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Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy.

ABSTRACT: Mitochondria are critical metabolic and signaling hubs, and dysregulated mitochondrial homeostasis is implicated in many diseases. Degradation of damaged mitochondria by selective GABARAP/LC3-dependent macro-autophagy (mitophagy) is critical for maintaining mitochondrial homeostasis. To identify alternate forms of mitochondrial quality control that functionally compensate if mitophagy is inactive, we selected for autophagy-dependent cancer cells that survived loss of LC3-dependent autophagosome formation caused by inactivation of ATG7 or RB1CC1/FIP200. We discovered rare surviving autophagy-deficient clones that adapted to maintain mitochondrial homeostasis after gene inactivation and identified two enhanced mechanisms affecting mitochondria including mitochondrial dynamics and mitochondrial-derived vesicles (MDVs). To further understand these mechanisms, we quantified MDVs via flow cytometry and confirmed an SNX9-mediated mechanism necessary for flux of MDVs to lysosomes. We show that the autophagy-dependent cells acquire unique dependencies on these processes, indicating that these alternate forms of mitochondrial homeostasis compensate for loss of autophagy to maintain mitochondrial health.

SUBMITTER: Towers CG

PROVIDER: S-EPMC8319140 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy.

Towers Christina G CG Wodetzki Darya K DK Thorburn Jacqueline J Smith Katharine R KR Caino M Cecilia MC Thorburn Andrew A

Developmental cell 20210624 14

Mitochondria are critical metabolic and signaling hubs, and dysregulated mitochondrial homeostasis is implicated in many diseases. Degradation of damaged mitochondria by selective GABARAP/LC3-dependent macro-autophagy (mitophagy) is critical for maintaining mitochondrial homeostasis. To identify alternate forms of mitochondrial quality control that functionally compensate if mitophagy is inactive, we selected for autophagy-dependent cancer cells that survived loss of LC3-dependent autophagosome ...[more]

PMID: 34171288

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Project description:Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroautophagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy. Abbreviations: ACTB/β-actin: actin beta; Atg8: autophagy-related 8; CL: cardiolipin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DTT: DL-dithiothreitol; FKBP8: FKBP prolyl isomerase 8; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; IMM: inner mitochondrial membrane; LUV/LUVs: large unilamellar vesicle/s; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NME4/NDPK-D/Nm23-H4: NME/NM23 nucleoside diphosphate kinase 4; O/A: oligomycin A + antimycin A; OMM: outer mitochondrial membrane; PA: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; PINK1: PTEN induced putative kinase 1; PtdIns4P: phosphatidylinositol-4-phosphate; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SUV/SUVs: small unilamellar vesicle/s.

Project description:Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation. Abbreviations: ACTB: actin beta; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy-related; CYCS: cytochrome c, somatic; CLQ: chloroquine; COX: cyclooxygenase; CTR: control; GFP: green fluorescent protein; HIFIA/Hif alpha: hypoxia inducible factor 1 subunit alpha; IMS: intermembrane space; LIR: LC3 interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR/mTOR: mechanistic target of rapamycin kinase; OA: oligomycin and antimycin A; OMM: outer mitochondrial membrane; PHB: prohibitin; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SHC: src Homology 2 domain-containing transforming protein; TMRM: tetramethylrhodamine, methyl ester; TOMM: translocase of outer mitochondrial membrane; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Dataset Information

Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy.

Publications

Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy.

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