Cortical actin nanodynamics determines nitric oxide release in vascular endothelium

The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (K_cortex) of living endothelial cells. It is hypothesized that cortical actin dynamics determines K_cortex and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between K_cortex and bulk cell stiffness (K_bulk) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that K_cortex softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ~100 nm phase of the submembranous actin web, without affecting K_bulk, regulate NO release and thus determines endothelial function.