![Figure 3. Rab recruitment to and cycling on WPBs: evidence for association of EGFP-Slp4-a with Rab3s on mature WPBs. (A) Left: Images from time-lapse videos showing Rab acquisition by immature VWF-tdT containing WPBs in HUVECs coexpressing either mEGFP-Rab3B (top) or EGFP-Rab27A (bottom). Times are indicated below the panels. Right: Example time courses of mEGFP-Rab3B (○, τrecr 709 seconds) or EGFP-Rab27A (▴, τrecr 1070 seconds) recruitment to VWF-tdT–labeled WPBs. Bar plot represents the mean values of τrecr for WPB-mEGFP-Rab3B (1730 ± 355 seconds, n = 7) and WPB-EGFP-Rab27A (2260 ± 365 seconds, n = 9) were not significantly different (P = .35). (B) Left: Images from FRAP experiments with WPB-EGFP-Rab27A (top; scale bar represents 2 μm) and WPB-mEGFP-Rab3B (bottom; scale bar represents 1 μm) show FI recovery after complete WPB bleaching (FRAP, t = 0 seconds). Times are indicated below frames. Right: Average WPB mEGFP-Rab3B (○) and EGFP-Rab27A (▴) FIs normalized to the prebleach levels. Graphs were fitted with exponential association functions A × [1 − exp(−t/τass)]. Unconstrained fits to fluorescence recovery reached 0.80 for Rab3B and 0.63 for Rab27A. (C) Left: Images from iFRAP experiments with EGFP-Rab27A (top) and mEGFP-Rab3B (bottom). Scale bars represent 10 μm. Regions indicated by boxes were continuously bleached for 5 minutes, then FI of unbleached WPBs was measured. Fluorescence of neighboring cells was used for control. Times after bleaching are indicated below frames. Right: Average FIs of WPB-mEGFP-Rab3B (○) and WPB-EGFP-Rab27A (▴) were normalized to the initial postbleach levels. Graphs were fitted with exponential decay functions A × exp(−t/τdiss). (D) Average cycling time constants determined for each EGFP-Rab by FRAP (τass, gray) and iFRAP (τdiss, white) were as follows: EGFP-Rab27A, 1970 ± 550 seconds (n = 8) and 4020 ± 1090 seconds (n = 6); mEGFP-Rab3B, 196 ± 18 seconds (n = 6) and 468 ± 211 seconds (n = 5); mEGFP-Rab3D, 496 ± 79 seconds (n = 8) and 1300 ± 296 seconds (n = 8), respectively. The τass (FRAP) for mEGFP-Rab3B and Rab3D were significantly faster than for EGFP-Rab27A (P = .0007 and P = .002, respectively). The τdiss (iFRAP) for mEGFP-Rab3B was also significantly faster than for EGFP-Rab27A (P = .017). (E) Average τass for EGFP-Slp4-a coexpressed with RFP-tagged Rab27A (n = 8), Rab3B (n = 7), Rab3D (n = 8), or alone (n = 14) as indicated. τass for EGFP-Slp4-a coexpressed with Rab3B or Rab3D were not different from that for EGFP-Slp4-a alone (P = .31 and P = .43, respectively), whereas they were significantly faster than τass with mRFP-Rab27A coexpression.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/120/13/10.1182_blood-2012-05-429936/4/zh89991297070003.jpeg?Expires=1722820552&Signature=NQAuNkespOhD2OQVWHDZOW9djTZKAnFNn47jtvbBsECQ-vSkvl2wvTt55JuqQ9HyFGHze9sT1JbZ8ENMiZefoj6QV4nOR1eruPHsGs2oGNPg8VKqE63AbJYmFsO-2KbFAwO8sn1cHdVKoTdQuf5VGFDOdgMNqYEfvgBnePlGj3EQLwVCHids5EAbFg5dvn67rnpZtZXp799F4tzU7jSUjR-hSrjiihomGU74yGbx3fafnmvdY4cT0JtAdw8BXLkzjdexaIerKDnAxNfJ5Boy7h-MYNLUiBdEVBIq7lYNOihEoUDprXLEWB76LYM8UHFplC-ceUxpxf5le7pz4Smhfg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Rab recruitment to and cycling on WPBs: evidence for association of EGFP-Slp4-a with Rab3s on mature WPBs. (A) Left: Images from time-lapse videos showing Rab acquisition by immature VWF-tdT containing WPBs in HUVECs coexpressing either mEGFP-Rab3B (top) or EGFP-Rab27A (bottom). Times are indicated below the panels. Right: Example time courses of mEGFP-Rab3B (○, τrecr 709 seconds) or EGFP-Rab27A (▴, τrecr 1070 seconds) recruitment to VWF-tdT–labeled WPBs. Bar plot represents the mean values of τrecr for WPB-mEGFP-Rab3B (1730 ± 355 seconds, n = 7) and WPB-EGFP-Rab27A (2260 ± 365 seconds, n = 9) were not significantly different (P = .35). (B) Left: Images from FRAP experiments with WPB-EGFP-Rab27A (top; scale bar represents 2 μm) and WPB-mEGFP-Rab3B (bottom; scale bar represents 1 μm) show FI recovery after complete WPB bleaching (FRAP, t = 0 seconds). Times are indicated below frames. Right: Average WPB mEGFP-Rab3B (○) and EGFP-Rab27A (▴) FIs normalized to the prebleach levels. Graphs were fitted with exponential association functions A × [1 − exp(−t/τass)]. Unconstrained fits to fluorescence recovery reached 0.80 for Rab3B and 0.63 for Rab27A. (C) Left: Images from iFRAP experiments with EGFP-Rab27A (top) and mEGFP-Rab3B (bottom). Scale bars represent 10 μm. Regions indicated by boxes were continuously bleached for 5 minutes, then FI of unbleached WPBs was measured. Fluorescence of neighboring cells was used for control. Times after bleaching are indicated below frames. Right: Average FIs of WPB-mEGFP-Rab3B (○) and WPB-EGFP-Rab27A (▴) were normalized to the initial postbleach levels. Graphs were fitted with exponential decay functions A × exp(−t/τdiss). (D) Average cycling time constants determined for each EGFP-Rab by FRAP (τass, gray) and iFRAP (τdiss, white) were as follows: EGFP-Rab27A, 1970 ± 550 seconds (n = 8) and 4020 ± 1090 seconds (n = 6); mEGFP-Rab3B, 196 ± 18 seconds (n = 6) and 468 ± 211 seconds (n = 5); mEGFP-Rab3D, 496 ± 79 seconds (n = 8) and 1300 ± 296 seconds (n = 8), respectively. The τass (FRAP) for mEGFP-Rab3B and Rab3D were significantly faster than for EGFP-Rab27A (P = .0007 and P = .002, respectively). The τdiss (iFRAP) for mEGFP-Rab3B was also significantly faster than for EGFP-Rab27A (P = .017). (E) Average τass for EGFP-Slp4-a coexpressed with RFP-tagged Rab27A (n = 8), Rab3B (n = 7), Rab3D (n = 8), or alone (n = 14) as indicated. τass for EGFP-Slp4-a coexpressed with Rab3B or Rab3D were not different from that for EGFP-Slp4-a alone (P = .31 and P = .43, respectively), whereas they were significantly faster than τass with mRFP-Rab27A coexpression.
