The dynamics of the actin cytoskeleton and its connection to endothelial cell–cell junctions determine the barrier function of endothelial cells. The proper regulation of barrier opening/closing is necessary for the normal function of vessels, and its dysregulation can result in chronic and acute inflammation leading to edema formation. By using atomic force microscopy, we show here that thrombin‐induced permeability of human umbilical vein endothelial cells, associated with actin stress fiber formation, stiffens the cell center. The depletion of the MEK/ERK kinase BRAF reduces thrombin‐induced permeability prevents stress fiber formation and cell stiffening. The peripheral actin ring becomes stabilized by phosphorylated myosin light chain, while cofilin is excluded from the cell periphery. All these changes can be reverted by the inhibition of ROCK, but not of the MEK/ERK module. We propose that the balance between the binding of cofilin and myosin to F‐actin in the cell periphery, which is regulated by the activity of ROCK, determines the local dynamics of actin reorganization, ultimately driving or preventing stress fiber formation.

Mechanical forces acting on cell–cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell–cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the stress-induced response. Control cells responded to external forces by organizing and stabilizing actin cables in the stretched cell junctions. This was accompanied by an increase in intercellular gap formation, which was prevented in BRAF knockdown monolayers. In the absence of BRAF, there was excess stress fiber formation due to the enhanced reorganization of actin fibers. Our findings suggest that stretch-induced intercellular gap formation, leading to a decrease in barrier function of blood vessels, can be reverted by BRAF RNAi. This is important when the endothelium experiences changes in external stresses caused by high blood pressure, leading to edema, or by immune or cancer cells in inflammation or metastasis.

Variants in ACTB gene encoding for cytoplasmic β-actin result in a group of rare disorders called non-muscle actinopathies (NMA). A subgroup of NMA patients with ACTB pLoF (predicted loss-of-function) disorder have various congenital malformations, such as microcephaly and intellectual disability. We investigated the cellular effects of an ACTB pLoF missense variant, G302A, and a four-amino-acid deletion, S338-I341 in cytoskeletal β-actin, in patient-derived fibroblast cells. We found that neither of the mutations affected the organization of actin or the width of the actin-filament bundles, while the mutation G302A reduced the stiffness of the cells as measured by using atomic force microscopy. The latter effect might be associated with the misorganization of tubulin and with the increased size and number of focal adhesions. When we challenged the cells by monolayer stretching and followed the reorganization of the actin cytoskeleton, we found that G302A mutant cells showed denser actin filament bundles within the cells compared to wild type cells. At the same time, the extent of cofilin reorganization from the cell periphery was increased upon stretch, and this correlated with an increased cofilin phosphorylation. In the case of deletion, while the extent of cofilin phosphorylation increased, the extent of reorganization was unaltered; rather, the phosphorylation of myosin light chain, important in counteracting external force, was reduced by 75%. We could partially rescue this effect by overexpressing the active form of the formin mDia1. Our findings open the possibility to verify the cellular phenotype in the most affected patients' cells, in neurons.