, our results identified CRB3, a polarity protein, as a regulator of epithelial monolayer mechanics during EMP.

cell biology↗