Additional file 1: Figure S1. of A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

The FLPout system in Drosophila. (A-B) The recombination between FRT sites by the FLP recombinase under the control of a heat shock promoter (A) results in excision of the CD2- STOP cassette and expression of GAL4 (B) which in turn activates the expression of the transgenes downstream the UAS promoter, including a fluorescent reporter (GFP, or GFP-tagged protein). No recombination between the FRT leaves the CD2-STOP cassette in place, thus preventing GAL4 expression (C). (TIF 676 kb

Additional file 3: Figure S3. of A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

Validation of defects in the endosomal pathway by Texas Red-Avidin uptake. (A-B) The endocytic tracer TR-avidin fails to be internalized in clonal cells expressing either ShiK44A (A) or Rab5-IR (B). Clones were detected by the co-expression of the autophagy marker GFP-Atg8a. (C-G) Internalized TR-avidin fails to be transported to the lysosomes when late stages of the endocytic process are defective. Clonal cells were detected by the expression of the lysosomal marker GFP-LAMP1. Occasional colocalization between the endocytic tracer TR-avidin and the lysosomes are observed in control cells (D) but not in cells expressing Rab4SN (E), Chmp1-IR (F) or Rab7TN (G). Quantification of the colocalization between the TR-avidin and GFP-LAMP1 using the Pearson’s Correlation Coefficient (PCC) is shown in C. Bars denote mean ± s.d. Statistical significance was determined using one-way ANOVA: *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. Genotypes: (A) y w hs-FLP/UAS-ShiK44A; UAS-GFP-Atg8a/+; Ac > CD2 > Gal4/ UAS-ShiK44A, (B) y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac > CD2 > Gal4/UAS-Rab5-IR, (C) y w hs-FLP/+; UAS-GFP-LAMP1/+; Ac > CD2 > Gal4/+, (D) y w hs-FLP/+; UAS-GFP-LAMP1/+; Ac > CD2 > Gal4/UAS-Rab4SN, (E) y w hs-FLP/+; UAS-GFP-LAMP1/+; Ac > CD2 > Gal4/UAS-Chmp1-IR, (F) y w hs-FLP/+; UAS-GFP-LAMP1/UAS-Rab7TN; Ac > CD2 > Gal4/+. (TIF 1831 kb

iLIR database: A web resource for LIR motif-containing proteins in eukaryotes

<p>Atg8-family proteins are the best-studied proteins of the core autophagic machinery. They are essential for the elongation and closure of the phagophore into a proper autophagosome. Moreover, Atg8-family proteins are associated with the phagophore from the initiation of the autophagic process to, or just prior to, the fusion between autophagosomes with lysosomes. In addition to their implication in autophagosome biogenesis, they are crucial for selective autophagy through their ability to interact with selective autophagy receptor proteins necessary for the specific targeting of substrates for autophagic degradation. In the past few years it has been revealed that Atg8-interacting proteins include not only receptors but also components of the core autophagic machinery, proteins associated with vesicles and their transport, and specific proteins that are selectively degraded by autophagy. Atg8-interacting proteins contain a short linear LC3-interacting region/LC3 recognition sequence/Atg8-interacting motif (LIR/LRS/AIM) motif which is responsible for their interaction with Atg8-family proteins. These proteins are referred to as LIR-containing proteins (LIRCPs). So far, many experimental efforts have been carried out to identify new LIRCPs, leading to the characterization of some of them in the past 10 years. Given the need for the identification of LIRCPs in various organisms, we developed the iLIR database (<a href="https://ilir.warwick.ac.uk" target="_blank">https://ilir.warwick.ac.uk</a>) as a freely available web resource, listing all the putative canonical LIRCPs identified in silico in the proteomes of 8 model organisms using the iLIR server, combined with a Gene Ontology (GO) term analysis. Additionally, a curated text-mining analysis of the literature permitted us to identify novel putative LICRPs in mammals that have not previously been associated with autophagy.</p

A genetic screen identifies UBPY and USP12 as putative autophagy regulators.

<p>(A) Quantification of autophagy in the <i>Drosophila</i> larval fat body after silencing of the indicated DUB using the Cg-Gal4 driver line. Bars denote the proportion of autophagic cells from at least 6 animals. Cells were considered as “autophagic” if at least one GFP-LC3B vesicle was observed. (B) Quantification of autophagy after silencing by the FLPout method. Bars denote the proportion of autophagic cells from at least 6 animals. Statistical significance was determined using <i>one-way ANOVA</i>: **p<0.005. (C-G) Representative confocal sections after silencing of the indicated DUB in the larval fat body. (H-L) Clonal analysis of the four candidates after silencing by the FLPout method. One representative confocal section per genotype is shown. Actin is labelled with Phalloidin-Texas Red (red) and nuclei are labelled with Hoechst (blue). Scale bar: 10μm. Genotypes: (C) <i>Cg-Gal4/+; UAS-GFP-LC3B/+</i>, (D) <i>Cg-Gal4/ UAS-Uch-L3-IR; UAS-GFP-LC3B/+</i>, (E) <i>Cg-Gal4/ UAS-Usp45-IR; UAS-GFP-LC3B/+</i>, (F) <i>Cg-Gal4/+; UAS-GFP-LC3B/ UAS-Ubpy-IR</i>, (G) <i>Cg-Gal4/+; UAS-GFP-LC3B/ UAS-Usp12-IR</i>, (H) <i>y w hs-FLP/+; UAS-GFP-Atg8a/UAS-Luc-IR; Ac>CD2>Gal4/+</i>, (I) <i>y w hs-FLP/+; UAS-GFP-Atg8a/UAS-Uch-L3-IR; Ac>CD2>Gal4/+</i>, (J) <i>y w hs-FLP/+; UAS-GFP-Atg8a/UAS-Usp45-IR; Ac>CD2>Gal4/+</i>, (K) <i>y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac>CD2>Gal4/ UAS-Ubpy-IR</i>, <i>(L) y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac>CD2>Gal4/ UAS-Usp12-IR</i>.</p

UBPY silencing in HeLa cells activates autophagy.

<p>(A) The number of GFP-LC3 dots per cell was quantified in HeLa cells stably expressing the autophagy reporter GFP-LC3; cells were transfected with a control plasmid (pME-Flag) or plasmids expressing either the wild-type human UBPY protein (pME-UBPY^WT) or its catalytically inactive mutant (pME-UBPY^C>S). Bars denote mean ± s.d. Statistical significance was determined using <i>t-test</i>: ****p<0.0001 (B) The expression of UBPY was monitored by Western blot in GFP-LC3 HeLa cells stably transfected with a control shRNA or three different shRNAs targeting UBPY. (C) The number of GFP-LC3 dots per cell was quantified in GFP-LC3 HeLa cells stably transfected with a control shRNA or three different shRNAs targeting UBPY in absence (black bars) or in presence of bafilomycin A1 (BAF, gray bars). Bars denote mean ± s.d. Statistical significance was determined using <i>t-test</i>: ****p<0.0001; ***p<0.005 (D) The expression of the autophagy target protein p62 was monitored by Western blot in GFP-LC3 HeLa cells stably transfected with a control shRNA or three different shRNAs targeting UBPY. (E) Quantification of p62 levels in GFP-LC3 HeLa cells stably transfected with a control shRNA or three different shRNAs targeting UBPY from three independent Western blots. (F) The repartition of autolysosmes and autophagosomes was determined in mRFP-GFP-LC3 HeLa cells stably expressing either the control shRNA or the shUBPY #35 shRNA, in comparison with control transfected cells treated with bafilomycin A1.</p

<i>Ubpy</i> silencing induces lysomal defects.

<p>(A,B) Confocal sections of larval fat bodies clonally expressing the lysosomal markers GFP-Lamp1 alone (A) or in combination with the <i>Ubpy</i> silencing transgene (B). (C,D) Confocal sections of larval fat bodies clonally expressing the autophagy reporter GFP-Atg8a alone (C) or in combination with the <i>Ubpy</i> silencing transgene (D) after staining for the endogenous lysosomal hydrolase Cathepsin-L. Insets show the merged channels of the respective images and the clone boundaries are indicated as dotted lines (E). Quantification of GFP-Lamp1 dots size. (F) Quantification of the mean relative intensity of the Cathepsin-L staining in GFP-Atg8a expressing cells compared to the staining intensity of the adjacent wild-type neighboring cells. N>6 larvae per experimental condition. Bars denote mean ± s.d. Statistical significance was determined using <i>one-way Anova</i>: *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. Scale bar: 10μm (A-H), 50μm (J-Q). Genotypes: (A) <i>y w hs-FLP/+; UAS-GFP-Lamp1/+; Ac>CD2>Gal4/+</i>, (B) <i>y w hs-FLP/+; UAS-GFP-Lamp1/+; Ac>CD2>Gal4/UAS-Ubpy-IR</i>, (C) <i>y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac>CD2>Gal4/+</i> (D) <i>y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac>CD2>Gal4/ UAS-Ubpy-IR</i>.</p

Ultrastructural analysis of <i>Ubpy</i> silenced cells.

<p>Control fat body cells (A) contain large autolysosomes (black arrowhead). These vesicles are characterized by their heterogeneous content and organelle remnants. In contrast, <i>Ubpy</i> silenced cells (B-C) contain autophagosomes whith non-degraded organelles (white arrowhead) (a mitochondria in B and endoplasmic reticulum in C), small autolysosomes (black arrowhead) and vesicles with homogenous electron-dense content (asterisks). Scale bars: 1μm. (D-E) Quantification of lysosomal diameter (D) and number of vesicles with homogenous electron-dense content (E). Bars denote mean ± s.d. Statistical significance was determined using <i>t-test</i>: ****p<0.0001. Genotypes: (A) <i>Cg-Ggal4/+</i>, (B-C) <i>Cg-Gal4/+; UAS-Ubpy-IR/+</i>.</p

<i>Ubpy</i> loss-of-function blocks the autophagy flux.

<p>(A,B) Analysis of the autophagy flux using the tandem-tagged GFP-mCherry-Atg8a reporter in control cells from starved larvae (A) or in <i>Ubpy</i> silenced cells (B). Insets show an enlarged view for each condition (arrow: autophagosome, arrowhead: autolysosomes). Quantification of the colocalization of mCherry and GFP signals using the Pearson’s correlation coefficient is shown in F. (C,D) Lysotracker Red staining on fat bodies from fed (C) or starved (D) larvae silenced for <i>Ubpy</i>. Mutant cells were identified by the expression of the GFP-Atg8a marker (dotted lines and green channel in insets). (E) Confocal sections of larval fat bodies stained for the endogenous Ref(2)P protein. Mutant cells were identified by the expression of the GFP-Atg8a marker (dotted lines and green channel in insets). (G) Quantification of the size of the Ref(2)P aggregates. N>6 larvae per experimental condition. For all the quantifications, bars denote mean ± s.d. Statistical significance was determined using <i>one-way ANOVA</i>: *p<0.05, **p<0.005, ****p<0.0001. Scale bars: 20μm (A,B), 50μm (C-E). Genotypes: (A) <i>y w hs-FLP/+; UAS-GFP-mCherry-Atg8a/UAS-Luc-IR; Ac>CD2>Gal4/+</i>, (B) <i>y w hs-FLP/+; UAS-GFP-mCherry-Atg8a/+; Ac>CD2>Gal4/ UAS-Ubpy-IR</i>, (C-E) <i>y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac>CD2>Gal4/ UAS-Ubpy-IR</i>.</p

Expression of a catalytic inactive UBPY mutant blocks the autophagy flux.

<p>Expression of the wild-type form of UBPY has no effect on autophagy (Flag-UBPY^WT, A) whereas the UBPY catalytic mutant form induces accumulation of GFP-Atg8a dots (Flag-UBPY^C>S, B). Quantification of the number of GFP-Atg8a dots per cell is show in C. Confocal sections of larval fat bodies expressing the GFP-mCherry-Atg8a in combination with the wild-type (D) or catalytic inactive (E) forms of UBPY. Please note that the expressing the wild-type form of UBPY (D) were starved to induce autophagy and allow the observation of autophagosomes. (F) Quantification of the colocalization of mCherry and GFP signals using the Pearson’s correlation coefficient. Larvae expressing the wild-type (G) or the mutant (H) form of UBPY were starved to induce autophagy and stained with Lysotracker Red. Insets show the merged channels of the respective images and the clone boundaries are indicated as dotted lines. Scale bars: 20μm. N>6 larvae per experimental condition. For quantification, bars denote mean ± s.d. Statistical significance was determined using <i>one-way ANOVA</i>: ****p<0.0001, ns: not significant. Genotypes: (A, G) <i>y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac>CD2>Gal4/UAS-2xFlag-UBPY</i>^<i>WT</i>, (B, H) <i>y w hs-FLP/+; UAS-GFP-Atg8a/+; Ac>CD2>Gal4/ UAS-2xFlag-UBPY</i>^<i>C>S</i>, (D) <i>y w hs-FLP/+; UAS-GFP-mCherry-Atg8a/+; Ac>CD2>Gal4/UAS-2xFlag-UBPY</i>^WT, (E) <i>y w hs-FLP/+; UAS-GFP-mCherry-Atg8a/+</i>,<i>; Ac>CD2>Gal4/ UAS-2xFlag-UBPY</i>^<i>C>S</i>.</p

CHMP1B ubiquitination is controlled by USP8.

<p><b>A-C:</b> Wild-type or mutated GFP-CHMP1B constructs were transfected into HEK293T cells jointly with HA-ubiquitin (HA-Ub). In (B), cells were co-transfected with sh<i>Usp8</i> and in (C), cells were co-transfected with Flag-USP8, Flag-USP8^C748A or Flag-USP8^S680A constructs. Immunoprecipitations (IP) were carried out with anti-GFP antibodies after strong denaturation of the lysate and proteins were analyzed by immunoblot (IB) using either anti-HA (Ub) or anti-GFP (CHMP1B) antibodies. Whole cell lysates (WCL) were immunoblotted with anti-HA to reveal transfected Ub-HA and, in (B), with anti-USP8 to reveal endogenous USP8, and in (C), with anti-Flag to reveal Flag-USP8 constructs. Total protein amount is shown. <b>A’, B’, C’:</b> Quantification by densitometry of blots in A, B or C, respectively. Normalized signals are expressed as a fold-change over CHMP1B wild-type basal ubiquitination levels. Histograms represent the mean of two to three independent experiments. Error bars indicate the range. Values are mean ± SD. *p<0.05 (Student’s t-test).</p