Loss of vap disturbs PI(3)P homeostasis in fed and starved cells.

(A) Cg-Gal4-driven expression of the GFP:FYVE biosensor was performed in control, w- and mutant, vap¹ fat cells, and images recorded from stage-matched early/ mid-3^rd instar live tissues form fed or 3h-starved animals. PI(3)P specific sub-pools comprising perinuclear early-endosomes (EE) and cytoplasmic-dispersed autophagy sites (AP sites), were delimited by red rings used for quantification following a setup described in [34] and S2C Fig, and graphed in details in S1B Fig. As noticed previously [34], starvation for 3h caused an elevation of the GFP:FYVE staining of both PI(3)P sub-pools, as found here in control, w- cells and in mutant, vap¹ cells. Scale bars = 20 μm. (A’) Relative changes of cytoplasmic vs perinuclear PI(3)P were scored here. The respective GFP+ areas in selected acquisitions encompassing 3–4 cells and 7–9 depth sections each (corrected for nuclei numbers), were plotted. Underlined numbers are the mean relative percentage of cytoplasmic PI(3)P fractions in indicated categories. Control, w- fed: 1.9% (+/- 0.37) and starved: 2.8% (+/- 0.40). Mutant vap¹ fed: 10.3% (+/- 3.9) and starved: 7.3% (+/- 1.2). The cytoplasmic PI(3)P fractions of vap¹ fed and starved cells is significantly increased compared to controls: Fed, vap¹ vs control, w- = 5 fold increase (p<0.0001). Starved, vap¹ vs control, w- = 3 fold increase (p<0.001). In controls, starvation produced low but significant elevation of the relative cytoplasmic PI(3)P fraction (2.8% vs 1,9%; p<0.002). No starvation mediated elevation or clear decline is observed in vap¹ cells (7,3% vs 10,3%; p>0.05). Note the dispersion of values in mutants as often the case. Significances are from Student’s t-tests. (B) Clonal expression of an UAS-vap wild-type transgene in fed larval fat body cells in the presence of the GFP:FYVE biosensor was induced using the Act>CD2>Gal4 flipout cassette method. When compared to control, w- clones, clonal excess of Vap produced a complete absence of perinuclear GFP with rare remaining PI(3)P spots at the cell periphery. Scale bars = 20 μm. (C) Clonal expression of an UAS-vap(wt) transgene in the presence of the autophagosome marker GFP:Atg8a in larval fat cells was obtained as above, but larvae were starved for 3h and cells were additionally stained with LysoTracker red. Clones with excess Vap (inset and delimited by white lines) shows fewer or not any stained lysosomes (Top and bottom right panels respectively) compared to the wild-type neighboring cells, whereas GFP:Atg8a-labeled autophagomes is reduced to tiny GFP:Atg8a-positive structures (green arrow) as compared to starvation-induced autolysosomes (AL, yellow arrow) in control, w- clones. Scale bars = 20 μm. Genotypes. (A) Control: w¹¹¹⁸/Y; cg-GAL4/ UAS-GFP:myc:2xFYVE. Assay: vap¹/Y; cg-GAL4/ UAS-GFP:myc:2xFYVE. (B) Control: w¹¹¹⁸/ hsFLP¹²; UAS-GFP:myc:2xFYVE, Act>CD2>GAL4/+. Assay: w¹¹¹⁸/ hsFLP¹²; UAS-Vap:myc^16.4/+; UAS-GFP:myc:2xFYVE, Act>CD2>GAL4/+. (C) Control: w¹¹¹⁸/ hsFLP¹²; Act>CD2>GAL4, UAS-GFP:Atg8a/+. Assay: w¹¹¹⁸/ hsFLP¹²; UAS-Vap:myc^16.4/+; Act>CD2>GAL4, UAS-GFP:Atg8a/+.