Rab5 is required for early events during autophagosome biogenesis.

(A) Clones of fat body cell deprived of any Rab5 product were generated by heat shock-Flp/FRT mitotic recombination of the Rab52-null, deletion allele, in fed or 2h-starved animals in presence of myc-tagged PI(3)P biosensor (GFP:myc:FYVE) expressed in every fat-body cells. Anti-myc immunostaining (in green) was used to detect the biosensor in fixed tissue. Inset: identified GFP-, Rab5-/- clones. In fed cells, both perinuclear and dispersed cytoplasmic PI(3)P is absent from mutant cells (arrows pointing two plans of same clones). Little but detectable PI(3)P labeling persists in the Rab5-/- cells of 2h-starved animals. Scale bars = 20 μm. S8 Fig for single channels. (B, B’) The myc labeling of tagged GFP:FYVE of surface or middle cell plans of Rab5-/- clones was quantified relative to neighboring control cells in both fed and starved cells samples of A (fed GFP- n = 3, fed GFP+ n = 6; sta GFP- n = 3, sta GFP+ n = 6). Fed, Rab5-null cells has negligible amount of PI(3)P. 2h-starved cell shows 1/3 to 1/8 of residual PI(3)P labeling in the Rab5 mutant cells (surface or middle plans respectively). Error bars are standard errors; significances are from Student’s t-tests. (C) The cell size of Rab5-null cells is increased in identified clone of fed animals compared to neighboring wild-type cells. Data from A were quantified as in Fig 3E (Ctl n = 23; Rab5-/- n = 10). Error bars are mean differences; significance is from Student’s t-tests. (D-D’) Clones of Rab5-CA, vap(RI) or Rab5-DN cells were generated together with the GFP:Atg8a or GFP:FYVE markers expression, using the Act>CD2>Gal4 flipout cassette method. Fed or 3h-starved fat cells of early/mid-3rd instar were analyzed in fixed tissues. Fluorescent GFP and phalloïdin are shown in panel D, and phalloïdin-labeled actin structures are shown panel D’. In fed animals, Rab5-CA clones induces excessive actin-labeled phagophore network (or PhAS, white arrow in D’). These overlapped with GFP:Atg8a-labeled isolation membranes (yellow arrows in D or green arrows in inset). In the same conditions, vap(RI) clones leads to equivalent excess of actin-labeled PhAS formation (arrow). Upon starvation, Rab5-CA clones formed plenty of induced-autophagosomes. Those were abnormal in shape (inset: enlarged picture) and were not overlapping with the large PhAS network (arrow in D’). Analysis of the Rab5-CA-induced green-vesicle signal (Materials and Methods) revealed that mutant cells has an 2.8 time higher density in autophagosomes compared to wild-type controls; median size: Rab5-CA, Mdn = 0,931 μm2 (n = 411); control, w- Mdn = 1,238 μm2 (n = 158). Total amount of autophagy-membranes areas are thus doubling in the Rab5-CA expressing cells. Inhibition of Rab5 in Rab5-DN expressing clones prevents the formation of starvation-induced autophagosomes and that of endogenous actin-labeled PhAS (arrow in D’). Scale bars in all panels = 20 μm. Genotypes. (A) Assay: w1118/ hsFLP12; Rab52, FRT40A/ 2xUAS-EGFP, FRT40A, Fb-GAL4; UAS-GFP:myc:2xFYVE/+. (D) Assay: w1118/ hsFLP12; UAS-GFP:myc:Atg8a, Act>CD2>GAL4/ UAS-Rab5CA. w1118/ hsFLP12; UAS-vap(RI-KK)/ +; Act>CD2>GAL4, UAS-GFP:myc:2xFYVE/+. w1118/ hsFLP12; UAS-GFP:Atg8a/, Act>CD2>GAL4/ UAS-Rab5DN.