Figure 1.
Figure 1. Spinning-disk confocal microscope assay for monitoring fusion of—and VWF release from—individual WPBs in live cells. (A–E) HUVECs were nucleofected with the mCherry–P-selectinLum domain and GFP-VWF and imaged with a spinning-disk confocal microscope system in the absence (A) or presence (B–E) of 100 ng/ml PMA. Time on the panels indicates total time in media (A), media with PMA (B), or relative to fusion (0 s; C–E). Z stacks were acquired at a spacing of 0.5 µm every 5 s for 10 min, and images shown represent maximum intensity projections. (A, unstimulated) mCherry–P-selectinLum (Psel.lum.mcherry) and GFP-VWF are colocalized in WPBs, and there is a similar number of WPBs 605 s later. Individual WPBs move within the cytoplasm over time. Smaller images show individual channels of the 5-s merged image. (B, PMA stimulated) mCherry–P-selectinLum and GFP-VWF remain colocalized within WPBs by 5 s of PMA stimulation. Smaller images show individual channels of the 5-s merged image. By 605 s, many WPBs have exocytosed (asterisks indicate WPBs that fully exocytose during the time course of imaging). (C) Quantification of the intensity of fluorescence of mCherry–P-selectinLum and GFP-VWF for the individual organelle shown in D. Note that decrease in mCherry–P-selectinLum fluorescence (−5 to 0 s) occurs before the decrease in GFP-VWF fluorescence (−5 to 25 s) in the same WPB. (D) Still images of a video of exocytosis of the WPB in the box in B. The WPB becomes rounded in shape (compare GFP-VWF at −5 and 0 s), and this is linked to loss of mCherry fluorescence intensity (compare −5 and 0 s). (E) Quantification of the interval between formation of a rounded GFP-VWF shape and loss of mCherry fluorescence intensity for the population of exocytosing WPBs at 5-s time resolution (plotted from 51 events in five cells). (F) Schematic of exocytosis of full-length P-selectin and the P-selectin luminal domain. Bars: (A and B) 10 µm; (D) 4 µm.

Spinning-disk confocal microscope assay for monitoring fusion of—and VWF release from—individual WPBs in live cells. (A–E) HUVECs were nucleofected with the mCherry–P-selectinLum domain and GFP-VWF and imaged with a spinning-disk confocal microscope system in the absence (A) or presence (B–E) of 100 ng/ml PMA. Time on the panels indicates total time in media (A), media with PMA (B), or relative to fusion (0 s; C–E). Z stacks were acquired at a spacing of 0.5 µm every 5 s for 10 min, and images shown represent maximum intensity projections. (A, unstimulated) mCherry–P-selectinLum (Psel.lum.mcherry) and GFP-VWF are colocalized in WPBs, and there is a similar number of WPBs 605 s later. Individual WPBs move within the cytoplasm over time. Smaller images show individual channels of the 5-s merged image. (B, PMA stimulated) mCherry–P-selectinLum and GFP-VWF remain colocalized within WPBs by 5 s of PMA stimulation. Smaller images show individual channels of the 5-s merged image. By 605 s, many WPBs have exocytosed (asterisks indicate WPBs that fully exocytose during the time course of imaging). (C) Quantification of the intensity of fluorescence of mCherry–P-selectinLum and GFP-VWF for the individual organelle shown in D. Note that decrease in mCherry–P-selectinLum fluorescence (−5 to 0 s) occurs before the decrease in GFP-VWF fluorescence (−5 to 25 s) in the same WPB. (D) Still images of a video of exocytosis of the WPB in the box in B. The WPB becomes rounded in shape (compare GFP-VWF at −5 and 0 s), and this is linked to loss of mCherry fluorescence intensity (compare −5 and 0 s). (E) Quantification of the interval between formation of a rounded GFP-VWF shape and loss of mCherry fluorescence intensity for the population of exocytosing WPBs at 5-s time resolution (plotted from 51 events in five cells). (F) Schematic of exocytosis of full-length P-selectin and the P-selectin luminal domain. Bars: (A and B) 10 µm; (D) 4 µm.

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