Examination of K78 mono-ubiquitylation with respect to Dronc’s catalytic activity.

<p>(<b>A</b>) <i>da>Flag-Dronc</i>^<i>wt</i>, <i>da>Flag-Dronc</i>^<i>K78R</i> and <i>da>Flag-Dronc</i>^{<i>K78RC318A</i>} can rescue the lethality of <i>dronc</i>^<i>I29</i> null mutants, whereas <i>da>Flag-Dronc</i>^<i>C318A</i> cannot. (<b>B</b>) Quantification of the number of additional interommatidial cells (IOC) shown in (C-G). Genotypes are indicated. MARCM was used to express transgenic <i>Flag-Dronc</i> constructs in <i>dronc</i>^<i>I29</i> mutant cell clones. n = 10 for <i>dronc</i>^<i>I29</i> MARCM clones, n = 11 for <i>Flag-Dronc</i>^<i>wt</i> in <i>dronc</i>^<i>I29</i> clones, n = 7 for <i>Flag-Dronc</i>^<i>K78R</i> in <i>dronc</i>^<i>I29</i> clones, n = 11 for <i>Flag-Dronc</i>^{<i>K78RC318A</i>} in <i>dronc</i>^<i>I29</i> clones, n = 8 for <i>Flag-Dronc</i>^<i>C318A</i> in <i>dronc</i>^<i>I29</i> clones. Each n corresponds to an average of extra IOC of 3 clones. ns—not significant. (<b>C-G</b>) Pupal retinae 48h after puparium formation expressing the indicated <i>Flag-Dronc</i> constructs in <i>dronc</i>^<i>I29</i> MARCM clones. Clones are marked by GFP and are enclosed by white dashes in the right panels. Examples of extra IOC are marked with yellow arrows. <i>Flag</i>-<i>Dronc</i>^<i>wt</i> and <i>Flag</i>-<i>Dronc</i>^<i>K78R</i> rescue the IOC phenotype of <i>dronc</i> null mutants. However, <i>Flag</i>-<i>Dronc</i>^{<i>K78RC318A</i>} and <i>Flag</i>-<i>Dronc</i>^<i>C318A</i> fail to rescue this phenotype. Quantified in (B). <b>(H)</b> Immunoblotting of lysates of each Flag-Dronc construct in the <i>dronc</i>^<i>I24</i><i>/dronc</i>^<i>I29</i> background shows similar expression levels. For quantifications, the student’s t-test was used. Error bars are SD. * P<0.05; ** P<0.01; *** P<0,001; **** P<0.0001. ns—not significant.</p

Biochemical characterization of Dronc^K78R.

<p>(<b>A</b>) Bacterially expressed 6xHis-Dronc^wt and 6xHis-Dronc^K78R constructs display similar auto-processing activities. 6xHis-Dronc^C318A and 6xHis-Dronc^K78RC318A do not show any auto-processing. (<b>B</b>) <i>In vitro</i> caspase cleavage assays show that 6xHis-Dronc^wt and 6xHis-Dronc^K78R cleave Myc-Drice^C211A with similar activities. 6xHis-Dronc^C318A and 6xHis-Dronc^K78RC318A cannot cleave Myc-Drice^C211A. <b>(C,C’)</b> 3^rd instar lysates of <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>wt</i> and <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>K78R</i> show that in the presence of Dark, Flag-Dronc^K78R is processed significantly more than Flag-Dronc^wt. In (C’), the average of 4 immunoblots is plotted. (<b>D</b>) GFP-Dark interacts with Flag-Dronc^wt and Flag-Dronc^K78R. GFP-immunoprecipitates of 3^rd instar larval extracts from <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>wt</i>, <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>K78R</i> and <i>da</i>><i>GFP-Dark</i>+<i>EV</i> (<i>Flag-Empty Vector</i>) animals, probed with anti-GFP antibody (upper panel) and anti-Flag antibody (lower panel). There is a stronger interaction between GFP-Dark and Flag-Dronc^K78R, resulting in significantly more efficient procession of Flag-Dronc^K78R compared to Flag-Dronc^wt. (<b>D’</b>) Relative ratio of processed and unprocessed Flag-Dronc proteins in the Dark apoptosome. Flag-Dronc^K78R is more efficiently processed than Flag-Dronc^wt. The average of 3 immunoblots is plotted. For quantifications, the student’s t-test was used. Error bars are SD. * P<0.05.</p

Additional file 7: Figure S7. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

Functional tests of the RNAi lines targeting dAtg1, dAg3, dAtg8a, and dAtg8b. (A–E) Starvation assay of fat bodies from third instar larvae. Formation of autophagosomes was visualized by mCherry-Atg8 (red in A–E; grey in A’–E’). Cells expressing RNAi constructs are labeled by GFP and outlined by yellow dotted lines. (A) Wildtype fat body displaying mCherry-Atg8 puncta both in clone cells and surrounding cells. (B–E) Cells expressing dAtg1, dAtg3, dAtg8a, and dAtg8b RNAi (GFP +) fail to form mCherry-Atg8 marked autophagosomes. The loss of mCherry-Atg8 signals by dAtg8a and dAtg8b RNAi in (D) and (E) also demonstrates that these RNAi lines target mCherry-Atg8 transcripts. (F–J) Adult eyes expressing eyeful and indicated RNAi transgenes. As previously reported [77], loss of autophagy strongly enhances the eyeful phenotype. The functionality of dAtg1, dAtg3, dAtg8a, and dAtg8b RNAi transgenes is confirmed by enhancement of the eyeful phenotype. (TIF 8420 kb

Additional file 3: Figure S3. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

Loss of dAtg1 does not suppress apoptosis. (A–B’) Mosaic late third instar wing discs with hid-p35-expressing clones positively marked by GFP. Simultaneous expression of hid and p35 in clones induces strong cCasp3 labeling (A, A’, arrows). Similar cCasp3 labeling persists in dAtg1 mutant clones (B, B’, arrows). (C) Quantification of cCasp3 labeling intensity in hid-p35-expressing clones and hid-p35-expressing dAtg1 mutant clones (mean ± SE). No significant difference of cCasp3 labeling was observed. (D–D’) A representative late third instar GMR-hid eye disc with dAtg1 mutant clones negatively marked by GFP (highlighted by yellow dotted lines). The wave of apoptosis (arrow) induced by GMR-hid persists in dAtg1 mutant clones. (E, F) Representative adult eyes of the indicated genotypes. GMR-hid-induced eye ablation phenotype (E) is not altered by RNAi knockdown of dAtg1 (F). (TIF 6416 kb

Imaginal discs predominantly mutant for <i>vps22</i>, <i>vps25</i>, or <i>vps36</i> are apoptotic.

<p>Shown are predominantly mutant eye-antennal imaginal discs. Phalloidin (green) or DAPI (green) is used to mark the overall shape of the tissue. Scale bars represent 50 µm. (<b>A–D</b>) Cleaved Caspase-3 (Cas-3*; red and grayscale) labelings show that apoptosis is increased in discs predominantly mutant for <i>vps22</i> (B), <i>vps25</i> (C), or <i>vps36</i> (D), as compared to apoptosis in control discs (A,A’). (<b>E–H</b>) TUNEL (red and grayscale) labelings show that apoptosis is increased in discs predominantly mutant for <i>vps22</i> (F), <i>vps25</i> (G), or <i>vps36</i> (H), as compared to apoptosis in control discs (E,E’). <b>Genotypes</b>: (<b>A</b>) <i>eyFLP; FRT42D y⁺/FRT42D cl.</i> (<b>B,F</b>) <i>eyFLP;; FRT82B vps22^5F3-8/FRT82B cl.</i> (<b>C,G</b>) <i>eyFLP; FRT42D vps25^N55/FRT42D cl.</i> (<b>D,H</b>) <i>eyFLP;; vps36^Δ69 FRT80B/cl FRT80B.</i> (<b>E</b>) <i>eyFLP;; FRT82B/FRT82B cl.</i></p

Additional file 4: Figure S4. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

Expression of dAtg1 is not sufficient to induce growth signals for AiP. Late third instar eye discs labeled with wg-lacZ (red in B, C and grey in A, B’, C’), dpp-lacZ (red in E, F and grey in D, E’,F’) or kekkon-lacZ (kek-lacZ, red in H, I and grey in G, H’, I’). Anterior is to the left. DE-Gal4 tub-Gal80 ts (DE ts ) was used to control expression of UAS-transgenes at 29 °C for 12 h in the dorsal portion of eye discs, followed by 24 h of recovery at 18 °C (TS12hR24h). Compared to control discs (A, D, G), temporal expression of hid leads to apoptosis, indicated by the cCasp3 labeling (green in B), and ectopic induction of wg-lacZ (B’, arrow), dpp-lacZ (E’, arrow) and kek-lacZ (H’, arrow) which are markers of the growth signaling pathways mediating AiP. In contrast, expression of dAtg1 under the same conditions (TS12hR24h) does not activate ectopic wg, dpp or kek (compare C’, F’, I’ to B’, E’, H’) although a low level of apoptosis is induced (cCasp3-labeling, green in C). (TIF 7173 kb

Additional file 5: Figure S5. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

Specificity of in situ probes to detect dAtg1 transcripts. In situ hybridization of late third instar larval eye discs with DIG-labeled probes detected with Tyramide Signal Amplification. (A) Endogenous dAtg1 is expressed at low level in wildtype eye discs. (B, C) Labeling of GMR > dAtg1 discs using sense probes (B) and anti-sense dAtg1 probes (C). The dAtg1 antisense probes recognize high levels of dAtg1 transcripts driven by GMR-Gal4 (C, the GMR domain expressing dAtg1 is highlighted). (TIF 1140 kb

Additional file 2: Figure S2. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

Expression of dAtg1 enhances caspase activity and apoptosis. Late third instar larval eye discs labeled with the cleaved Caspase-3 antibodies (cCasp3, green in A, B, grey in A’, B’, blue in C, and grey in C’), anterior is to the left. (A–B’) Compared to ey > hid-p35 discs (A, A’), cCasp3 labeling indicating activity of Dronc is not affected by expression of dAtg1 which enhances ey > hid-p35-induced overgrowth phenotype (B, B’). (C–C”’) Expression of dAtg1 under control of DE-Gal4 and tub-Gal80ts (DEts) and indicated by GFP. Expression of dAtg1 by a temperature shift (ts) to 29 °C for 48 h induces apoptosis as indicated by cCasp3 labeling (C’, arrow) and developmental defects in the eye disc indicated by the affected pattern of ELAV labeling (C”, arrow). (TIF 4775 kb

Notch, JAK/STAT, and JNK signaling are upregulated in <i>vps22</i>, <i>vps25</i>, and <i>vps36</i> mutant tissues.

<p>Shown are predominantly mutant eye-antennal imaginal discs. Phalloidin (green) is used to mark the overall shape of the tissue. <i>Gbe-Su(H)-lacZ</i>, and <i>E(spl)m8 2.61-lacZ</i> are detected by β-gal labeling (red or grayscale). Scale bars represent 50 µm. (<b>A,E</b>) Imaginal discs predominantly mutant for <i>vps25</i> induce high levels of <i>Gbe-Su(H)-lacZ</i> (E), as compared to control discs (A,A’). (<b>B,C,E</b>) Imaginal discs predominantly mutant for <i>vps22</i> (C) or <i>vps36</i> (E) induce high levels of <i>E(spl)m8 2.61-lacZ</i>, as compared to control discs (B,B’). (F–I) Imaginal discs predominantly mutant for <i>vps22</i> (G), <i>vps25</i> (H), or <i>vps36</i> (I) induce high levels of <i>10X-STAT-GFP</i>, as compared to control discs (F,F’). (J–M) Imaginal discs predominantly mutant for <i>vps22</i> (K), <i>vps25</i> (L), or <i>vps36</i> (M) show high levels of phosphorylated JNK protein, as compared to control discs (J,J’). <b>Genotypes</b>: (<b>A</b>) <i>eyFLP; FRT42D y⁺/FRT42D cl; Gbe-Su(H)-lacZ/+.</i> (<b>B</b>) <i>eyFLP; E(Spl)m8 2.61-lacZ/+; FRT82B/FRT82B cl.</i> (<b>C</b>) <i>eyFLP; E(spl)m8 2.61-lacZ/+; FRT82B vps22^5F3-8/FRT82B cl.</i> (<b>D</b>) <i>eyFLP; FRT42D vps25^N55 y⁺/FRT42D cl; Gbe-Su(H)-lacZ/+.</i> (<b>E</b>) <i>eyFLP; E(spl)m8 2.61-lacZ/+; vps36^Δ69 FRT80B/cl FRT80B.</i> (<b>F</b>) <i>eyFLP; FRT42D y⁺/FRT42D cl; 10X-STAT-GFP/+.</i> (<b>G</b>) <i>eyFLP; 10X-STAT-GFP/+; FRT82B vps22^5F3-8/FRT82B cl.</i> (<b>H</b>) <i>eyFLP; FRT42D vps25^N55 y⁺/FRT42D cl; 10X-STAT-GFP/+.</i> (<b>I</b>) <i>eyFLP; 10X-STAT-GFP/+; vps36^Δ69 FRT80B/cl FRT80B.</i> (<b>J</b>) <i>eyFLP; FRT42D y⁺/FRT42D cl.</i> (<b>K</b>) <i>eyFLP;; FRT82B vps22^5F3-8/FRT82B cl.</i> (<b>L</b>) <i>eyFLP; FRT42D vps25^N55 y⁺/FRT42D cl.</i> (<b>M</b>) <i>eyFLP;; vps36^Δ69 FRT80B/cl FRT80B.</i></p

Animals with imaginal discs predominantly mutant for ESCRT-II components die as headless pharate pupae.

<p>Animals with predominantly mutant tissues are generated with the <i>eyFLP-cl</i> system. (<b>A,B</b>) Animals with eye-antennal imaginal discs predominantly mutant for <i>vps22</i> (A) or <i>vps25</i> (B) die as pharate pupae that lack heads. <b>Genotypes</b>: (<b>A</b>) <i>eyFLP;; FRT82B vps22^5F3-8/FRT82B cl.</i> (<b>B</b>) <i>eyFLP; FRT42D vps25^N55 y⁺/FRT42D cl</i>.</p