Monitoring mitochondrial translation in living cells

Yousefi, Roya; Fornasiero, Eugenio F.; Cyganek, Lukas; Montoya, Julio; Jakobs, Stefan; Rizzoli, Silvio O.; Rehling, Peter; Pacheu-Grau, David

doi:10.15252/embr.202051635

Cited by 53 publications

(41 citation statements)

References 56 publications

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“…1C), as reported in earlier work (Estell et al 2017), but also succeeded in human HEK293 and HeLa S3 cells (Fig. 1D), suggesting the versatility of in situ mito-FUNCAT in a wide array of cells (Yousefi et al 2021;Zorkau et al 2021).…”

Section: S1bsupporting

confidence: 88%

“…Note that we used a lower concentration of HPG (50 μM) than earlier reports (Estell et al 2017;Yousefi et al 2021;Zorkau et al 2021) (0.5-1 mM) to prevent the introduction of biases and/or artifacts with high doses of this compound. Under these conditions, we observed incremental accumulation of newly synthesized proteins in mitochondria over time (Supplemental Fig.…”

Section: S1bmentioning

confidence: 99%

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Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Kimura

Saito

Osaki

et al. 2022

Preprint

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Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

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Section: S1bsupporting

confidence: 88%

Section: S1bmentioning

confidence: 99%

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Kimura

Saito

Osaki

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…This exceeds the lifetime of many short-lived mitochondrial proteins (Vincow et al, 2013). Likewise, proteins of the respiratory chain encoded by the mitochondrial genome are constantly synthesized in axonal and dendritic mitochondria (Yousefi et al, 2021), but how the complementary nuclear-encoded subunits are supplied is unknown. Local translation of mitochondrial mRNAs provides a possible resolution of this problem.…”

Section: Introductionmentioning

confidence: 99%

Neuronal mitochondria transport Pink1 mRNA via Synaptojanin 2 to support local mitophagy

Harbauer

Wanderoy

et al. 2021

Preprint

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PTEN-induced kinase 1 (PINK1) is a very short-lived protein that is required for the removal of damaged mitochondria through Parkin translocation and mitophagy. Because the short half-life of PINK1 limits its ability to be trafficked into neurites, local translation is required for this mitophagy pathway to be active far from the soma. The Pink1 transcript is associated with and cotransported with neuronal mitochondria. In concert with translation, the mitochondrial outer membrane protein Synaptojanin 2 binding protein (SYNJ2BP) and Synaptojanin 2 (SYNJ2) are required for tethering Pink1 mRNA to mitochondria via an RNA-binding domain in SYNJ2. This neuron-specific adaptation for local translation of PINK1 provides distal mitochondria with a continuous supply of PINK1 for activation of mitophagy.

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“…Mitochondrial translation activity was monitored using Click-iT TM HPG Alexa Fluor TM 488 Protein synthesis Assay Kit (Invitrogen, Carlsbad, CA) as described previously with a slight modification [ 31 ]. To label newly synthesized proteins at mitoribosome, cytosolic translation activity was inhibited by preincubating cells with 20 μg/ml emetine (Merck Millipore, Burlington, MA) for 1 h. Then, cells were incubated in methionine-free RPMI medium (Invitrogen) supplemented with 50 μM L-homopropargylglycine (Click-iT TM HPG, Invitrogen) for 1 h. After washed twice with PBS, the cells were lysed in lysis buffer (1% SDS, 50 mM Tris, pH 8.0, 1 mM NaF, 1 mM Na 3 VO 4 , 1 mM PMSF, 1.5 μg/ml pepstatin A, 1.5 μg/ml leupeptin).…”

Section: Methodsmentioning

confidence: 99%

MRPS31 loss is a key driver of mitochondrial deregulation and hepatocellular carcinoma aggressiveness

et al. 2021

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Deregulated mitochondrial energetics is a metabolic hallmark of cancer cells. However, the causative mechanism of the bioenergetic deregulation is not clear. In this study, we show that somatic copy number alteration (SCNA) of mitoribosomal protein (MRP) genes is a key mechanism of bioenergetic deregulation in hepatocellular carcinoma (HCC). Association analysis between the genomic and transcriptomic profiles of 82 MRPs using The Cancer Genome Atlas-Liver HCC database identified eight key SCNA-dependent MRPs: MRPS31, MRPL10, MRPL21, MRPL15, MRPL13, MRPL55, and DAP3. MRPS31 was the only downregulated MRP harboring a DNA copy number (DCN) loss. MRPS31 loss was associated specifically with the DCN losses of many genes on chromosome 13q. Survival analysis revealed a unique dependency of HCC on the MRPS31 deficiency, showing poor clinical outcome. Subclass prediction analysis using several public classifiers indicated that MRPS31 loss is linked to aggressive HCC phenotypes. By employing hepatoma cell lines with SCNA-dependent MRPS31 expression (JHH5, HepG2, Hep3B, and SNU449), we demonstrated that MRPS31 deficiency is the key mechanism, disturbing the whole mitoribosome assembly. MRPS31 suppression enhanced hepatoma cell invasiveness by augmenting MMP7 and COL1A1 expression. Unlike the action of MMP7 on extracellular matrix destruction, COL1A1 modulated invasiveness via the ZEB1-mediated epithelial-to-mesenchymal transition. Finally, MRPS31 expression further stratified the high COL1A1/DDR1-expressing HCC groups into high and low overall survival, indicating that MRPS31 loss is a promising prognostic marker. Significance Our results provide new mechanistic insight for mitochondrial deregulation in HCC and present MRPS31 as a novel biomarker of HCC malignancy.

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Monitoring mitochondrial translation in living cells

Cited by 53 publications

References 56 publications

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Neuronal mitochondria transport Pink1 mRNA via Synaptojanin 2 to support local mitophagy

MRPS31 loss is a key driver of mitochondrial deregulation and hepatocellular carcinoma aggressiveness

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