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Blood, Vol. 111, Issue 11, 5282-5290, June 1, 2008
Selective release of molecules from Weibel-Palade bodies during a lingering kiss
Blood Babich et al.
111: 5282
Supplemental materials for: Babich et al
Files in this Data Supplement:
- Figure S1. Effect of WPB alkalinisation on EGFP and Red Fluorescent Protein fluorescence within WPB (JPG, 206 KB)
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Panel Ai shows images of a HUVEC co-expressing VWF-EGFP (top panels) and Proregion-mRFP (lower panels) prior to (control) and during exposure to a solution containing 20mM NH4Cl at pH 7.4. Exposure to NH4Cl solutions causes the intra-WPB pH to rise from its resting level of ∼pH 5.5 to >pH 7.4 {Erent, 2007 #114}. Images represent a 3 frame average taken from a time-lapse movie acquired at 20 frames per second. Panel Aii shows in more detail the time-course for the fluorescence change of VWF-EGFP (solid circles) and Proregion-mRFP (open circles) within a single WPB (montage above) during exposure to NH4Cl; scale bar is 2µm. Fluorescence in each case was normalised to the peak after NH4Cl addition. Panel Aiii summarises the fold increase in fluorescence of VWF-EGFP and Proregion-mRFP for 20 WPB analysed from 3 HUVEC exposed to NH4Cl. Panel Bi shows the same experiment as in A for HUVEC co-expressing Proregion-EGFP (top panel; green in merged image) and IL-8-mCherry (middle panel; red in merged image) prior to (control) or during exposure to NH4Cl. Scale bar is 10µm. Images are individual frames taken from a time-lapse movie acquired at 0.83 frames per second. Panel Bii shows the time-course for the fluorescence change of Proregion-EGFP (solid circles) and IL-8-mCherry (open circles) within a single WPB (montage above), during exposure to NH4Cl; scale bar is 2µm. Images are shown at 5 frame intervals. The image montage in panel Biii shows the effect of NH4Cl on the fluorescence of Proregion-EGFP (Pro-EGFP) and mCherry fused to VWF within a single WPB; scale bar is 2µm. Lower panel in Biii summarises the mean fold increase in fluorescence of various co-expressed EGFP, mRFP and mCherry fusion proteins as indicated following exposure to NH4Cl (n=20 WPB in each case from 3-4 HUVEC). Histogram in panel Aiii is reproduced here for ease of comparison.
- Figure S2. IL-8-mCherry expression labels WPB in living HUVEC (JPG, 74.2 KB)
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Panel A shows immunoblots for IL-8 immunoreactivity (left panel and green in merged panel), and red fluorescent protein immunoreactivity (middle panel and red in merged panel) for a sample of human recombinant IL-8 (rhIL-8) and for cell lysates from CHO cells Nucleofected with expression vectors for cytosolic mCherry (mCherry) or IL-8-mCherry. Predicted sizes for IL-8, mCherry and IL-8-mCherry are indicated by arrows on the right. Panel B shows an image of a living HUVEC co-expressing Proregion-EGFP (Pro-EGFP; green in merged image) and IL-8-mCherry (red in merged image). Scale bar is 10µm. Images are a two frame average from a two-colour time-lapse movie taken at 30 frames per second.
- Figure S3. Fluorescence of VWF-mCherry is maintained during a lingering kiss (JPG, 86.8 KB)
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Panel (i) shows a living HUVEC co-expressing Proregion-EGFP (left panel and green in merged image) and VWF-mCherry (middle panel and red in merged image). Scale bar is 10µm. Panels ii and iii show examples of the fluorescence changes for Proregion-EGFP (solid circles) and VWF-mCherry (open circles) during a complete (ii) or lingering kiss (iii) fusion event evoked by 1µM histamine. Images of the WPBs from which the data plots were made are shown in the montages above each plot. Images were acquired at 0.83 frames per second and are displayed at 4 frame intervals.
- Figure S4. Co-expressed Proregion-EGFP and P-selectin-mRFP or mRFP-CD63 label WPB in living HUVEC (JPG, 117 KB)
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Fluorescence images of PFA fixed HUVEC 24h post-Nucleofection with Proregion-EGFP (left panels; green in merged image) and P-selectin-mRFP (Top middle panel; red in merged image) or mRFP-CD63 (bottom middle panel; red in merged image). Scale bar is 10µm.
- Figure S5. Fusion pore flicker and WPB EGFP fluorescence (JPG, 88.9 KB)
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Panel shows an example of step wise accumulation of extracellular alexa-647 (6µM) into a single WPB following stimulation with histamine. The black trace shows the fluorescence changes of intra-WPB Proregion-EGFP fluorescence and the red trace shows the fluorescence intensity of alexa-647 in the same WPB. Asterisks indicate the decline in EGFP fluorescence during periods in which accumulation of alexa-647 fluorescence terminated abruptly indicating fusion pore closure. The sequence of events indicated by this experiment are summarised in the cartoon below. Data in B and C were acquired on a Leica SP2 confocal microscope as described in the methods.
- Figure S6. Partial recovery of EGFP-CD63 fluorescence following stimulus-evoked collapse of WPB (JPG, 81.8 KB)
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Panel A: Co-expression of VWF-mCherry and EGFP-CD63 label WPB. Fluorescence images of a HUVEC 24h post nucleofection with VWF-mCherry (left panel and red in merged image) and EGFP-CD63 (middle panel and green in merged image). Panels Bi and ii show images of the WPB in ROI2 of movie 7 (supplemental materials). The WPB undergoes a lingering kiss indicated by a marked morphological change, retention of VWF-mCherry and loss of EGFP-CD63 fluorescence (i and iii); the time-course for changes in VWF-mCherry (solid circles) and EGFP-CD63 (open circles) are shown in panel Biii. Images in the montage in panel Bii show frames taken at intervals of approximately 300 s following fusion and show a slow partial recovery of EGFP-CD63 fluorescence (see red arrows in images).
- Video 1. Simultaneous imaging of VWF-EGFP and Proregion-mRFP release during complete WPB exocytosis (MOV, 299 KB)
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Video shows a single WPB containing both VWF-EGFP (top panel; green in merged image) and Proregion-mRFP (middle panel; red in merged image) during stimulation with 1µM histamine. Images were acquired using an Olympus IX70 epi-fluorescence microscope equipped with an ×100 1.35 NA objective (Olympus) at 0.83 frames per second. Time elapsed from the start of movie is indicated in the top left corner. Scale bar is 2µm.
- Video 2. Retention of VWF-EGFP and Proregion-mRFP within stimulus-evoked collapsed WPB (MOV, 2.15 MB)
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Video shows a group of VWF-EGFP (left panel and green in merge panel) and Proregion-mRFP (middle panel and red in merged panel) co-expressing WPB during stimulation (indicated by a yellow square in the top left corner) with 1µM histamine. Images were acquired using an Olympus IX70 epi-fluorescence microscope equipped with an ×100 1.35 NA objective (Olympus) at 0.83 frames per second and the time elapsed from the start of movie is indicated in the top left corner. The positions of two WPB that undergo stimulus-evoked collapse are marked by white circles. Scale bar is 5µm.
- Video 3. Accumulation of the extracellular marker FM4-64 within the WPB membrane during plasma membrane fusion (MOV, 608 KB)
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Video shows a single proregion-EGFP containing WPB in a HUVEC during stimulation with 1µM ionomycin in the presence of 10µM extracellular FM4-64. The top panel shows EGFP fluorescence, the middle panel FM4-64 fluorescence and the bottom panel a merged image (EGFP; green, FM4-64; red). The image sequence was acquired using a Leica SP2 confocal microscope equipped with an ×100 1.40 NA objective (Leica). Images were acquired at 7.4 frames per second and the time elapsed from the start of movie is indicated in the top left corner.
- Video 4. Discrimination of fusion events in which full pore expansion results in complete or partial loss of major core proteins (MOV, 1.72 MB)
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Video shows a group of three proregion-EGFP containing WPB in a HUVEC during stimulation with 100µM histamine. The image sequence was acquired using an Olympus IX70 microscope equipped with an ×100 1.3NA objective (Olympus). Images were acquired at 1.6 frames per second and the time elapsed from the start of movie is indicated in the top right corner.
- Video 5. Accumulation of 3kDa dextran-TRITC within WPB during a lingering kiss fusion event (MOV, 877 KB)
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Video shows a single proregion-EGFP containing WPB in a HUVEC during stimulation with 1µM ionomycin in the presence of 7µM extracellular alexa-647 and 1mM 3kDa dextran-TRITC. The top panel shows EGFP fluorescence, the second panel the alexa-647 fluorescence, the third panel dextran-TRITC fluorescence and the bottom panel a merged image (EGFP; green, alexa-647; red, dextran-TRITC; blue). The image sequence was acquired using a Leica SP2 confocal microscope equipped with an ×100 1.40 NA objective (Leica). Images were acquired at 7.4 frames/s and the time elapsed from the start of movie is indicated in the top left corner.
- Video 6. Exclusion of of 70kDa dextran-TRITC from WPB during a lingering kiss fusion event (MOV, 1.96 MB)
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Video shows a single proregion-EGFP containing WPB in a HUVEC during stimulation with 1µM ionomycin in the presence of 7µM extracellular alexa-647 and 1mM 70kDa dextran-TRITC. The top panel shows EGFP fluorescence, the second panel the alexa-647 fluorescence, the third panel dextran-TRITC fluorescence and the bottom panel a merged image (EGFP; green, alexa-647; red, dextran-TRITC; blue). The image sequence was acquired using a Leica SP2 confocal microscope equipped with an ×100 1.40 NA objective (Leica). Images were acquired at 7.4 frames/s and the time elapsed from the start of movie is indicated in the top left corner.
- Video 7. Loss of EGFP-CD63 during stimulus-evoked WPB collapse (MOV, 1.63 MB)
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Video shows a sub-region of a HUVEC co-expressing EGFP-CD63 (green) and VWF-mCherry (red) during stimulation with 1µM histamine. The image sequence was acquired using an Olympus IX70 epi-fluorescence microscope equipped with an ×100 1.35 NA objective (Olympus). From time = 0 to 61.7s dual-color images were acquired at 0.83 frames per second. At times >61.7s dual-color images were acquired at 0.2 frames per second to help reduce photo-bleaching. The time elapsed from the start of movie is indicated in the top left corner. The positions of two WPB that undergo stimulus-evoked collapse and loss of EGFP-CD63 are marked by the white circles ROI1 and ROI2 respectively. Scale bar is 5µm.
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