Actin is recruited to WPBs during exocytosis. (A–G) HUVECs coexpressing either Lifeact-GFP, a marker for actin filaments, and the mCherry–P-selectinLum (P.sel.lum.mcherry) domain (A, B, and D–F) or Lifeact-Ruby and GFP-VWF (C and G) stimulated with 100 ng/ml PMA and imaged live with a spinning-disk confocal microscope. Z stacks at a spacing of 0.5 µm were acquired every 2 s (A, B, and D–F) or 5 s (C and G) for 5 min, and images are all maximum intensity projections, except Lifeact channels in B and C, which are a single 0.5-µm-deep slices (see Materials and methods for a further explanation). Fusion is identified either by loss of mCherry fluorescence (A, D, and E) or by formation of rounded WPB structures (B, C, F, and G; assigned to 0 s in D–G). (A) Quantification of WPB fusion events in which Lifeact-GFP was recruited (plotted as the percentage of 143 total fusion events in nine cells). (B) Still images from a video that probes an individual WPB fusion event. The asterisk denotes a WPB that does not exocytose in the time course shown. (C) Still images from a video that probes an individual WPB fusion event and release of GFP-VWF contents. (D) Lifeact-GFP lifetime on individual WPBs (total of 43 WPBs in five cells). Actin filament ring total lifetime is defined as total time to reach peak fluorescence intensity and subsequent decay of signal. (E) Lag between WPB fusion and recruitment of Lifeact-GFP (total of 43 WPB fusion events in five cells). Initial actin recruitment is defined as the frame relative to fusion in which Lifeact fluorescence is first identified associated with the exocytosing WPB. (F) Change in mean fluorescence intensity of mCherry–P-selectinLum and Lifeact-GFP with the times of two WPB fusion events (the granule shown in B is plotted on the left trace). (G) For the WPB shown in C, change in mean fluorescence intensity with time of GFP-VWF and Lifeact-Ruby of the fusion and release of VWF content events. The dotted lines show the time points of WPB fusion. Bars, 2 µm.