BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization

Abstract

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.

BRAF increases endothelial cell stiffness through reorganization of the actin cytoskeleton

“…Subsequently, actin formed thinner stress fibers, and thicker ones were assembled 15 min following treatment. BRAF-deficient HUVECs showed a thicker peripheral actin ring (Figure 2A) compared to control cells, as we previously observed upon BRAF depletion in human, 23 and in mouse endothelial 15 cells. In addition, actin did not form stress fibers in the cell center upon thrombin treatment (Figure 2D).…”

“…Figure 6A shows the experimental arrangement of the AFM combined with an epifluorescence microscope which was used to identify the EGFP‐expressing cells co‐expressing either control or BRAF shRNAs. BRAF‐knockdown cells were identified by their EGFP expression as described in Hollósi et al 23 Figure 6B illustrates the phase‐contrast image of the fixed monolayer, the fluorescence image of the same monolayer, as well as the high‐resolution AFM deflection image. The effect of thrombin was investigated on fixed cells, since with our AFM setup there is no possibility to inject thrombin after setting up the system, therefore on the short timescale of thrombin treatment we cannot produce significant amount of data to analyze the effect of thrombin.…”

“…AIMV medium was supplemented with 1% FBS, 1 ng/ml bFGF, 2 ng/ml EGF and 7.5 U/ml heparin. HEK293T cells used for lentiviral production were cultured and transfected with the appropriate plasmids as described in Hollósi et al 23 …”