Figure 6.
Figure 6. Myosin II activity is required for the release of VWF contents. (A) Untransfected HUVECs were stimulated for 5 min with 100 ng/ml PMA and then fixed in formaldehyde with a procedure optimal for imaging the actin cytoskeleton and myosin II (see Materials and methods), costained for pan–myosin II (panMyoII; blue), VWF (red), and actin (green), and imaged on a confocal microscope. The magnified insets on the right show an exocytosing WPB with an actin ring and a WPB in a similar part of the cell yet to exocytose with no actin ring. Images shown are maximum intensity projections. The arrows highlight the recruitment of actin and myosin to the exocytosing VWF in the first three images. But in a situation in which VWF is not released (the next three images), actin and myosin are not recruited. Bar, 10 µm. (B) Western blot analysis of myosin II isotypes in HUVECs. (C) HUVECs expressing the mCherry–P-selectinLum domain were stimulated with 100 ng/ml PMA for 10 min with or without a 5-min preincubation with 25 µM blebbistatin. Mean number of individual WPB fusion events in 300 s (control: n = 11 cells; blebbistatin: n = 6 cells; **, P = 0.004; ns, P = 0.863; t test). Error bars show SDs. (D) Quantification of VWF secretion at different blebbistatin concentrations. Error bars represent SDs. (E–H) HUVECs coexpressing VWF-GFP and Lifeact-Ruby were imaged before treatment with blebbistatin, preincubated with 25 µM blebbistatin for 2 min, and then stimulated with 100 ng/ml PMA for 10 min (in the continued presence of blebbistatin). During blebbistatin treatment, time-lapse images were only acquired from the Lifeact-Ruby (i.e., no blue light was present). A final image was then acquired from both markers. (E) Still images from a video of individual WPB fusion and blebbistatin-inhibited release of VWF in live cells. Bar, 2 µm. (F) Actin filament ring total lifetime, defined as total time to reach peak fluorescence intensity and subsequent decay of signal, in blebbistatin-treated cells (plotted from 39 actin-positive fusion events in six cells). (G) Quantification of intensity of fluorescence of Lifeact-Ruby for the individual organelle shown in E. The dotted line shows the time point of WPB fusion. (H) Quantification of WPB fusion events in which Lifeact-Ruby was recruited (plotted as the percentage of 82 total fusion events in six cells) to WPBs in the presence of blebbistatin. Note that blocking myosin II has no effect on the fusion of WPBs yet blocks subsequent secretion of VWF from the same fused organelle and extends the lifetime of the actin filament ring. In all parts of this figure, blebbistatin treatment of cells was performed in the absence of any blue light, except for the final image in E (labeled post).

Myosin II activity is required for the release of VWF contents. (A) Untransfected HUVECs were stimulated for 5 min with 100 ng/ml PMA and then fixed in formaldehyde with a procedure optimal for imaging the actin cytoskeleton and myosin II (see Materials and methods), costained for pan–myosin II (panMyoII; blue), VWF (red), and actin (green), and imaged on a confocal microscope. The magnified insets on the right show an exocytosing WPB with an actin ring and a WPB in a similar part of the cell yet to exocytose with no actin ring. Images shown are maximum intensity projections. The arrows highlight the recruitment of actin and myosin to the exocytosing VWF in the first three images. But in a situation in which VWF is not released (the next three images), actin and myosin are not recruited. Bar, 10 µm. (B) Western blot analysis of myosin II isotypes in HUVECs. (C) HUVECs expressing the mCherry–P-selectinLum domain were stimulated with 100 ng/ml PMA for 10 min with or without a 5-min preincubation with 25 µM blebbistatin. Mean number of individual WPB fusion events in 300 s (control: n = 11 cells; blebbistatin: n = 6 cells; **, P = 0.004; ns, P = 0.863; t test). Error bars show SDs. (D) Quantification of VWF secretion at different blebbistatin concentrations. Error bars represent SDs. (E–H) HUVECs coexpressing VWF-GFP and Lifeact-Ruby were imaged before treatment with blebbistatin, preincubated with 25 µM blebbistatin for 2 min, and then stimulated with 100 ng/ml PMA for 10 min (in the continued presence of blebbistatin). During blebbistatin treatment, time-lapse images were only acquired from the Lifeact-Ruby (i.e., no blue light was present). A final image was then acquired from both markers. (E) Still images from a video of individual WPB fusion and blebbistatin-inhibited release of VWF in live cells. Bar, 2 µm. (F) Actin filament ring total lifetime, defined as total time to reach peak fluorescence intensity and subsequent decay of signal, in blebbistatin-treated cells (plotted from 39 actin-positive fusion events in six cells). (G) Quantification of intensity of fluorescence of Lifeact-Ruby for the individual organelle shown in E. The dotted line shows the time point of WPB fusion. (H) Quantification of WPB fusion events in which Lifeact-Ruby was recruited (plotted as the percentage of 82 total fusion events in six cells) to WPBs in the presence of blebbistatin. Note that blocking myosin II has no effect on the fusion of WPBs yet blocks subsequent secretion of VWF from the same fused organelle and extends the lifetime of the actin filament ring. In all parts of this figure, blebbistatin treatment of cells was performed in the absence of any blue light, except for the final image in E (labeled post).

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