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.