ECs use Myo1c as part of the WPB exocytic machinery. (A) Super-resolution imaging and immunofluorescent localization of endogenous Myo1c (green), actin (magenta), and VWF (blue) in unstimulated or PMA (100 ng/mL) stimulated HUVEC in the presence and absence of 1 μM of the actin polymerization inhibitor cytochalasin E (CCE). Scale bar, 10 μm. Inset, 1 μm. Myo1c is recruited independently of actin but was dependent on stimulation with PMA. (B) Myo1c-GFP encapsulates WPB after fusion as determined by live cell super-resolution spinning disk imaging of PMA stimulated (100 ng/mL) HUVEC coexpressing a Myo1c-GFP and the WPB fusion marker P.sel.lum.mCherry. Scale bar, 1 μm. Arrows indicate point of collapse/fusion of vesicle (C) Live cell imaging of LifeAct-GFP and P.sel.lum.mCherry expressing HDMEC indicated the utility of actin rings to expel VWF after stimulation. Scale bar, 10 μm. Inset, 1 μm. Z stacks of 0.5 μm were acquired continuously for 10 minutes (Zeiss LSM 800). (D) Confocal imaging and IF analyses of endogenous Myo1c in HDMEC that were left untreated or stimulated with PMA, CCE, or CCE and PMA. Arrows illustrate where Myo1c is recruited to fused/collapsed WPB. Scale bar, 10 μm.