Fat cell clones of manipulated <i>vap</i> activity showed growth competitive phenotypes.

<p>(A) Clonal loss of <i>vap</i> in fat bodies of well-fed animals was generated by the MARM, GFP-positive labeling technique and analyzed in mid-3<sup>rd</sup> larvae. Clonal <i>vap</i><sup><i>1</i></sup> cells (inset and arrows) grown for ca. 88h, shows autonomous cell size reduction compared to wild-type neighboring cells used as controls (Ctl). (B) An extreme case of disfavored growth of a <i>vap</i><sup><i>1</i></sup> mutant clone in the process of active elimination (size diminished by 60% and corresponding reduction of nucleus size). (B’) Mutant cell lost part of the phalloïdin-labeled actin cytoskeleton at a cellular contact (arrowhead). (B‘‘) A similar clone in the process of nuclear fragmentation is extruded from the tissue. (C) Doubly mutant clones of <i>vap</i><sup><i>1</i></sup> and <i>tubulin-Gal4</i> driven <i>Atg1(RI)</i> cells, were generated in fed animals and analyzed as in A (inset: a distinct clone). If anything, these shows enhanced cell size reduction rather than suppression of cell growth. Such a synergism could be related to the autophagy-independent requirement of Drosophila <i>Atg1</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.ref043" target="_blank">43</a>]. <i>Atg1(RI)</i> expression alone in these conditions has not detectable effects (E). See validation of experimental setting and used <i>Atg1(RI)</i> construct in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s003" target="_blank">S3A Fig</a></b>. (D) Clonal expression of an <i>UAS-vap(wt)</i> transgene in larval fed fat body cells was achieved using the <i>Act>CD2>Gal4</i> flipout cassette method. A mild and autonomous increase in cell size is observed. Scale bars in all panels = 20 μm. (E) Relative cell-size changes of manipulated cell clones in A-D were quantified. Lengths of the cell contours (in μm) were determined from images of the clones and compared to wild-type cells contours in the same images. MARCM and flipout genetic setting resulted in normal sized GFP-positive cells. (Clt n = 23 /<i>vap</i><sup><i>1</i></sup> n = 13; Ctl n = 14 /<i>vap</i><sup><i>1</i></sup>, <i>Atg1(RI</i>) n = 10; Ctl n = 8 /<i>Atg1(RI)</i> n = 8; Ctl n = 10 /<i>UAS-vap</i> n = 12). Error bars are mean differences; significances are from Student’s <i>t</i>-tests Genotypes. (A-B”) <i>vap</i><sup><i>1</i></sup>, <i>FRT19A / tub-GAL80</i>, <i>hsFLP1</i>, <i>FRT19A; UAS-CD8</i>:<i>GFP/+; tub-GAL4/+</i>. (C, E) Control: <i>FRT19A / tub-GAL80</i>, <i>hsFLP1</i>, <i>FRT19A; UAS-CD8</i>:<i>GFP/+; tub-GAL4/ UAS-Atg1(RI)</i>. Assay: <i>vap</i><sup><i>1</i></sup>, <i>FRT19A / tub-GAL80</i>, <i>hsFLP1</i>, <i>FRT19A; UAS-CD8</i>:<i>GFP/+; tub-GAL4/ UAS-Atg1(RI)</i>. (D) Assay: <i>w</i><sup><i>1118</i></sup><i>/ hsFLP</i><sup><i>12</i></sup><i>; UAS-Vap</i>:<i>myc</i><sup><i>16</i>.<i>4</i></sup><i>/+; Act>CD2>GAL4</i>, <i>UAS-GFP/ +</i>.</p>