Sprint appears to be dispensable for starvation-induced autophagy.
(A) Clones of spri(RI) fat cell or control, w- were generated together with GFP:Atg8a autophagosome marker expression, using the Act>CD2>Gal4 flipout cassette method. Larvae were starved for 3h, and fat body stained with LysoTracker red when needed. spri mutant cells shows autophagosomes of reduced size compared to that of control, w- cells. (B) spri(RI) clonal cells (delimited by a white line) shows small-sized or tiny lysosomes as stained with LysoTracker red. (C) A close view of autophagy vesicles shows fused ‘yellow’ autolysosomes (arrows) in control, w-whereas mutant spri(RI) forms small but fused autolysosomes (arrows) and more sporadically, none-fused but tethered autophagosome and lysosome vesicles (arrows and S1 Table). (D) spri(RI) clonal cells were stained for the ESCRT-0 early endosome marker Hrs, showing perinuclear accumulation of these structures (arrowhead) as compared to control nearby cells (arrow). This either resulted from blocked progression into endosomal MVB (as intraluminal vesicles formation of MVB requires PI(3)P for scission from the surface ), or else from ineffective maturating fusion of autophagosome to the MVB . (E) Clones of spri(RI) fat cell or control, w- were generated together with the GFP:FYVE biosensor, using the Act>CD2>Gal4 flipout cassette method. Fed or 3h-starved early/mid-3rd instar larvae were analyzed in fixed tissues. Fed spri-silenced cells form fine PI(3)P foci at the periphery and the perinuclear pools of PI(3)P is reduced compared to control (red circles for delimitation of the pools, see S2C Fig). Free GFP:FYVE fluorescent probe remains in the cytosol and nucleus as in the case of UAS-vap wild-type transgene expression (Fig 2B). Upon starvation, spri-silenced cells partly recovered from these defects, including the formation of new perinuclear PI(3)P pools. The phenotype is subjected to variation (S1 Table). A detailed quantification is found in S5B Fig. (F, F’) Clonal spri(RI) cells developed in normally fed animal has mild increased size. Cell size was quantified relative to neighboring control cells as in Fig 3. (Clt n = 24; spri- n = 21). Error bars are mean differences; significance is from Student’s t-tests. Scale bars in all panels = 20 μm. Genotypes. (A-D, F, F’) Control: w1118/ hsFLP12; +/+; Act>CD2>GAL4, UAS-GFP:Atg8a/+. Assay: w1118/ hsFLP12; UAS-spri(RI)/ +; Act>CD2>GAL4, UAS-GFP:Atg8a/+. (E) Control: w1118/ hsFLP12; UAS-GFP:myc:2xFYVE, Act>CD2>GAL4/+. Assay: w1118/ hsFLP12; UAS-spri(RI)/ +; UAS-GFP:myc:2xFYVE, Act>CD2>GAL4/+.