Galectin-3 Coordinates a Cellular System for Lysosomal Repair and Removal

Jia et al. show that Galectin-3 recruits ESCRT components to damaged lysosomes for repair and restoration of their function. During sustained lysosomal injury, galectins induce autophagy and lysosomal biogenesis for a staged repair, removal, and replacement program. This response is deployed during damage with neurotoxic tau or Mycobacterium tuberculosis infection

MERIT, a cellular system coordinating lysosomal repair, removal and replacement

Membrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal membrane repair, removal and replacement. Specifically, lysosomal membrane damage induces, in succession, ESCRT-dependent membrane repair, macroautophagy/autophagy-dominant removal of damaged lysosomes, and initiation of lysosomal biogenesis via transcriptional programs. The MERIT system is governed by galectins, a family of cytosolically synthesized lectins recognizing β-galactoside glycans. We found in this study that LGALS3 (galectin 3) detects membrane damage by detecting exposed lumenal glycosyl groups, recruits and organizes ESCRT components PDCD6IP/ALIX, CHMP4A, and CHMPB at damaged sites on the lysosomes, and facilitates ESCRT-driven repair of lysosomal membrane. At later stages, LGALS3 cooperates with TRIM16, an autophagy receptor-regulator, to engage autophagy machinery in removal of excessively damaged lysosomes. In the absence of LGALS3, repair and autophagy are less efficient, whereas TFEB nuclear translocation increases to compensate lysosomal deficiency via de novo lysosomal biogenesis. The MERIT system protects endomembrane integrity against a broad spectrum of agents damaging the endolysosomal network including lysosomotropic drugs, Mycobacterium tuberculosis, or neurotoxic MAPT/tau. Abbreviations: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; ESCRT: endosomal sorting complexes required for transport; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; TFEB: transcription factor EB; TFRC: transferrin receptor; TRIM16: tripartite motif-containing 16

Etude du rôle de la protéine autophagique ATG9A dans les cancers du sein

Autophagy is an intracellular process which contributes to the maintenance of cell homeostasis. The deregulation of this complex process, which requires more than 40 ATG proteins, has been shown to be involved in tumor development. In our laboratory, we analyzed a cohort of 80 breast cancers and demonstrated that ATG9A gene expression is increased in triple negative breast cancer samples compared to adjacent healthy tissues. We then studied the role of ATG9A in the triple negative breast cancer cell line MDA-MB-436 using two extinction models created with the sh-RNA or the CRISPR-Cas9 technology. Our two extinction models presented a blockade of autophagy, due to a decrease of autophagosome degradation. We also observed a decrease of in vitro and in vivo cancer phenotypes, such as proliferation, invasion or in vivo tumor growth, of sh-ATG9A cells compared to control cells. However, we did not observe any difference of cancer phenotypes between the CRISPR-CAS9 cells and the control ones. Since we still detected the presence of the ATG9A mRNA in the CRISPR models but not in the sh-RNA models, we hypothesized that this mRNA might play a role in the maintenance of breast cancer phenotypes in these cells, either by the expression of a truncated isoform of the ATG9A protein from the mutated ATG9A mRNA obtained after the action of the CRISPR-Cas9 system, or its interaction with non-coding mRNAs. If proven, this could establish ATG9A mRNA as a potential therapeutic target in triple negative breast cancers for which no targeted therapy is currently available.L’autophagie est un mécanisme cellulaire complexe, nécessitant plus de 40 protéines ATGs (AuTophaGy related), impliqué dans le maintien de l’homéostasie cellulaire. Sa dérégulation a été décrite comme une cause possible de la tumorigénèse. Nos travaux ont montré dans une cohorte de 80 patientes atteintes de cancer du sein, que l’expression du gène codant la protéine ATG9A, jouant un rôle dans les étapes précoces de l’autophagie, est plus importante dans les tissus cancéreux des patientes de type triple négatif. Afin d’étudier le rôle d’ATG9A dans la lignée de cancer du sein triple négatif MDA-MB-436, nous avons développé deux modèles d’extinction du gène ATG9A à l’aide de sh-ARN ou de la technique CRISPR-Cas9. Ces modèles d’extinction présentent un blocage de l’autophagie via une diminution de la dégradation des autophagosomes. Nous avons également montré une inhibition des phénotypes cancéreux in vitro et in vivo des cellules sh-ATG9A comparé aux cellules contrôles. Cependant, nous n’avons observé aucune différence de phénotypes cancéreux entre le modèle CRISPR-Cas9, contrairement au modèle sh-RNA, nous avons émis l’hypothèse que l’ARNm d’ATG9A pourrait jouer un rôle dans la maintenance des phénotypes cancéreux via l’expression d’une isoforme de la protéine ATG9A, exprimée après mutation de la séquence d’ATG9A par le système CRISPR-Cas9 ou via son interaction avec des ARN non codants régulateurs. Si cette hypothèse est confirmée, cet ARNm pourrait devenir une cible thérapeutique dans les cancers du sein triple négatif pour lesquels aucune thérapie ciblée n’existe actuellement

Study of the role of ATG9A, an autophagic protein, in breast cancer

L’autophagie est un mécanisme cellulaire complexe, nécessitant plus de 40 protéines ATGs (AuTophaGy related), impliqué dans le maintien de l’homéostasie cellulaire. Sa dérégulation a été décrite comme une cause possible de la tumorigénèse. Nos travaux ont montré dans une cohorte de 80 patientes atteintes de cancer du sein, que l’expression du gène codant la protéine ATG9A, jouant un rôle dans les étapes précoces de l’autophagie, est plus importante dans les tissus cancéreux des patientes de type triple négatif. Afin d’étudier le rôle d’ATG9A dans la lignée de cancer du sein triple négatif MDA-MB-436, nous avons développé deux modèles d’extinction du gène ATG9A à l’aide de sh-ARN ou de la technique CRISPR-Cas9. Ces modèles d’extinction présentent un blocage de l’autophagie via une diminution de la dégradation des autophagosomes. Nous avons également montré une inhibition des phénotypes cancéreux in vitro et in vivo des cellules sh-ATG9A comparé aux cellules contrôles. Cependant, nous n’avons observé aucune différence de phénotypes cancéreux entre le modèle CRISPR-Cas9, contrairement au modèle sh-RNA, nous avons émis l’hypothèse que l’ARNm d’ATG9A pourrait jouer un rôle dans la maintenance des phénotypes cancéreux via l’expression d’une isoforme de la protéine ATG9A, exprimée après mutation de la séquence d’ATG9A par le système CRISPR-Cas9 ou via son interaction avec des ARN non codants régulateurs. Si cette hypothèse est confirmée, cet ARNm pourrait devenir une cible thérapeutique dans les cancers du sein triple négatif pour lesquels aucune thérapie ciblée n’existe actuellement.Autophagy is an intracellular process which contributes to the maintenance of cell homeostasis. The deregulation of this complex process, which requires more than 40 ATG proteins, has been shown to be involved in tumor development. In our laboratory, we analyzed a cohort of 80 breast cancers and demonstrated that ATG9A gene expression is increased in triple negative breast cancer samples compared to adjacent healthy tissues. We then studied the role of ATG9A in the triple negative breast cancer cell line MDA-MB-436 using two extinction models created with the sh-RNA or the CRISPR-Cas9 technology. Our two extinction models presented a blockade of autophagy, due to a decrease of autophagosome degradation. We also observed a decrease of in vitro and in vivo cancer phenotypes, such as proliferation, invasion or in vivo tumor growth, of sh-ATG9A cells compared to control cells. However, we did not observe any difference of cancer phenotypes between the CRISPR-CAS9 cells and the control ones. Since we still detected the presence of the ATG9A mRNA in the CRISPR models but not in the sh-RNA models, we hypothesized that this mRNA might play a role in the maintenance of breast cancer phenotypes in these cells, either by the expression of a truncated isoform of the ATG9A protein from the mutated ATG9A mRNA obtained after the action of the CRISPR-Cas9 system, or its interaction with non-coding mRNAs. If proven, this could establish ATG9A mRNA as a potential therapeutic target in triple negative breast cancers for which no targeted therapy is currently available

Detection of SQSTM1/LC3B, SQSTM1/GL1, NIX/GL1 and NIX/LC3B interactions by P-LISA.

<p>(<b>A</b>) MDA-MB-436 cells were cultured for 24 h at 37°C and 5% CO₂, fixed, permeabilized, blocked with 5% BSA, incubated with rabbit anti-LC3B, rabbit anti-GL1, mouse anti-SQSTM1 or/and mouse anti-NIX antibodies overnight at 4°C and then with an Alexa Fluor 488 goat anti-rabbit and an Alexa Fluor 555 goat anti-mouse, respectively, for 1 h. The cells were then analyzed using a confocal microscope. (<b>B</b>) For P-LISA, the protocol was performed according to the manufacturer’s recommendations using the same antibodies as described above. Nuclei were stained with DAPI. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. Scale bars: 20μm.</p

Increase of SQSTM1/GL1 and NIX/GL1 interactions quantified by P-LISA in MCF-7 overexpressing FLAG-GABARAPL1-6His and GFP-NIX.

<p>(A) Expression of GL1 protein was analyzed using western blotting in MCF-7 expressing or not FLAG-GL1-6His. (<b>B</b>) MCF-7 Control or MCF-7 FLAG-GL1-6His cells were cultured for 24 h at 37°C and 5% CO₂, fixed, permeabilized, blocked with 5% BSA, incubated with mouse anti-SQSTM1 and rabbit anti-GL1 antibodies antibodies overnight at 4°C and then with an Alexa Fluor 555 goat anti-mouse and an Alexa Fluor 488 goat anti-rabbit, respectively, for 1 h. The cells were then analyzed using a confocal microscope (top panel). For P-LISA, the protocol was performed according to the manufacturer’s recommendations using mouse anti-SQSTM1 and rabbit anti-GL1 antibodies (bottom panel). (<b>C</b>) MCF-7 FLAG-GL1-6His cells were cultured for 24 h at 37°C and 5% CO₂ and transfected with pEGFP-NIX plasmid and then fixed and permeabilized. P-LISA was performed as precognized by the manufacturer using rabbit anti-GL1 and mouse anti-NIX antibodies. Nuclei were stained with DAPI. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. Scale bar: 20μm.</p

Technical controls demonstrating the specificity of P-LISA signals.

<p>(<b>A</b>) MDA-MB-436 cells were cultured for 24 h at 37°C and 5% CO₂. P-LISA were performed according to the manufacturer’s recommendations. No primary antibodies were added before performing P-LISA with PLA R+ (anti-rabbit) and PLA M- (anti-mouse) (left panel); P-LISA SQSTM1/LC3B was also performed with PLA R+ (anti-rabbit) against LC3B and with PLA G- (anti-goat) unable to recognize SQSMT1 (left panel). Similar controls were performed for P-LISA SQSTM1/GL1, P-LISA LC3B/NIX and P-LISA GL1/NIX. (<b>B</b>) Quantification of <i>LC3B</i> mRNA expression in MDA-MB-436 cells following <i>LC3B</i> siRNA transfection analyzed using qRT-PCR (top panel). Quantification of SQSTM1/LC3 interactions detected by P-LISA in MDA-MB-436 cells transfected or not with <i>LC3B</i> siRNA (bottom panel) according to the manufacturer’s recommendations using rabbit anti-LC3B and mouse anti-SQSTM1 antibodies. (<b>C</b>) Absence of SQSTM1/LC3B P-LISA signals in murine cells since the anti-SQSTM1 antibody is specific of the human SQSTM1 protein and restoration of P-LISA signals when these cells were transfected with a vector coding the human HA-SQSTM1 protein. (<b>D</b>) Absence of GL1 protein was validated using western blotting in MDA-MB-436 cells expressing or not a <i>GABARAPL1</i> shRNA (<b>E</b>) Quantification of NIX/GL1 interactions was performed by P-LISA in MDA-MB-436 cells expressing or not a <i>GABARAPL1</i> shRNA using rabbit anti-GL1 and mouse anti-NIX antibodies.</p

Proximity Ligation <i>In situ</i> Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction

<div><p>Macroautophagy is a highly regulated intracellular degradation process which has been extensively studied over the last decade. This pathway has been initially described as a non selective process inducing the degradation of parts of the cytoplasm as well as organelles at random. Nevertheless, over the last few years, new research highlighted the existence of a more selective autophagy pathway specifically recruiting some organelles or aggregates to the autophagosomes in order to induce their degradation. These selective autophagy pathways such as aggrephagy, mitophagy, pexophagy or xenophagy, involve the intervention of a cargo, the material to be degraded, cargo adapters, the molecules allowing the recruitment of the cargo to the autophagosome, and the proteins of the ATG8 family which link the cargo adapters to the autophagosome. One of the main questions which now remain is to develop new techniques and protocols able to discriminate between these different types of induced autophagy. In our work, we studied the possibility to use the P-LISA technique, which has been recently developed to study endogenous <i>in vivo</i> protein interactions, as a new technique to characterize the ATG proteins specifically involved in bulk or selective autophagy. In this manuscript, we indeed demonstrate that this technique allows the study of endogenous ATG protein interactions in cells following autophagy induction, but more interestingly that this technique might be used to characterize the ATG proteins involved in selective autophagy.</p></div

Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins

Autophagy is a conserved eukaryotic process with metabolic, immune, and general homeostatic functions in mammalian cells. Mammalian autophagosomes fuse with lysosomes in a SNARE-driven process that includes syntaxin 17 (Stx17). How Stx17 translocates to autophagosomes is unknown. In this study, we show that the mechanism of Stx17 recruitment to autophagosomes in human cells entails the small guanosine triphosphatase IRGM. Stx17 directly interacts with IRGM, and efficient Stx17 recruitment to autophagosomes requires IRGM. Both IRGM and Stx17 directly interact with mammalian Atg8 proteins, thus being guided to autophagosomes. We also show that Stx17 is significant in defense against infectious agents and that Stx17–IRGM interaction is targeted by an HIV virulence factor Nef