In Situ Landscape of Focal Adhesions and Cytoskeletal Integration Revealed by Cryo-Electron Tomography

ABSTRACT Focal adhesions (FAs) are dynamic hubs for mechanotransduction, linking the extracellular matrix to actin fibers, intermediate filaments, and microtubules. Using cryo–electron tomography combined with 3-dimensional segmentation and subtomogram averaging, we visualize in situ the structural architecture of the FA environment at the leading edge of human fibroblasts. Our analysis reveals a rich architectural diversity within the FA landscape, where the spatial organisation and interplay of FA protein clusters, actin, vimentin, and microtubules change from the actin bundle core to its tip and across adjacent regions. Notably, we reveal diverse arrangements and connections of vimentin filaments, supporting their multifaceted role in the control and mechanics of adhesions. Together, these findings establish a structural framework for FA maturation and cytoskeletal integration, extending classical lamellipodial adhesion models and providing mechanistic insight into how FAs coordinate force transmission during cell migration.