BRAF increases endothelial cell stiffness through reorganization of the actin cytoskeleton

• DOI: 10.1096/fj.202200344r
• Publication Date: 2022-08-02T07:47:12Z
• Authors: Anna Hollósi; Katalin Pászty; Bálint Levente Bunta; Tamás Bozó; Miklós Kellermayer; Márta Lídia Debre...; László Cervenak; Manuela Baccarini; Andrea Varga
• Keywords: Proto-Oncogene Proteins B-raf; 0301 basic medicine; Mitogen-Activated Protein Kinase Kinases/metabolism; actin cytoskeleton; endothelial monolayer; Myosin Light Chains; Cells; 03 medical and health sciences; 106023 Molekularbiologie; Human Umbilical Vein Endothelial Cells; Actin Depolymerizing Factors/metabolism; Humans; Phosphorylation; 106052 Cell biology; Human Umbilical Vein Endothelial Cells/metabolism; Cells, Cultured; Actin Cytoskeleton/metabolism; Mitogen-Activated Protein Kinase Kinases; Cultured; Thrombin/metabolism; atomic force microscopy; Thrombin; 106023 Molecular biology; BRAF RNAi; Actins; intercellular gaps; Actin Cytoskeleton; Proto-Oncogene Proteins B-raf/metabolism; Q1 Science (General) / természettudomány általában; Actin Depolymerizing Factors; Actins/metabolism; Myosin Light Chains/metabolism; 106052 Zellbiologie; permeability

AbstractThe 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.