Rab5 is required for early events during autophagosome biogenesis.

<p>(A) Clones of fat body cell deprived of any Rab5 product were generated by heat shock-Flp/FRT mitotic recombination of the <i>Rab5</i><sup><i>2</i></sup>-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-, <i>Rab5</i>-/- 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. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s008" target="_blank">S8 Fig</a></b> for single channels. (B, B’) The <i>myc</i> labeling of tagged GFP:FYVE of surface or middle cell plans of <i>Rab5</i>-/- 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, <i>Rab5</i>-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 <i>Rab5</i> mutant cells (surface or middle plans respectively). Error bars are standard errors; significances are from Student’s <i>t</i>-tests. (C) The cell size of <i>Rab5</i>-null cells is increased in identified clone of fed animals compared to neighboring wild-type cells. Data from A were quantified as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.g003" target="_blank">Fig 3E</a></b> (Ctl n = 23; <i>Rab5</i>-/- n = 10). Error bars are mean differences; significance is from Student’s <i>t</i>-tests. (D-D’) Clones of <i>Rab5-CA</i>, <i>vap(RI)</i> or <i>Rab5-DN</i> cells were generated together with the <i>GFP</i>:<i>Atg8a</i> or GFP:FYVE markers expression, using the <i>Act>CD2>Gal4</i> flipout cassette method. Fed or 3h-starved fat cells of early/mid-3<sup>rd</sup> 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, <i>Rab5-CA</i> 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, <i>vap(RI)</i> clones leads to equivalent excess of actin-labeled PhAS formation (arrow). Upon starvation, <i>Rab5-CA</i> 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: <i>Rab5-CA</i>, Mdn = 0,931 μm<sup>2</sup> (n = 411); control, <i>w-</i> Mdn = 1,238 μm<sup>2</sup> (n = 158). Total amount of autophagy-membranes areas are thus doubling in the <i>Rab5-CA</i> expressing cells. Inhibition of Rab5 in <i>Rab5-DN</i> 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: <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; Rab5</i><sup><i>2</i></sup>, <i>FRT40A/ 2xUAS-EGFP</i>, <i>FRT40A</i>, <i>Fb-GAL4; UAS-GFP</i>:<i>myc</i>:<i>2xFYVE/+</i>. <i>(D)</i> Assay: <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; UAS-GFP</i>:<i>myc</i>:<i>Atg8a</i>, <i>Act>CD2>GAL4/ UAS-Rab5</i><sup><i>CA</i></sup>. <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; UAS-vap(RI-KK)/ +; Act>CD2>GAL4</i>, <i>UAS-GFP</i>:<i>myc</i>:<i>2xFYVE/+</i>. <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; UAS-GFP</i>:<i>Atg8a/</i>, <i>Act>CD2>GAL4/ UAS-Rab5</i><sup><i>DN</i></sup>.</p>