Cell migration involves coordination of protrusion, adhesion and contraction. Central to this process is the spatiotemporal regulation of focal adhesion (FA) assembly, maturation and disassembly. Myosin II-mediated contraction in the actin cytoskeleton drives FA maturation, which is characterized by FA growth and changes in FA protein composition and phosphorylation state. Epidermal growth factor (EGF), a physiological stimulus for cell migration, is well-studied for its effects on cell protrusion, but how it temporally controls contraction leading to traction generation and FA maturation and disassembly is less clear.

We observed that epidermal keratinocytes stimulated with EGF show a protrusion phase followed by a temporally delayed retraction phase. Using microscopic and biochemical assays, we quantified the temporal ordering of biochemical and mechanical processes with respect to these morphological changes. We found that myosin-II activation occurs very quickly after stimulation. Lagging behind this response was protrusion, traction generation and the formation of small FAs rich in phosphorylated paxillin. Much later, paxillin was dephosphorylated, marking the maturation of FAs, and retraction occurred. Inhibition of myosin-II not only blocked the EGF-stimulated contraction leading to cell retraction; it also eliminated the delay in paxillin dephosphorylation and FA maturation. This suggests that EGF-mediated activation of myosin-II acts as a mechanical signal to promote a period of adhesion maturation that delays paxillin dephosphorylation and adhesion weakening relative to increases in traction generation. This may serve as a mechanism by which the cell temporally segregates protrusion, adhesion and traction generation from retraction during EGF-stimulated cell migration.