10.1371/journal.pone.0209759.g006
Marc Bourouis
Marc
Bourouis
Magali Mondin
Magali
Mondin
Aurore Dussert
Aurore
Dussert
Pierre Leopold
Pierre
Leopold
Sprint appears to be dispensable for starvation-induced autophagy.
Public Library of Science
2019
basal autophagy rate
GEF
endocytic Rab 5-containing vesicles
PI
pre-autophagosome structures formation
cell surface-derived flow
vacuolar peduncle Basal autophagy
basal autophagy
cell energy balance
Drosophila vacuolar peduncle
GAP
phenotype
vap adult brains
Rab 5 activity
2019-02-08 21:08:24
Figure
https://plos.figshare.com/articles/figure/Sprint_appears_to_be_dispensable_for_starvation-induced_autophagy_/7698020
<p>(A) Clones of <i>spri(RI)</i> fat cell or control, <i>w-</i> were generated together with GFP:Atg8a autophagosome marker expression, using the <i>Act>CD2>Gal4</i> flipout cassette method. Larvae were starved for 3h, and fat body stained with LysoTracker red when needed. <i>spri</i> mutant cells shows autophagosomes of reduced size compared to that of control, <i>w-</i> cells. (B) <i>spri(RI)</i> 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, <i>w-</i>whereas mutant <i>spri(RI)</i> forms small but fused autolysosomes (arrows) and more sporadically, none-fused but tethered autophagosome and lysosome vesicles (arrows and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s009" target="_blank">S1 Table</a></b>). (D) <i>spri(RI)</i> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.ref053" target="_blank">53</a>]), or else from ineffective maturating fusion of autophagosome to the MVB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.ref078" target="_blank">78</a>]. (E) Clones of <i>spri(RI)</i> fat cell or control, <i>w-</i> were generated together with the GFP:FYVE biosensor, using the <i>Act>CD2>Gal4</i> flipout cassette method. Fed or 3h-starved early/mid-3<sup>rd</sup> instar larvae were analyzed in fixed tissues. Fed <i>spri</i>-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 <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s002" target="_blank">S2C Fig</a></b>). Free GFP:FYVE fluorescent probe remains in the cytosol and nucleus as in the case of <i>UAS-vap</i> wild-type transgene expression (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.g002" target="_blank">Fig 2B</a></b>). Upon starvation, <i>spri</i>-silenced cells partly recovered from these defects, including the formation of new perinuclear PI(3)P pools. The phenotype is subjected to variation (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s009" target="_blank">S1 Table</a>).</b> A detailed quantification is found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s005" target="_blank">S5B Fig</a></b>. (F, F’) Clonal <i>spri(RI</i>) cells developed in normally fed animal has mild increased size. Cell size was quantified relative to neighboring control cells as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.g003" target="_blank">Fig 3</a>.</b> (Clt n = 24; <i>spri-</i> n = 21). Error bars are mean differences; significance is from Student’s <i>t</i>-tests. Scale bars in all panels = 20 μm. Genotypes. (A-D, F, F’) Control: <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; +/+; Act>CD2>GAL4</i>, <i>UAS-GFP</i>:<i>Atg8a/+</i>. Assay: <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; UAS-spri(RI)/ +; Act>CD2>GAL4</i>, <i>UAS-GFP</i>:<i>Atg8a/+</i>. (E) Control: <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; UAS-GFP</i>:<i>myc</i>:<i>2xFYVE</i>, <i>Act>CD2>GAL4/+</i>. Assay: <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; UAS-spri(RI)/ +; UAS-GFP</i>:<i>myc</i>:<i>2xFYVE</i>, <i>Act>CD2>GAL4/+</i>.</p>