Figure 9.
Acute loss of V-ATPase function rapidly collapses apical Rab8 tubules into vacuoles that fuse with apical carriers.(A) p75-GFP was imaged in mRFP-Rab8a expressing larvae after a 1-h TGN release in the presence of 750 nM bafilomycin or DMSO as a control. In controls, p75-GFP overlaps with mRFP-Rab8a in post-Golgi tubules (flat arrows) and apical tubules (concave arrows). Upon loss of V-ATPase function, mRFP-Rab8a condenses into vacuoles (asterisks) surrounded by p75-GFP (arrowheads). Scale bars are 10 µm and 2 µm in magnified insets. (B) Colocalization analysis of p75-GFP and mRFP-Rab8a from ROIs of the apical cytoplasm from ~5 µm z-series shown as a Gardner-Altman plot of Pearson’s coefficient (R). Left, mean ± SD is plotted. Right, mean difference (Baf-DMSO) plotted on a floating axis as a bootstrap sampling distribution with the mean difference depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. n ≥ 10 ROIs from at least two animals. P = 9.6e-6, Mann-Whitney test. (C) Optical section taken from a 5-µm z-series of a group of mRFP-Rab8a vacuoles (asterisks) and p75-GFP tubules (arrowheads), some of which appear to be fusing with vacuoles (arrow), after a 1-h TGN release in the presence of 750 nM bafilomycin. Scale bar is 1 µm. (D) 3D reconstruction of the z-series from C. Highlighted structures correspond approximately to those in E. (E) Optical section taken from a 7-µm z-series of an mRFP-Rab8a vacuole (asterisk) and p75-GFP (arrowheads) after an 8-h TGN release in the presence of 500 nM bafilomycin. Scale bar is 1 µm. (F) 3D reconstruction of the z-series from E. Highlighted structures correspond approximately to those in E. (G) mRFP-Rab8a expressing larvae were treated as in E and stained for endogenous Rab11. In bafilomycin-treated larvae, Rab11 (arrows) remains enriched subapically and does not localize to mRFP-Rab8a vacuoles (arrowheads). Scale bars are 5 µm and 2 µm in magnified insets. n ≥ 10 larvae. (H–J) Loss of V-ATPase activity acutely impairs Rab8 dynamics along tubular carriers. (H) Experimental strategy. 5-dpf larvae expressing mRFP-Rab8a were treated with either 1 µM bafilomycin or DMSO for 45 min before live imaging. (I) Overlay of dynamic mRFP-Rab8a tracks (multicolored lines) superimposed on still frames from live imaging videos. Scale bars are 5 µm. (J) Magnified insets of still frames from videos represented in I. Arrowheads mark Rab8 moving along tubules in controls, and arrows show Rab8 vacuoles deficient in tubulation during bafilomycin treatment. Scale bars are 1 µm. See also Video 2, Video 3, and Video 4. n ≥ 6 larvae. Baf, bafilomycin.

Acute loss of V-ATPase function rapidly collapses apical Rab8 tubules into vacuoles that fuse with apical carriers. (A) p75-GFP was imaged in mRFP-Rab8a expressing larvae after a 1-h TGN release in the presence of 750 nM bafilomycin or DMSO as a control. In controls, p75-GFP overlaps with mRFP-Rab8a in post-Golgi tubules (flat arrows) and apical tubules (concave arrows). Upon loss of V-ATPase function, mRFP-Rab8a condenses into vacuoles (asterisks) surrounded by p75-GFP (arrowheads). Scale bars are 10 µm and 2 µm in magnified insets. (B) Colocalization analysis of p75-GFP and mRFP-Rab8a from ROIs of the apical cytoplasm from ~5 µm z-series shown as a Gardner-Altman plot of Pearson’s coefficient (R). Left, mean ± SD is plotted. Right, mean difference (Baf-DMSO) plotted on a floating axis as a bootstrap sampling distribution with the mean difference depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. n ≥ 10 ROIs from at least two animals. P = 9.6e-6, Mann-Whitney test. (C) Optical section taken from a 5-µm z-series of a group of mRFP-Rab8a vacuoles (asterisks) and p75-GFP tubules (arrowheads), some of which appear to be fusing with vacuoles (arrow), after a 1-h TGN release in the presence of 750 nM bafilomycin. Scale bar is 1 µm. (D) 3D reconstruction of the z-series from C. Highlighted structures correspond approximately to those in E. (E) Optical section taken from a 7-µm z-series of an mRFP-Rab8a vacuole (asterisk) and p75-GFP (arrowheads) after an 8-h TGN release in the presence of 500 nM bafilomycin. Scale bar is 1 µm. (F) 3D reconstruction of the z-series from E. Highlighted structures correspond approximately to those in E. (G) mRFP-Rab8a expressing larvae were treated as in E and stained for endogenous Rab11. In bafilomycin-treated larvae, Rab11 (arrows) remains enriched subapically and does not localize to mRFP-Rab8a vacuoles (arrowheads). Scale bars are 5 µm and 2 µm in magnified insets. n ≥ 10 larvae. (H–J) Loss of V-ATPase activity acutely impairs Rab8 dynamics along tubular carriers. (H) Experimental strategy. 5-dpf larvae expressing mRFP-Rab8a were treated with either 1 µM bafilomycin or DMSO for 45 min before live imaging. (I) Overlay of dynamic mRFP-Rab8a tracks (multicolored lines) superimposed on still frames from live imaging videos. Scale bars are 5 µm. (J) Magnified insets of still frames from videos represented in I. Arrowheads mark Rab8 moving along tubules in controls, and arrows show Rab8 vacuoles deficient in tubulation during bafilomycin treatment. Scale bars are 1 µm. See also Video 2, Video 3, and Video 4. n ≥ 6 larvae. Baf, bafilomycin.

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