Figure 5. AP-3 RNAi dysregulates the... | Rockefeller University Press

Figure 5.

$Figure 5. AP-3 RNAi dysregulates the exocytosis of VMAT2. PC12 cells were transfected with 50 nM AP-3δ or control siRNA, cotransfected 2 d later with the same siRNA and VMAT2 containing a lumenal pHluorin (VMAT2-pHluorin), then imaged live by TIRF microscopy after an additional 2 d. Basal exocytosis of VMAT2-pHluorin was measured in Tyrode’s solution containing 2.5 mM K+, and release was stimulated in Tyrode’s with 90 mM K+ for 60 s. (A) Representative images acquired before and after depolarization show increased baseline fluorescence and fewer stimulated events (arrowheads) after transfection with AP-3 siRNA (Videos 1 and 2). (B) Total VMAT2 fluorescence was revealed by alkalinization in Tyrode’s solution containing 50 mM NH4Cl, pH 7.4 (top), and surface VMAT2-pHluorin revealed by acidification in Tyrode’s with 25 mM MES, pH 6.5 (bottom). AP-3δ RNAi increases the surface fraction of VMAT2 in cells despite expression equivalent to transfection with control siRNA. *, P < 0.05 relative to control by two-tailed Student’s t test (n = 12 for control and 14 for AP-3 siRNA). (C) Whole cell fluorescence expressed as a percentage of total fluorescence (revealed in NH4Cl) shows a robust response to depolarization with 90 mM K+ (arrow) in control cells but a greatly impaired response after AP-3 siRNA. The traces indicate the mean values of 12 individual traces for control and 14 for AP-3 RNAi cells. (D) The quantification of individual exocytotic events shows increased basal VMAT2-pHluorin exocytosis with AP-3 RNAi but a reduction in stimulated exocytosis. *, P < 0.02; **, P < 0.002 (relative to control by two-tailed Student’s t test; control, n = 31; AP-3, n = 19). Error bars indicate SEM. Bars, 5 µm.$

AP-3 RNAi dysregulates the exocytosis of VMAT2. PC12 cells were transfected with 50 nM AP-3δ or control siRNA, cotransfected 2 d later with the same siRNA and VMAT2 containing a lumenal pHluorin (VMAT2-pHluorin), then imaged live by TIRF microscopy after an additional 2 d. Basal exocytosis of VMAT2-pHluorin was measured in Tyrode’s solution containing 2.5 mM K⁺, and release was stimulated in Tyrode’s with 90 mM K⁺ for 60 s. (A) Representative images acquired before and after depolarization show increased baseline fluorescence and fewer stimulated events (arrowheads) after transfection with AP-3 siRNA (Videos 1 and 2). (B) Total VMAT2 fluorescence was revealed by alkalinization in Tyrode’s solution containing 50 mM NH₄Cl, pH 7.4 (top), and surface VMAT2-pHluorin revealed by acidification in Tyrode’s with 25 mM MES, pH 6.5 (bottom). AP-3δ RNAi increases the surface fraction of VMAT2 in cells despite expression equivalent to transfection with control siRNA. *, P < 0.05 relative to control by two-tailed Student’s t test (n = 12 for control and 14 for AP-3 siRNA). (C) Whole cell fluorescence expressed as a percentage of total fluorescence (revealed in NH₄Cl) shows a robust response to depolarization with 90 mM K⁺ (arrow) in control cells but a greatly impaired response after AP-3 siRNA. The traces indicate the mean values of 12 individual traces for control and 14 for AP-3 RNAi cells. (D) The quantification of individual exocytotic events shows increased basal VMAT2-pHluorin exocytosis with AP-3 RNAi but a reduction in stimulated exocytosis. *, P < 0.02; **, P < 0.002 (relative to control by two-tailed Student’s t test; control, n = 31; AP-3, n = 19). Error bars indicate SEM. Bars, 5 µm.