Mechanically induced clathrin-dependent endocytosis of integrin in endothelial cells.

Abstract

The detachment of integrin in focal contacts (FCs) is crucial for this morphological change. This study investigated how mechanical stimuli regulate the detachment of integrin in cultured HUVECs. Mechanical stimulation was made by displacing laterally a fibronectin coated glass bead attached on the apical cell surface for a few micrometers. As the FCs spotaneously formed underneath the bead were linked to the FCs at the basal surface of the cell via actin stress fibers, the tension in the actin fibers generated by the bead displacement was transmitted to the basal FCs. HUVECs were stained with an Alexa-conjugated antibody against the extracellular domain of the β1 integrin of which dynamics was live-imaged by a multi-mode fluorescence microscope. When the bead was displaced, integrin clusters constituting the basal FCs gradually disappeared within 10 min. These integrins appeared to be associated with clathrin and internalized into the cytoplasm. They were also associated with certain structures that were stained with FM4-64, a fluorescent endocytosis marker. The disappearance of integrin clusters was inhibited by the depletion of external K⁺, which procedure is known to inhibit the clathrin-dependent endocytosis. These results suggest that mechanical stimuli to the FCs via actin stress fibers facilitate clathrin-dependent endocytosis of integrins in the basal FCs. [J Physiol Sci. 2007;57 Suppl:S132]