将自噬成分人工靶向线粒体揭示了常规和非常规的丝裂吞噬途径。

Katharina C Lorentzen, Alan R Prescott, Ian G Ganley
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引用次数: 0

摘要

大自噬/自噬能使溶酶体降解各种细胞内物质。这一过程对细胞的正常功能至关重要,其失调与许多疾病有关。有鉴于此,人们对了解自噬的作用机制产生了浓厚的兴趣,以便确定如何才能最好地针对自噬进行治疗。在有丝分裂(通过自噬选择性地降解线粒体)过程中,线粒体首先需要被信号激活,以便招募核心自噬机制,推动在目标线粒体周围局部形成自噬体。为了确定招募不同的自噬核心成分如何能驱动有丝分裂,我们利用了mito-QC有丝分裂测定(线粒体外膜定位的串联mCherry-GFP标签)。通过用抗mCherry(或抗GFP)纳米抗体标记自噬蛋白,我们可以将它们招募到线粒体,同时监测有丝分裂的水平。我们发现,靶向 ULK1、ATG16L1 和不同的 Atg8 家族蛋白足以诱导有丝分裂。线粒体招募ULK1和Atg8家族蛋白会诱导传统的有丝分裂途径,需要RB1CC1/FIP200、PIK3C3/VPS34活性和ATG5。令人惊讶的是,ATG16L1 招募后的有丝分裂途径与 ATG5 无关,但仍需要 RB1CC1 和 PIK3C3/VPS34 的活性。在后一种途径中,线粒体通过摄取到早期内体而被运送到溶酶体。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Artificial targeting of autophagy components to mitochondria reveals both conventional and unconventional mitophagy pathways.

Macroautophagy/autophagy enables lysosomal degradation of a diverse array of intracellular material. This process is essential for normal cellular function and its dysregulation is implicated in many diseases. Given this, there is much interest in understanding autophagic mechanisms of action in order to determine how it can be best targeted therapeutically. In mitophagy, the selective degradation of mitochondria via autophagy, mitochondria first need to be primed with signals that allow the recruitment of the core autophagy machinery to drive the local formation of an autophagosome around the target mitochondrion. To determine how the recruitment of different core autophagy components can drive mitophagy, we took advantage of the mito-QC mitophagy assay (an outer mitochondrial membrane-localized tandem mCherry-GFP tag). By tagging autophagy proteins with an anti-mCherry (or anti-GFP) nanobody, we could recruit them to mitochondria and simultaneously monitor levels of mitophagy. We found that targeting ULK1, ATG16L1 and the different Atg8-family proteins was sufficient to induce mitophagy. Mitochondrial recruitment of ULK1 and the Atg8-family proteins induced a conventional mitophagy pathway, requiring RB1CC1/FIP200, PIK3C3/VPS34 activity and ATG5. Surprisingly, the mitophagy pathway upon recruitment of ATG16L1 proceeded independently of ATG5, although it still required RB1CC1 and PIK3C3/VPS34 activity. In this latter pathway, mitochondria were alternatively delivered to lysosomes via uptake into early endosomes.Abbreviation: aGFP: anti-GFP nanobody; amCh: anti-mCherry nanobody; ATG: autophagy related; ATG16L1: autophagy related 16 like 1; AUTAC/AUTOTAC: autophagy-targeting chimera; BafA1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; COX4/COX IV: cytochrome c oxidase subunit 4; DFP: deferiprone; DMSO: dimethyl sulfoxide; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; HRP: horseradish peroxidase; HTRA2/OMI: HtrA serine peptidase 2; IB: immunoblotting; IF: immunofluorescence; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NBR1: NBR1 autophagy cargo receptor; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; OPTN: optineurin; (D)PBS: (Dulbecco's) phosphate-buffered saline; PD: Parkinson disease; PFA: paraformaldehyde; POI: protein of interest; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAB, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase 1; VPS: vacuolar protein sorting; WIPI: WD repeat domain, phosphoinositide interacting.

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