Fat cell clones of manipulated vap activity showed growth competitive phenotypes.
(A) Clonal loss of vap in fat bodies of well-fed animals was generated by the MARM, GFP-positive labeling technique and analyzed in mid-3rd larvae. Clonal vap1 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 vap1 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 vap1 and tubulin-Gal4 driven Atg1(RI) 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 Atg1 . Atg1(RI) expression alone in these conditions has not detectable effects (E). See validation of experimental setting and used Atg1(RI) construct in S3A Fig. (D) Clonal expression of an UAS-vap(wt) transgene in larval fed fat body cells was achieved using the Act>CD2>Gal4 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 /vap1 n = 13; Ctl n = 14 /vap1, Atg1(RI) n = 10; Ctl n = 8 /Atg1(RI) n = 8; Ctl n = 10 /UAS-vap n = 12). Error bars are mean differences; significances are from Student’s t-tests Genotypes. (A-B”) vap1, FRT19A / tub-GAL80, hsFLP1, FRT19A; UAS-CD8:GFP/+; tub-GAL4/+. (C, E) Control: FRT19A / tub-GAL80, hsFLP1, FRT19A; UAS-CD8:GFP/+; tub-GAL4/ UAS-Atg1(RI). Assay: vap1, FRT19A / tub-GAL80, hsFLP1, FRT19A; UAS-CD8:GFP/+; tub-GAL4/ UAS-Atg1(RI). (D) Assay: w1118/ hsFLP12; UAS-Vap:myc16.4/+; Act>CD2>GAL4, UAS-GFP/ +.