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

ALIX co-localizes with Shrub during cytokinesis in <i>Drosophila</i> female germline stem cells.

<p><b>(A)</b> Schematic overview of ALIX and Shrub domain structures and conserved interaction motifs. <b>(B)</b><i>Drosophila</i> Dmel cells transiently expressing GFP or Shrub-GFP were subjected to GFP trap immunoprecipiation analysis. ALIX and GFP were detected by immunoblotting. A representative result is shown. <b>(C-F)</b> ALIX and GFP-Shrub co-localize at MRs and MBs during fGSC cytokinesis. GFP-Shrub was expressed under the control of <i>Nanos-GAL4</i> (<i>Nos-GAL4</i>). ALIX co-localizes with GFP-Shrub at MRs in G1/S (C, arrow), S phase (D, arrow) and at MBs during abscission in G2 (E-F, arrows). Ovaries were fixed and stained with antibodies against ALIX (red) and hts-F (white), and with GFP Booster (green) and Hoechst (blue). CC, cap cell. Scale bars represent 5 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s009" target="_blank">S9 Fig</a>.</p

ALIX and Shrub coordinately control abscission in <i>Drosophila</i> female germline stem cells.

<p><b>(A-D)</b> RNAi-mediated depletion of Shrub and ALIX causes abscission defects in fGSCs. (A) Control fGSC-CB pair in cytokinesis (MB, arrowhead). <i>alix-RNAi</i> (B), <i>shrub-RNAi</i> (C) and combined <i>shrub-</i> and <i>alix-RNAi</i> (D) gives rise to fGSCs connected to chains of daughter cells via MRs (green, arrows) and fusome (red). The asterisk in (D) shows a part of the fusome in the fGSC of the stem cyst, a part of which is enlarged. Ovaries were fixed and stained with antibodies against Cindr (green) and hts-F (red) and with Hoechst (blue). CC, cap cell. Scale bars represent 10 µm (full germaria) and 5 µm (enlarged images). <b>(E)</b> Graph showing the average percentages of fGSCs with the indicated phenotypes from the genotypes in (A-D). Control, five independent experiments, n = 97 fGSCs, 37 germaria; <i>alix-RNAi</i>, five independent experiments, n = 103 fGSCs, 42 germaria; <i>shrub-RNAi</i>, four independent experiments, n = 94 fGSCs, 41 germaria; <i>shrub & alix-RNAi</i>, three indepdendent experiments, 39 fGSCs, 25 germaria. A systematically significant difference between <i>control</i> and either <i>alix</i>-RNAi, <i>shrub</i>-RNAi or <i>shrub-RNAi & alix-RNAi</i> was detected in each experiment (p<0.005, Fisher’s exact test). CB, cystoblast; MR, midbody ring; MB, midbody. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s010" target="_blank">S10 Fig</a>.</p

Loss of ALIX gives rise to egg chambers with increased number of germ cells during <i>Drosophila</i> oogenesis.

<p><b>(A)</b> Schematic of the <i>Drosophila alix</i> gene locus and the <i>alix¹</i> and <i>alix³</i> alleles. The <i>alix</i> gene (<i>CG12876</i>) is located on 3R, band 98B1, and is encoded by six exons. <b>(B)</b> Overview of <i>Drosophila</i> oogenesis, female germline stem cell (fGSC) and germ cell divisions. Each fGSC in the stem cell niche in the anterior tip of the germarium divides with complete cytokinesis to give rise to another fGSC and a daughter cell, a cystoblast (CB). Cytokinetic abscission occurs as the MR closes to form an MB and the fusome (red) is cut in two unequal parts. The CB leaves the niche and undergoes four mitotic divisions with incomplete cytokinesis, giving rise to a 16-cell cyst in which the cells are interconnected by ring canals (RCs). One of the cells with four RCs will be specified as the oocyte. The 16-cell cyst becomes encapsulated by follicle cells to form an egg chamber, each of which will undergo 14 developmental stages to form an egg. <b>(C, F)</b> Western blots showing the lack of ALIX protein in (C) <i>alix¹</i> and (F) <i>alix³</i> mutant ovaries, respectively. α-tubulin was used as a loading control. <b>(D, G)</b><i>alix¹</i> (D) and <i>alix³</i> (G) mutant egg chambers (ECs) frequently contain 32 germ cells (GCs). Upper panels: <i>Wild type</i> ECs with four RCs (arrows) to the oocyte. Lower panels: (D) <i>alix¹</i> and (G) <i>alix³</i> mutant ECs with five RCs (arrows) to the oocyte. Ovaries were fixed and stained to visualize F-actin (white) and nuclei (blue). Scale bars represent 20 µm (left and middle images in each panel) and 10 µm (right images in each panel). <b>(E, H)</b> Graphs showing the average percentage of ECs with 16, 32 or more GCs from three independent experiments from <i>wild type</i> and <i>alix¹</i> or <i>alix³</i> mutant flies, respectively. (E) <i>Wild type</i>, n = 548 ECs; <i>alix¹</i>, n = 273 ECs. (H) <i>Wild type</i>, n = 222 ECs; <i>alix³</i>, n = 228 ECs. Data are based on three independent experiments and presented as mean ± STD in both (E) and (H). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s004" target="_blank">S4</a> Figs.</p

ALIX interacts with Shrub to promote abscission in <i>Drosophila</i> female germline stem cells.

<p><b>(A)</b><i>Drosophila</i> Dmel cells transiently expressing GFP, <i>wild type</i> GFP-ALIX (GFP-ALIX-wt) or the two GFP-ALIX variants containing mutations of amino acids F198 (F198D) or I211 (I211D) were used for GFP trap analysis. Co-immunoprecipitated endogenous Shrub was detected by immunoblotting. Anti-GFP was used to validate the expression levels and levels of precipitated GFP-tagged proteins. A representative result is presented. <b>(B)</b> Ovaries of the indicated genotypes were fixed and stained with antibodies against Cindr (red) and hts-F (white) and with Hoechst (blue). fGSC-CB pairs and stem cysts are outlined and MRs indicated with arrows. CC, cap cell. Scale bars represent 5 µm. <b>(C)</b> Graph showing the average percentages of the indicated fGSC phenotypes in germaria of females of the genotypes in (B) from three independent experiments. <i>Nanos-GAL4/+</i>, n = 43 fGSCs, 15 germaria; <i>Nanos-GAL4/UASp-GFP-ALIX</i>, 41 fGSCs, 15 germaria; <i>Nanos-GAL4/UASp-GFP-ALIX-F198D</i>, 40 fGSCs, 15 germaria; <i>Nanos-GAL4/UASp-GFP-ALIX-I211D</i>, 42 fGSCs, 15 germaria; <i>alix¹</i>, 59 fGSCs, 29 germaria; <i>Nanos-GAL4/UASp-GFP-ALIX; alix¹</i>, 59 fGSCs, 28 germaria; <i>Nanos-GAL4/UASp-GFP-ALIX-F198D</i>; <i>alix¹</i>, 54 fGSCs, 30 germaria; <i>Nanos-GAL4/UASp-GFP-ALIX-I211D; alix¹</i>, 52 fGSCs, 29 germaria. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s011" target="_blank">S11 Fig</a>.</p

ALIX controls abscission in <i>Drosophila</i> female germline stem cells.

<div><p><b>(A-C)</b> Loss of ALIX causes abnormal fGSC division. (A) <i>Wild type</i> Nanos-positive fGSCs show normal spectrosome/fusome morphologies (fGSCs and fGSC-CB pairs are outlined). Nanos-positive fGSCs in the anterior tip of <i>alix¹</i> (B) and <i>alix³</i> (C) mutant germaria are interconnected to chains of daughter cells via abnormally long fusomes (outlined). Ovaries were fixed and stained with antibodies against hts-F (red), Nanos (green) and Vasa (white), and with Hoechst (blue). CC, cap cell. Scale bars represent 10 µm. <b>(D-G)</b> Loss of ALIX function causes abscission defects in fGSCs. (D-E) <i>Wild-type</i> fGSCs show normal morphologies: single fGSCs with a spectrosome (red, asterisks), fGSC-CB pairs with an MR (green, arrows) and fusing fusomes (red) and an fGSC-CB pair in abscission with a MB (green, arrowhead) and a fusome with exclamation point morphology (red). (F-G) <i>alix³</i> mutant fGSCs display abnormal morphologies: fGSCs in linear (F) or branched (G) chains via MRs (green, arrows) and fusome (red). Ovaries were fixed and stained with antibodies against Cindr (green) and hts-F (red), and with Hoechst (blue). Scale bars represent 10 µm. <b>(H)</b> Graph showing the average percentage of fGSCs with the fGSC phenotypes as described in (D-G) and the Materials and Methods from <i>wild type</i> and <i>alix³</i> mutant females. <i>Wild type</i>, three independent experiments, n = 61 fGSCs, 22 germaria; <i>alix³</i>, three independent experiments, n = 60 fGSCs, 29 germaria. CB, cystoblast; MR, midbody ring; MB, midbody. Data are presented as mean ± STD. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s014" target="_blank">S3 Table</a>.</p> <p><b>(I)</b> Left: <i>Wild type</i> fGSC in mitosis. Right: Four dividing cells in <i>alix¹</i> mutant germairum, one of which is an fGSC. Ovaries were fixed and stained with antibodies against phospho-Histone H3 (PH3, green), γ-tubulin (red), with phalloidin to visualize F-actin (blue), and with Hoechst (white). Scale bars represent 5 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s005" target="_blank">S5–S7</a> Figs.</p></div

ALIX localizes at the midbody ring during cytokinesis in <i>Drosophila</i> S2 cells.

<p><b>(A)</b> Schematic of <i>Drosophila</i> and human ALIX domain structures. The <i>Drosophila</i> ALIX protein shows ~60% homology to human ALIX and contains an N-terminal Bro1 domain (BRO1), a central coiled-coil (CC) and a C-terminal proline-rich domain (PRD). <b>(B-E)</b> ALIX localizes at centrosomes in (B) metaphase, (C) early anaphase, (D) late anaphase and (E) early telophase. <b>(F)</b> In mid telophase, ALIX localizes at the ICB in two pools that overlap with α-tubulin on each side of the central region of the MB. <b>(G)</b> In late telophase/cytokinesis, ALIX localizes at the MR. In (B-G), S2 cells stably expressing GFP-α-tubulin (green) were fixed and stained with a guinea pig anti-ALIX antibody (red), and with Hoechst (blue). Scale bars represent 5 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004904#pgen.1004904.s001" target="_blank">S1 Fig</a>.</p