Myosin light chain kinase activity is required for the release of VWF contents. (A–G) HUVECs coexpressing GFP-VWF and the mCherry–P-selectinLum domain were stimulated with 100 ng/ml PMA for 10 min with or without a 15-min preincubation with 50 µM ML-7 (A and C–G) or treated with Y27632 (B). Fusion is assigned to 0 s. (A) Quantification of VWF secretion at different ML-7 concentrations. (B) Quantification of VWF secretion at different Y27632 concentrations. (C) Lag between WPB fusion and full release of GFP-VWF for individual WPBs in live PMA-stimulated cells in the presence (137 fusion events in nine cells) or absence (83 fusion events in 11 cells) of ML-7. 300+ s indicates that VWF failed to release during 600 s of time-lapse filming. (D) Still images from a video of an individual WPB fusion event in ML-7–inhibited cells cotransfected with mCherry–P-selectinLum (P.sel.lum.mcherry) and Lifeact-GFP. Bar, 2 µm. (E) Actin filament ring total lifetime, defined as total time to reach peak fluorescence intensity and subsequent decay of signal, in ML-7–treated cells (plotted from 63 actin positive fusion events from six cells). (F) Quantification of intensity of fluorescence of mCherry–P-selectinLum and Lifeact-GFP for the individual organelle shown in D. The dotted line shows the time point of WPB fusion. (G) Quantification of WPB fusion events in which Lifeact-GFP was recruited (plotted as the percentage of 142 total fusion events in six cells) to WPBs in the presence of ML-7. Error bars represent SDs.