American Society of Hematology

Figure 6

$Figure 6. The probability of WPB exocytosis is maintained over a wide range of WPB-Rab27A concentrations. (A) Mean intensities of WPB-IR for endogenous Rab27A (squares), MyRIP (triangles), and Slp4-a (circles) as a function of WPB numbers per cell. Data scaled to the mean intensity for the < 50 WPBs per cell. Numbers of cells analyzed: Rab27A, 124-218; MyRIP, 17-73; Slp4-a, 16-39. For all cells with > 50 WPBs, Rab27A and MyRIP intensities of WPB-IR were significantly lower than for cells with < 50 WPBs (P < .0001 in all cases). For Slp4-a, corresponding WPB-Slp4-a-IR differences were not significant (P = .14). (B) Probabilities of WPB exocytosis, Pr, for ionomycin or histamine stimulation as indicated, plotted against the mean number of fluorescent Proregion-EGFP–containing WPBs, binned similarly to panel A. Data from 17-51 (ionomycin), 15-32 (100μM histamine), and 3-10 (1μM histamine) cells. (C) Quantification of Rab27A levels in HUVECs seeded at 1.0 × 105 cells per 3.5-cm dish and cultured in HUVEC growth medium for 24-120 hours. Growth media was replaced after 72 hours. At indicated times, the cells were counted, lysates made, and protein content determined (supplemental Figure 6B top). The intracellular content of Proregion (red), Rab27A (blue), α-tubulin (light gray), and actin (dark gray) at each time point was determined by immunoblotting, band intensities quantified in ImageJ, and corrected for cell number at corresponding time points. Cellular protein levels are presented as a percentage of those at 24 hours (data from 3 experiments, each performed in triplicate). (D) Changes in exocytosis of Proregion-EGFP–positive WPBs with time after nucleofection, same abscissa in subpanels i through iii. (Di) Increase in mean number of fluorescent WPBs per cell. (Dii) Mean percentage of Proregion-EGFP–positive WPBs displaying endogenous WPB-Slp4-a-IR (●) or WPB-MyRIP-IR (○) at the times indicated after nucleofection. (Diii) Probability of WPB exocytosis Pr for 100μM histamine stimulus (●). (○), Pr values scaled by the mean fraction of Slp4-a–positive WPBs (from Figure 6Dii, ∼ 7, 12, 24, and 48 hours). Sixteen to 29 cells were analyzed at each time point.$

The probability of WPB exocytosis is maintained over a wide range of WPB-Rab27A concentrations. (A) Mean intensities of WPB-IR for endogenous Rab27A (squares), MyRIP (triangles), and Slp4-a (circles) as a function of WPB numbers per cell. Data scaled to the mean intensity for the < 50 WPBs per cell. Numbers of cells analyzed: Rab27A, 124-218; MyRIP, 17-73; Slp4-a, 16-39. For all cells with > 50 WPBs, Rab27A and MyRIP intensities of WPB-IR were significantly lower than for cells with < 50 WPBs (P < .0001 in all cases). For Slp4-a, corresponding WPB-Slp4-a-IR differences were not significant (P = .14). (B) Probabilities of WPB exocytosis, P_r, for ionomycin or histamine stimulation as indicated, plotted against the mean number of fluorescent Proregion-EGFP–containing WPBs, binned similarly to panel A. Data from 17-51 (ionomycin), 15-32 (100μM histamine), and 3-10 (1μM histamine) cells. (C) Quantification of Rab27A levels in HUVECs seeded at 1.0 × 10⁵ cells per 3.5-cm dish and cultured in HUVEC growth medium for 24-120 hours. Growth media was replaced after 72 hours. At indicated times, the cells were counted, lysates made, and protein content determined (supplemental Figure 6B top). The intracellular content of Proregion (red), Rab27A (blue), α-tubulin (light gray), and actin (dark gray) at each time point was determined by immunoblotting, band intensities quantified in ImageJ, and corrected for cell number at corresponding time points. Cellular protein levels are presented as a percentage of those at 24 hours (data from 3 experiments, each performed in triplicate). (D) Changes in exocytosis of Proregion-EGFP–positive WPBs with time after nucleofection, same abscissa in subpanels i through iii. (Di) Increase in mean number of fluorescent WPBs per cell. (Dii) Mean percentage of Proregion-EGFP–positive WPBs displaying endogenous WPB-Slp4-a-IR (●) or WPB-MyRIP-IR (○) at the times indicated after nucleofection. (Diii) Probability of WPB exocytosis P_r for 100μM histamine stimulus (●). (○), P_r values scaled by the mean fraction of Slp4-a–positive WPBs (from Figure 6Dii, ∼ 7, 12, 24, and 48 hours). Sixteen to 29 cells were analyzed at each time point.

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