Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells. MIC10 KO cells show a drastic reduction in MIC13, MIC26 and MIC27 protein levels, while protein levels of other MICOS components remain unchanged. MIC60 KO cells show a drastic reduction in protein levels of all MICOS components. B Representative electron micrographs of WT, MIC10 KO and MIC60 KO HAP1 cells. Scale bar 500 nm. C Quantification of CJs per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value. Data from n = 55–69 mitochondria (from two independent experiments) are shown as data points in the boxplots. **** P < 0.0001 (using unpaired Student's t ‐test). D Quantification of cristae per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value. Data from n = 55–69 mitochondria (from two independent experiments) are shown as data points in the boxplots. **** P < 0.0001 (using unpaired Student's t ‐test). E Comparison of oxygen consumption rates (pmol O 2 /s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non‐mitochondrial respiration in WT and MIC10 KO cells obtained from three independent experiments. The data are normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. * P = 0.034 for basal (using one‐sample t‐ test) and * P = 0.018 for maximum respiration (using unpaired Student's t ‐test). F Comparison of oxygen consumption rates (pmol O 2 /s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non‐mitochondrial respiration in WT and MIC60 KO cells obtained from three independent experiments. The data are normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from three independent experiments. * P = 0.01 for basal (using one‐sample t‐ test), *** P = 0.0004 for maximum respiration and * P = 0.012 for non‐mitochondrial respiration (using unpaired Student's t ‐test).

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Western Blot, Comparison, Staining

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Scheme of investigated marker proteins, subjected to FRAP, located at different mitochondrial subcompartments. B, C FRAP curves (curve fitted) of WT (B) and MIC13 KO HeLa cells (C) expressing GFP‐tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (three independent experiments, 6–10 mitochondria for each experiment). Error bars for each time point representing SEM are shown. D Histogram showing average percentage of mobile fractions, obtained from curve fits of all mitochondria expressing GFP‐tagged versions of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM) in WT and MIC13 KO HeLa cells (data calculated from FRAP curves shown in B and C that are obtained from three independent experiments, 6–10 mitochondria from each experiment). E Diffusion coefficients of the above‐mentioned proteins are shown in the table.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Marker, Expressing, Diffusion-based Assay

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Western blot analysis of WT and MIC13 KO HeLa cells showing a reduction in MIC10, MIC26 and MIC27 protein levels in MIC13 KOs. B Representative electron micrographs of WT and MIC13 KO HeLa cells show loss of CJs in MIC13 KOs. Scale bar 500 nm. C Boxplot showing quantification of CJs per mitochondrial section from different mitochondria in WT and MIC13 KO HeLa cells represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value. Data from n = 70–90 mitochondria (from two independent experiments) are shown as data points in the boxplots. **** P < 0.0001 (WT versus MIC13 KO), unpaired Student's t ‐test. D Boxplot showing quantification of cristae per mitochondrial section in WT and MIC13 KO HeLa cells represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value. Data from n = 70–90 mitochondria (from two independent experiments) are shown as data points in the boxplots. **** P < 0.0001, unpaired Student's t ‐test. E Representative STED super‐resolution images of WT HeLa cells stained with anti‐MIC13 antibody shows punctate distribution of MIC13. Scale bar 500 nm.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Western Blot, Staining

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A–D Representative single‐particle tracks of cells expressing MIC60‐ (A) and MIC10‐SNAP (B) in WT HeLa cells and MIC60‐ (C) and MIC10‐SNAP (D) in MIC13 KO HeLa cells, stained with silicon rhodamine dye and imaged at a rate of 33 ms/frame. Single tracks were colour‐coded according to temporal appearance. Scale Bar 5 μm. Insets in each image show zoomed‐in images of few representative tracks. E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO HeLa cells expressing MIC60‐ and MIC10‐SNAP stained with silicon rhodamine. Number of tracks analysed (obtained from two independent experiments): n = 2,541, 2,540, 3,560 and 1,441 in WT HeLa cells expressing MIC60‐SNAP and MIC10‐SNAP, MIC13 KO HeLa cells expressing MIC60‐SNAP and MIC10‐SNAP, respectively. (**** P ≤ 0.0001 for all possible comparisons, unpaired Student's t ‐test). F Percentage of subtracks having directed motion in WT and MIC13 KO HeLa cells expressing MIC60‐ and MIC10‐SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10‐SNAP. Number of corresponding subtracks analysed (obtained from two independent experiments): n = 2,561, 2,443, 3,402, 1,360, 561 and 1,110. Data are mean ± SEM. * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001, unpaired Student's t ‐test.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Single Particle, Expressing, Staining, Diffusion-based Assay

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A, B HEK293 cells expressing either MIC10‐SNAP/GFP (A) or MIC60‐SNAP/GFP (B) were used for coimmunoprecipitation using anti‐MIC13 antibody. Endogenous MIC13 could pull down MIC10‐SNAP or MIC10‐GFP (A) as well as MIC60‐SNAP or MIC60‐GFP (B). C Isolated mitochondria expressing MIC60‐SNAP or MIC60‐GFP were probed for proper incorporation of fusion proteins into the native MICOS complex. The complex containing MIC60‐GFP or MIC60‐SNAP runs at similar molecular weight as MICOS complex in native state marked by MIC60 or MIC10 antibody, confirming their incorporation into native complex. D MIC10 KO HAP1 cells expressing an empty vector (ev), 1 and 3 μg of MIC10‐SNAP/GFP and probed with antibodies against SNAP/GFP, MIC10 and MIC13. MIC13 protein levels in MIC10 KO HAP1 cells are rescued by the expression of MIC10‐SNAP (3 μg) and MIC10‐GFP (1 and 3 μg) showing that the GFP and SNAP tags do not interfere with the MIC10 function.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Expressing, Isolation, Molecular Weight, Plasmid Preparation

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A–D Representative live‐cell STED super‐resolution images ( t = 0 s) showing WT (A) and MIC13 KO (C) HeLa cells expressing MIC10‐SNAP stained with silicon rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D), respectively. Scale bar 500 nm. Time‐lapse image series of a mitochondrion expressing MIC10‐SNAP in WT (B) and MIC13 KO (D) HeLa cells (2.6 s/frame). Green and magenta asterisks show merging and splitting events of MIC10‐SNAP, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO HeLa cells expressing MIC10‐SNAP (three independent experiments, 5–8 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value. * P = 0.026 (merging and splitting events in WT cells) and **** P < 0.0001 (merging and splitting events of WT versus MIC13 KO), unpaired Student's t ‐test. F Scheme representing the dynamic nature of CJs in WT and MIC13 KO cells.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Expressing, Staining, Selection

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Representative live‐cell STED super‐resolution images ( t = 0 s) showing WT HeLa cells expressing MIC60‐SNAP stained with silicon rhodamine. Box in (A) marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time‐lapse image series of a mitochondrion expressing MIC60‐SNAP in WT HeLa cells (2.6 s/frame). Green and magenta asterisks show merging and splitting events of MIC60‐SNAP, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. C Representative live‐cell STED super‐resolution images ( t = 0 s) showing MIC13 KO HeLa cells expressing MIC60‐SNAP stained with silicon rhodamine. Box in (C) marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time‐lapse image series of a mitochondrion expressing MIC60‐SNAP in MIC13 KO HeLa cells (2.6 s/frame). Green and magenta asterisks show merging and splitting events of MIC60‐SNAP, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO HeLa cells expressing MIC60‐SNAP represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value. Data obtained from three independent experiments, 5–7 mitochondria for each experiment. *** P = 0.0008 (merging events WT versus MIC13 KO) and ** P = 0.0019 (splitting events WT versus MIC13 KO), unpaired Student's t ‐test.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Expressing, Staining, Selection

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Representative live‐cell STED super‐resolution images ( t = 0 s), showing WT HeLa cells expressing MIC13‐SNAP, from a time‐series of images acquired at a time interval of 2.5 s stained with silicon rhodamine. Box in (A) marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time‐lapse image series of a mitochondrion expressing MIC13‐SNAP imaged at a time interval of 2.5 s/frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13‐SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Cristae mergence events that form structure resembling letter “X” or “Y” are marked at 5 or 14.9 s, respectively. Scale bar 500 nm. C Representative live‐cell STED super‐resolution images (t=0 s), showing WT HeLa cells expressing MIC13‐SNAP, from a time‐series of images acquired at a time interval of 1.3 s stained with silicon rhodamine. Box in (C) marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time‐lapse image series of a mitochondrion expressing MIC13‐SNAP imaged at a time interval of 1.3 s/frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13‐SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E First image from (D) was used for showing the intensity profiles along the length of three cristae (numbered 1–3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM. Magenta boxes depict intensity maxima at IBM.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Expressing, Staining, Selection

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Raw data of live‐cell STED super‐resolution images ( t = 0 s) showing WT HeLa cells expressing MIC13‐SNAP stained with silicon rhodamine. Box in (A) marks selection shown as a zoom in panel (B). Raw data used for Fig A. Scale bar 500 nm. B Raw data of time‐lapse image series of a mitochondrion expressing MIC13‐SNAP in WT HeLa cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green “X” and “Y” represent X‐ and Y‐type imminent mergence events. Raw data used for Fig B. Scale bar 500 nm. C Raw data of live‐cell STED super‐resolution images ( t = 0 s) showing WT HeLa cells expressing MIC13‐SNAP stained with silicon rhodamine. Box in (C) marks selection shown as a zoom in panel (D). Raw data used for Fig C. Scale bar 500 nm. D Raw data of time‐lapse image series of a mitochondrion expressing MIC13‐SNAP in WT HeLa cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Raw data used for Fig D. Scale bar 500 nm. E Raw data of live‐cell STED super‐resolution images ( t = 0 s) showing WT HeLa cells expressing ATP5I‐SNAP stained with silicon rhodamine. Box in (E) marks selection shown as a zoom in panel (F). Raw data used for Fig A. Scale bar 500 nm. F Raw data of time‐lapse image series of a mitochondrion expressing ATP5I‐SNAP in WT HeLa cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green “X” and “Y” represent X‐ and Y‐type imminent merging events. Raw data used for Fig B. Scale bar 500 nm. G Raw data of live‐cell STED super‐resolution images ( t = 0 s) showing MIC13 KO HeLa cells expressing ATP5I‐SNAP stained with silicon rhodamine. Box in (G) marks selection shown as a zoom in panel (H). Raw data used for Fig C. Scale bar 500 nm. H Raw data of time‐lapse image series of a mitochondrion expressing ATP5I‐SNAP in MIC13 KO HeLa cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I‐SNAP, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Raw data used for Fig D. Scale bar 500 nm.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Expressing, Staining, Selection

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Representative live‐cell STED super‐resolution images ( t = 0 s) showing WT HeLa cells expressing ATP5I‐SNAP stained with silicon rhodamine. Box in (A) marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time‐lapse image series of a mitochondrion expressing ATP5I‐SNAP in WT HeLa cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I‐SNAP, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green “Y” represents Y‐type imminent mergence event. Scale bar 500 nm. C Representative live‐cell STED super‐resolution images ( t = 0 s) showing MIC13 KO HeLa cells expressing ATP5I‐SNAP stained with silicon rhodamine. Box in (C) marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time‐lapse image series of a mitochondrion expressing ATP5I‐SNAP in MIC13 KO HeLa cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I‐SNAP, respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E First image from (B) was used for showing the intensity profiles along the length of three cristae (numbered 1–3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM. The represented IBM area is superimposed from intensities of MIC13‐SNAP from Fig F. Magenta box depicts intensity maxima at cristae membrane apart from IBM. G Blind quantification of cristae merging and splitting events in WT and MIC13 KO HeLa cells expressing ATP5I‐SNAP (three independent experiments, 3–7 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value. **** P < 0.0001, unpaired Student's t ‐test.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Expressing, Staining, Selection, Membrane

Journal: EMBO Reports

Article Title: Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

doi: 10.15252/embr.201949776

Figure Lengend Snippet: A Mitochondrion in WT HeLa cells expressing ATP5I‐PAGFP was photoactivated, in the area shown by a blue box, at 0 s. Inset of white box at 0.98 s clearly shows fluorescent crista (red arrow), absent at 0.38, 0.59 and 0.78 s (red arrowhead showing the area enclosed by white dotted lines), confirming merging of non‐fluorescent cristae vesicle with the IBM. Threshold images of insets are shown above the whole images. Scale bar 500 nm. B Dual‐colour STED super‐resolution images of fused mitochondrion in WT HeLa cells (left panel) showing colocalization of cristae marked with ATP5I‐GFP (using anti‐GFP antibody) and ATP5I‐SNAP (stained with silicon rhodamine). (Right panel) Fused mitochondrion in MIC13 KO HeLa cells showing cristae marked with either ATP5I‐GFP (using anti‐GFP antibody) or ATP5I‐SNAP (stained with silicon rhodamine). Arrowheads show merged cristae, while arrows show cristae that maintain their individual identity. Scale bar 500 nm. C Boxplot showing quantification of colocalization events per μm of mitochondria in WT and MIC13 KO HeLa cells. Boxplots show median and interquartile range from 25 to 75 percentile, and whiskers represent minimum and maximum value (15 mitochondria were taken from WT and MIC13 KO cells from four and two independent experiments, respectively, * P = 0.03, unpaired Student's t ‐test). D Schematic illustration of the proposed “Cristae fission and fusion” (CriFF) as a working model and its link to CJ dynamics.

Article Snippet: 15% SDS–PAGE was performed, and separated proteins were subsequently blotted onto a PVDF membrane, and probed with indicated antibodies: MIC10 from Abcam (84969), MIC13 (custom‐made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625‐1‐AP), MIC25 (Proteintech, 20639‐1‐AP), MIC26 (Thermo Fisher, MA5‐15493), MIC27 (Atlas Antibodies, HPA000612), MIC60 (custom‐made, Pineda (Berlin)) against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), SNAP‐tag (P9310S, NEB) and β‐tubulin (Abcam, ab6046).

Techniques: Expressing, Staining