Rap localization and function in human umbilical vein endothelial cell monolayers. (A) HUVECs were retrovirally transduced with GFP-Rap1 or GFP vector and stained with an fluorochrome-conjugated VE-cadherin antibody. Live cells were imaged by confocal microscopy for GFP (green) or VE-cadherin (red). The upper panels depict a single 0.5-μm confocal slice showing GFP-Rap enriched in the lateral junctions as costained by VE-cadherin. Control GFP alone was evenly distributed throughout the cytoplasm. Bottom panels depict an orthogonal (xz) slice from the confocal stack above. GFP-Rap enrichment is likewise observed at junctions (arrows). The vertical (z) dimension has been exaggerated for clarity. Horizontal white scale bar = 10 μm. Vertical black scale bar = 2 μm. (B) HUVECs were retrovirally transduced with GFP-Spa1 or GFP-Spa1ΔGRD and cultured for 4 days. GFP was visualized by live-cell imaging and pictures taken and analyzed for the area (pixels) of the GFP-positive cells, the results of which were plotted. One of three representative experiments is shown for which more than 50 cells were analyzed per group. The frequency of the analyzed cells represents the percentage of cells with the indicated number of pixels in the x-axis. The difference in the median size of cells between the 2 groups, from 3 independent experiments, was statistically significant (median Spa1ΔGRD, 26 501 (n = 155); median Spa1, 20 592 (n = 154), P < .001). Representative pictures of Spa1 and Spa1ΔGRD-expressing cells are shown. For the analysis of monolayers expressing Spa1, only cells with discernable cellular structures were evaluated (arrows), while those that were completely round (*) were excluded to avoid inclusion of debris in the analysis.