Ultrastructural analysis of WPBs during exocytosis. (A–D) HUVECs were stimulated with 100 ng/ml PMA for 10 min (A and C) or pretreated with 1 µM CCE for 15 min before PMA stimulation (B and D) and then labeled for released VWF (15-nm gold [A and B] or 15-nm gold and Alexa Fluor 488 nm [C and D]). (A) Scanning EM of PMA-stimulated HUVECs shows a network of VWF strings (i) and marked membrane ruffling (ii and iii) that appear to be sites of WPB exocytosis and initial VWF string release. These strings and sites are extensively labeled with anti-VWF/gold (see Fig. S1 for backscatter images showing VWF gold labeling). (B, i) CCE-treated cells show a marked absence of VWF strings. Instead, only small patches of intense gold labeling are present with no membrane ruffling, indicating the WPBs have fused but failed to release VWF as strings. (ii and iii) Magnified regions of the areas described by the white boxes. (C and D, i) Differential interference contrast images overlayed with a fluorescent image of VWF labeling (green). A specific exocytic site is magnified in the white boxed region. (C and D, ii–v) Serial TEM sections of the exocytic site shown in C and D (i) with v being the highest section. (C and D, vi) 3D reconstruction of the serial sections shown above. The reconstructions are also shown as Videos 6 and 7, revealing the same exocytic structures from a variety of angles. For the reconstruction, the VWF is highlighted in green, whereas the remainder of the cell is purple. Note that the VWF is released normally in the untreated cell, whereas the VWF in the drug-treated cell appears to be incompletely exocytosed. Fluorescent images are maximum intensity projections. All the serial TEM sections are shown in Fig. S2. Bars: (A and B, i–iii) 1 µm; (C and D, i) 10 µm; (C and D, ii–v) 200 nm.