Actin polymerization and actomyosin contractility mediate increase of endothelial membrane tension and downstream signaling induced by ICAM-1 clustering. (A-G) HUVECs were preincubated without or with 10 µM cytochalasin D (CytoD) or 30 µM blebbistatin (Bleb) (A-D) or were transfected with control siRNA (siCtrl) or siRNA directed against the RNA encoding α-actinin-4 (siACTN4) or cortactin (siCTTN) (E-G) and were exposed to low flow alone, anti–ICAM-1 clustering antibodies (ICAM-1 XL) alone or both, and membrane tension was determined using FliptR (A-B,G; n = 20 measurements from 3 independent experiments), or immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC was performed (C-F). Bar diagrams show lifetime mean values (B,G) or immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC (D,F; 3 independently performed immunoblot experiments). (H) Schematic representation showing how fluid shear stress exerted by the flowing blood and leukocyte-induced ICAM-1 clustering synergistically activate PIEZO1 to induce downstream signaling events resulting in opening of the endothelial barrier. Shown are mean values ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001 (1-way ANOVA).