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

Mutations abolishing the 5′→3′ RNA helicase activity of Armi reduces its ability to trigger piRNA production.

<p>(A) Cartoon showing the domain organization of <i>Drosophila</i> Armi. Motifs required for ATP-related functions and the mutations made to these are indicated. Quality of purified recombinant Armi protein used in the RNA helicase assay. (B) Recombinant Armi was incubated with RNA duplexes containing different single-stranded overhangs, in the presence or absence of ATP. Only the smaller single-stranded RNA strand (ssRNA) is radioactively labelled at the 5′ end (shown in red). The native polyacrylamide gel shows the ATP-dependent unwinding activity of Armi as revealed by presence of the fast-migrating labelled ssRNA band. The ATPase mutant Armi^DQAG is inactive in this assay. (C) Indicated proteins were tethered to the reporter transcript in the somatic follicle cells of transgenic fly ovaries. Average amount of reporter-derived piRNAs produced is plotted with the error bars representing the range of values from two independent experiments. (D) The absolute levels of piRNAs produced are plotted as read coverage (rpm) along the reporter upon tethering of the indicated proteins. The amount of piRNAs produced from different reporter regions is also shown. (E) The 5′ nucleotide composition of produced piRNAs is given. The nucleotide composition of the reporter sequence is shown. (F) When triggered by NHA-Yb, piRNAs generated from the reporter (LacZ part) shows phased primary processing as revealed by the 5′-to-5′ end distance calculations. Peaks (red triangles) at regular intervals correspond to multiples of the average piRNA length of ~25 nt. The black triangle marks the piRNAs starting at neighbouring nucleotides (the distance equal to one). The 3′-to-5′ end distances are plotted. The piRNAs generated tend to be created one after the other in a non-overlapping manner, as demonstrated by the preferred 1 nt distance. The peak at 25–30 nt corresponds to the 5′ end of a piRNA produced further downstream.</p

Artificial tethering of Armi to a transcript identifies it as a primary piRNA precursor in the <i>Drosophila</i> ovarian follicle cells.

<p>(A) Cartoon representing a <i>Drosophila melanogaster</i> egg chamber shows the single layer of somatic follicle cells enclosing the germline. Ovarian soma-specific co-expression of fusion proteins and the reporter RNA is achieved by the use of the <i>traffic jam</i> (<i>tj</i>)-GAL4 driver in the transgenic flies. Ovarian somatic cells (OSCs) is a cell culture model derived from the ovarian follicle cells.(B) A <i>cis</i>-acting element called piRNA-trigger sequence (PTS) from the <i>flamenco</i> (<i>flam</i>) cluster transcript drives conversion of the downstream reporter transcript into piRNAs that are loaded into Piwi in OSCs. The fragment used corresponds to 1–718 nt of the original cluster transcript. Read coverage (rpm) along the reporter and the total amount of piRNAs produced from individual transcript parts are shown. The 5′ nucleotide composition of reporter-derived piRNAs is shown as a pie chart. (C) BoxB reporter transcript was specifically co-expressed with either NHA-Armi or HA-Armi in the follicle cells of <i>Drosophila</i> ovary. Mapping of the generated piRNAs to the reporter is shown. (D) The 5′ end nucleotide preference of reporter-derived piRNAs indicates a strong U1-bias. Nucleotide composition of the reporter sequence is shown. (E) The 5′-to-5′ end-distances between the piRNAs produced upon NHA-Armi tethering is shown for the LacZ part of the transcript. Many piRNAs start at neighbouring nucleotides as demonstrated by high proportion of piRNAs with a 5′-to-5′ distance equal to one (black triangle). However, LacZ-derived piRNAs also show enriched distances which are multiple of the average piRNA length (red triangles), indicating non-overlapping phased piRNA production. Fraction of piRNA pairs was plotted. The 3′-to-5′ end distances between produced piRNAs are plotted. Preferred 1 nt and 25–30 nt distances (red arrows) between LacZ-derived piRNAs indicate that these piRNAs are produced in a phased manner one after the another. (F) Individual piRNAs from a section of the LacZ part of the transcript, which are triggered by NHA-Armi tethering are shown. Only piRNAs with at least a read-count of 10 are plotted.</p

Armi tethering is sufficient to trigger piRNA production in the fly ovarian germ line.

<p>(A) A schematic representation of an egg chamber from <i>Drosophila melanogaster</i> ovary. The NGT-GAL4 expression system was used to co-express the NHA-tagged proteins with BoxB reporter transcript in the ovarian germ cells. All the three fly PIWI proteins were immunoprecipitated separately and deep sequencing libraries prepared with associated RNAs. (B) Presence of reporter-derived piRNAs in the three PIWI proteins (Aub, Ago3 and Piwi) under conditions where the indicated proteins were co-expressed. Armi and Shu tethering increased the piRNA production when compared to expression of HA-Armi, which cannot bind the reporter transcript. Yb did not induce piRNA production in the ovarian germline. (C) The 5′-to-5′ end-distances of piRNAs were compared for sequences arising from the LacZ region and shows the phased pattern of production. Note that the reporter co-expressed with HA-Armi (which cannot be tethered) also shows lower levels of piRNA production and these piRNAs also show the phasing pattern. (D) The frequencies of the 5′ nucleotide in reporter-derived piRNAs are shown. (E) Immunofluorescence detection of the ectopically expressed proteins in the fly ovarian germ cells. Co-localization of Armi, Shu and Yb with the endogenous Ago3 protein indicates their localization to the nuage.</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

Protein localization in the nuage is a prerequisite for its ability to trigger piRNA biogenesis.

<p>(A) Immunofluorescence analysis of NHA-tagged Armi wildtype and mutant versions (red) in transgenic fly ovaries. Single egg chambers are shown, with specific expression (<i>tj</i>-GAL4 driver) seen in the somatic follicle cells. Endogenous Yb (green) serves as marker for the cytoplasmic Yb bodies. Wildtype Armi co-localizes in the Yb bodies. Notice that the mutant Armi proteins are dispersed into numerous granules that are not co-stained with Yb. Scale bar is indicated. (B) Zoomed views with Armi or Armi mutant co-localization with endogenous Yb protein indicated (white arrowhead). Scale bars in all the panels correspond to 5μm. (C) NHA-Yb also localizes to the nuage of the ovarian follicle cells as shown by co-localization with endogenous Armi (white arrowhead). In contrast, NHA-Shu is diffused in the cytoplasm. (D) Tagged versions of <i>Drosophila</i> Armi and Piwi were co-expressed in insect cell cultures and subjected to a tandem affinity-purification strategy (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006956#pgen.1006956.s007" target="_blank">S1 Protocols</a>). Coomassie gel shows co-purification of the two proteins, indicating direct interaction. Bands were identified by mass spectrometry and Western blotting to detect the indicated tags.</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

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

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