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Figure 1 | Generating defined parkin-labelled mitochondria as substrates for autophagy. (a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right). (b) Diagram of light-induced mitophagy. Single mitochondrial tubules containing KR-dMito, when illuminated by 559 nm light, are selectively impaired, leading to surface recruitment of parkin and subsequent autophagic turnover. (c) Illuminating a selective region (white dotted circle, top) within a <t>KR-dMito/EBFP2-parkin-expressing</t> HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38–60 min after 559 illumination, white arrows). Scale bars (all panels), 10 mm.
Ebfp2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Figure 1 | Generating defined parkin-labelled mitochondria as substrates for autophagy. (a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right). (b) Diagram of light-induced mitophagy. Single mitochondrial tubules containing KR-dMito, when illuminated by 559 nm light, are selectively impaired, leading to surface recruitment of parkin and subsequent autophagic turnover. (c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38–60 min after 559 illumination, white arrows). Scale bars (all panels), 10 mm.

Journal: Nature communications

Article Title: Bit-by-bit autophagic removal of parkin-labelled mitochondria.

doi: 10.1038/ncomms3428

Figure Lengend Snippet: Figure 1 | Generating defined parkin-labelled mitochondria as substrates for autophagy. (a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right). (b) Diagram of light-induced mitophagy. Single mitochondrial tubules containing KR-dMito, when illuminated by 559 nm light, are selectively impaired, leading to surface recruitment of parkin and subsequent autophagic turnover. (c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38–60 min after 559 illumination, white arrows). Scale bars (all panels), 10 mm.

Article Snippet: EBFP2-parkin was generated by subcloning EBFP2 (Addgene plasmid 14,893)29 into the EYFP-parkin vector obtained from Addgene (plasmid 23,955)7.

Techniques: Staining, Expressing

Figure 2 | Bit-by-bit removal of parkin-labelled mitochondria by autophagy. (a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b–d) Magnified view of the photo-inactivated region in a 34–93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 mm.

Journal: Nature communications

Article Title: Bit-by-bit autophagic removal of parkin-labelled mitochondria.

doi: 10.1038/ncomms3428

Figure Lengend Snippet: Figure 2 | Bit-by-bit removal of parkin-labelled mitochondria by autophagy. (a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b–d) Magnified view of the photo-inactivated region in a 34–93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 mm.

Article Snippet: EBFP2-parkin was generated by subcloning EBFP2 (Addgene plasmid 14,893)29 into the EYFP-parkin vector obtained from Addgene (plasmid 23,955)7.

Techniques: Expressing, Sequencing

Figure 3 | Omegasomes form where ER and parkin-labelled mitochondria overlap. (a) A HeLa cell expressing KR-dMito (red), EGFP-DFCP1 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted circle region, leading to the generation of a long parkin-labelled mitochondria tubule (right, magnified view of the light-activated region) for autophagic turnover (white arrow). Scale bars, 10 mm. (b) After the generation of parkin-labelled mitochondrial tubules following treatments in a, discrete DFCP1 foci (green, middle panel, ER-associated omegasomes) formed along the parkin-labelled mitochondria (white arrows indicate DFCP1 spots that colocalized with parkin). Bottom panel: merged image of parkin and DFCP1. Scale bar, 10 mm. (c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 mm. (d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61b (green) and TagRFP-DFCP1 (red) were treated with 10 mM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61b) and impaired mitochondria (parkin) overlap. Scale bar, 10 mm. (e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin. (f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each ‘ þ ’ marks the times measured in individual HeLa cells (the red ‘&’ (square) denotes the average from five cells). P ¼ 0.0172 (Student’s t-test).

Journal: Nature communications

Article Title: Bit-by-bit autophagic removal of parkin-labelled mitochondria.

doi: 10.1038/ncomms3428

Figure Lengend Snippet: Figure 3 | Omegasomes form where ER and parkin-labelled mitochondria overlap. (a) A HeLa cell expressing KR-dMito (red), EGFP-DFCP1 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted circle region, leading to the generation of a long parkin-labelled mitochondria tubule (right, magnified view of the light-activated region) for autophagic turnover (white arrow). Scale bars, 10 mm. (b) After the generation of parkin-labelled mitochondrial tubules following treatments in a, discrete DFCP1 foci (green, middle panel, ER-associated omegasomes) formed along the parkin-labelled mitochondria (white arrows indicate DFCP1 spots that colocalized with parkin). Bottom panel: merged image of parkin and DFCP1. Scale bar, 10 mm. (c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 mm. (d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61b (green) and TagRFP-DFCP1 (red) were treated with 10 mM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61b) and impaired mitochondria (parkin) overlap. Scale bar, 10 mm. (e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin. (f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each ‘ þ ’ marks the times measured in individual HeLa cells (the red ‘&’ (square) denotes the average from five cells). P ¼ 0.0172 (Student’s t-test).

Article Snippet: EBFP2-parkin was generated by subcloning EBFP2 (Addgene plasmid 14,893)29 into the EYFP-parkin vector obtained from Addgene (plasmid 23,955)7.

Techniques: Expressing, Imaging

Figure 4 | Preferentially ubiquitinated foci signal bit-by-bit mitophagy. (a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61b (green) and mcherry-parkin (cyan) was treated with 10 mM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61b). (b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light- activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows). (d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2- parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 mm.

Journal: Nature communications

Article Title: Bit-by-bit autophagic removal of parkin-labelled mitochondria.

doi: 10.1038/ncomms3428

Figure Lengend Snippet: Figure 4 | Preferentially ubiquitinated foci signal bit-by-bit mitophagy. (a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61b (green) and mcherry-parkin (cyan) was treated with 10 mM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61b). (b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light- activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows). (d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2- parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 mm.

Article Snippet: EBFP2-parkin was generated by subcloning EBFP2 (Addgene plasmid 14,893)29 into the EYFP-parkin vector obtained from Addgene (plasmid 23,955)7.

Techniques: Expressing, Staining, Ubiquitin Proteomics