Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy

Human umbilical vein endothelial cells were stained with FITC-labeled anti- beta (1) integrin antibody and plated on a glass cover slip to elucidate th e mechanism of integrin clustering during focal contact formation. The proc ess of integrin clustering was observed by time-lapse total-internal-reflec tion fluorescence microscopy, which can selectively visualize the labeled i ntegrins at the basal surface of living cells. The clustering of integrins at focal contacts started at 1 hour after plating and individual clusters k ept growing for similar to6 hours. Most integrin clusters (similar to 80%) elongated towards the cell center or along the cell margin at a rate of 0.2 9 +/- 0.24 mum minute(-1). Photobleaching and recovery experiments with eva nescent illumination revealed that the integrins at the extending tip of th e clusters were supplied from the intracellular space. Simultaneous time-la pse imaging of exocytosis of integrin-containing vesicles and elongating fo cal contacts showed that most exocytosis occurred at or near the focal cont acts followed by their elongation. Double staining of F-actins and integrin s demonstrated that stress fibers were located near the integrin clusters a nd that intracellular punctate integrins were associated with these stress fibers. These results suggest that the clustering of integrins is mediated by actin-fiber-dependent translocation of integrins to the extending tip of focal contacts.