Fig. 1. Integrin clustering during FC formation. (A) HUVECs were fixed at 30 minutes (a-c), 1 hour (d-f) and 6 hours (g-i) after plating. The left-hand column shows EpiF images, the middle column TIRF images and the right-hand column DIC images of the cells at different time after plating; images in each line are from the same cell. At 30 minutes after plating (a-c), a uniform distribution of integrins is observed in >90% of the bottom area of the cell (b). At 1 hour (d-f), a dappled distribution of integrins (the region surrounded by a broken line in e) and clusters of integrin are seen. Arrows show typical clusters of integrins in the region surrounded by a continuous line in e. At 6 hours after plating (g-i), clusters of integrins cover the entire bottom surface of the cell (four typical examples are indicated by arrows in h); arrows in g and h show the same integrin clusters. Integrins in the bottom surface of the cell were imaged more clearly by TIRF microscopy than by EpiF microscopy. Arrowheads in a, d and g show the punctate spots of integrins near the cell nucleus. (B) Integrin clusters observed by TIRF (arrows in b) correspond to the dark areas in the RIC image (arrows in a) of a HUVEC at 2 hours after plating. (C) Integrin clusters observed by EpiF (a) correspond to the vinculin clusters in the EpiF image (b) of the same HUVECs at 2 hours after plating. Bars, 20 µm.