Focal Adhesions Undergo Longitudinal Splitting into Fixed-Width Units

Focal adhesions (FAs) and stress fibers (SFs) act in concert during cell motility and in response to the extracellular environment. Although the structures of mature FAs and SFs are well studied, less is known about how they assemble and mature de novo during initial cell spreading. In this study using live-cell Airyscan microscopy, we find that FAs undergo splitting'' during their assembly, in which the FA divides along its longitudinal axis. Before splitting, FAs initially appear as assemblies of multiple linear units (FAUs) of 0.3-mm width. Splitting occurs between FAUs, resulting in mature FAs of either a single FAU or of a small number of FAUs that remain attached at their distal tips. Variations in splitting occur based on cell type and extracellular matrix. Depletion of adenomatous polyposis coli (APC) or vasodilator-stimulated phosphoprotein (VASP) results in reduced splitting. FA-associated tension increases progressively during splitting. Early in cell spreading, ventral SFs are detected first, with other SF subtypes (transverse arcs and dorsal SFs) being detected later. Our findings suggest that the fundamental unit of FAs is the fixed-width FAU, and that dynamic interactions between FAUs control adhesion morphology.