Inhibition of Myo1 ATPase activity through pharmacological inhibition or point mutation (G108R) affects the actomyosin machinery associated with exocytosis. (A) Schematic of live cell imaging approach to study actin dynamics during WPB exocytosis. Scale bar, 1 μm. (B) PMA stimulated HUVEC coexpressing LifeAct-GFP and P.sel.lum.mCherry in the presence or absence of PCLP. The percentage of WPB fusion events that recruited an actin ring were unchanged in DMSO and PCLP (20 μM) treated cells. (C) The lifetime (seconds) of LifeAct-GFP signal at fusion sites was quantified in DMSO and PCLP-treated HUVEC. The distribution of frequency of events is presented here (n = 5 DMSO: 15 cells, 81 events; PCLP: 18 cells, 93 events; mean ± SEM). (D) Schematic of site-directed mutagenesis for the generation of a Myo1c rigor mutant. (E) HUVEC coexpressing GFP-tagged Myo1c constructs and P.sel.lum.mCherry were stimulated with PMA and the percentage of exocytic events that recruit GFP-Myo1c WT or G108R was quantified (n = 3; WT: 8 cells and 119 events; G108R: 8 cells and 58 events). (F) HUVEC coexpressing GFP-tagged Myo1c constructs and P.sel.lum.mCherry were stimulated with PMA and the duration of GFP signal in a ring shape forming at the site of WPB fusion was quantified. (n = 3; WT: 9 cells and 79 events; PCLP: 9 cells and 42 events). (G) The distribution of frequency closely resembles actin ring dynamics--panel C. For live cell confocal imaging experiments, 0.5 μm Z stacks were acquired continuously for 5 to 10 minutes (Zeiss LSM 800). (H) HUVEC expressing GFP, GFP-Myo1c (WT) or (G108R) were stimulated with PMA (100 ng/mL) for 10 minutes and labeled for external VWF (red) and total VWF (blue). Scale bar, 1 μm. (I) Quantification of the ratio of externalized VWF to total VWF. ∗P < .05, 1-way ANOVA; n = 3 NTC. Arrows indicate swollen intracellular VWF signal in cells expressing the G108R point mutant. NTC, nontransfected control.