Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration

Buffering of intracellular Ca2+ transients in human neutrophils leads to re duced motility due to defective uropod detachment on fibronectin and vitron ectin-coated surfaces. Since one potential target of a rise in [Ca2+](i) is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedion e monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926 ) resulted in impaired uropod retraction and a dose-dependent decrease in c hemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP), Treatme nt with ML-9 resulted in a redistribution of F-actin and talin to the non-r etracted uropods, mimicking the redistribution observed during [Ca2+](i) bu ffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin LT inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-c oated glass, At higher concentrations of ML-9, cell polarization was inhibi ted and pseudopod extension occurred radially. Using an antibody; specific for serine 19-phosphorylated regulatory light chain of myosin II, regions o f activated myosin II were found at the leading edge as well as the uropod in motile flMLP-stimulated cells. [Ca2+](i) depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II. suggesting that activa tion of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.