Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Schematic illustrating the PINK1–Parkin pathway reporter MFN2-Halo. ( B ) Representative confocal images of HeLa MFN2-Halo+mCh-Parkin cells transduced with a cassette co-expressing a dual sgRNA targeting MFN2 and TagBFP2 to mark transduced cells (white outline). Scale bar = 20 µm. ( C ) Flow cytometry of HeLa MFN2-Halo cells demonstrating loss of MFN-Halo signal in the presence of MFN2 sgRNA. Unpaired t test two-tailed **** P ≤ 0.0001 (exact P value P = 4.9e-07). Error bars mean +/− SD. N = 3 independent experiments. ( D ) Representative immunoblots of endogenously tagged and untagged MFN2 alleles (MFN2-Halo and MFN2-UT, respectively) of the same cells in (1B), showing that MFN2-Halo degradation following AO treatment is PINK1 dependent. N = 5 replicates on at least two occasions. ( E ) Representative histograms demonstrating the PINK1-dependent decrease in MFN2-Halo fluorescence following treatment with AO in HeLa MFN2-Halo+mCh-Parkin cells and quantification. ns = P = 0.8883, **** P ≤ 0.0001 (exact P values NT CTRL vs AO CTRL, P = 1.8e-14; AO CTRL vs AO PINK1, P = 1.8e-14; NT PINK1 vs AO PINK1, P = 8.27e-5) by two-way ANOVA with Tukey’s multiple comparisons test. Error bars mean +/− SD. N = 3 independent experiments. ( F ) Schematic illustrating the FACS-based genome-wide CRISPRi screening strategy. Six screens were performed in total, using three different cell lines with or without exposure to OXPHOS inhibitors. ( G ) Stacked bar graph represents the proportion of nuclear gene perturbations encoding mitochondrial (mito) vs. non-mitochondrial proteins (not mito). ( H ) Dot plots of gene scores (product of log 2 fold change and adjusted −log 10 P value) from screens described in ( F ). ( I ) One-sided volcano plots for screens described in ( F ). .

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Transduction, Expressing, Flow Cytometry, Two Tailed Test, Western Blot, Fluorescence, Genome Wide

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Schematic illustrating the core glycolysis pathway with screen hits in red and inhibitors tested in black. ( B ) Flow cytometry measurements in HeLa MFN2-Halo+mCh-Parkin cells (top), HeLa mt-Keima (middle), and HeLa PINK1-YFP (bottom) treated with CCCP 10 µM for 4 h, illustrating similar results between PINK1 and ENO1 sgRNA. **** P ≤ 0.0001 by Brown–Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons test (exact P values top graph—CTRL vs ENO1, P = 6.07e-09; CTRL vs PINK1, P = 2.17e-07; exact P values middle graph—CTRL vs ENO1, P = 4.55e-05; CTRL vs PINK1, P = 2.56e-08; exact P values bottom graph—CTRL vs ENO1, P = 3.26e-09; CTRL vs PINK1, P = 6.46e-09). Error bars mean +/− SD. N = 6 independent experiments from two separate transductions. ( C ) Representative immunoblots of PINK1 stabilization and activity in HeLa dCas9-BFP-ZIM3 cells with indicated glycolytic enzyme sgRNA treated with 10 µM CCCP + /− 10 mM 2-DG for 4 h. N = 3 independent experiments. ( D ) Representative immunoblots of PINK1 stabilization and activity in HeLa dCas9-BFP-ZIM3 cells treated with 10 µM CCCP, 10 µM HA, 10 mM 2-DG, and/or 50 µg/mL CHX for 4 h. N = 3 independent experiments. ( E ) Representative confocal image of i 3 Neurons immunostained for neuronal markers. Scale bar = 10 µm. ( F ) Representative immunoblot of lysates from i 3 Neurons, treated with 10 mM 2-DG, 10 µM HA, and/or 20 µM CCCP, with or without insulin withheld, probing for PINK1 stabilization and activity. *Denotes non-specific band. MFN2-ub1 = monoubiquitinated MFN2. N ≥ 6 total replicates from at least two independent differentiations. ( G ) Immunoblot SUNset assay (bottom) performed in HeLa PINK-YFP cells as indicated in the scheme (top), demonstrating block in overall translation and loss of PINK1 accumulation in the presence of 10 µM CCCP and 10 mM 2-DG for 4 h. Puro puromycylated proteins. N = 3 independent experiments. ( H ) Immunoblot of HeLa dCas9-BFP-ZIM3 cells illustrating cleaved PINK1 (PINK1-c) abundance, stabilized by proteasome inhibition (50 µM MG132), following inhibition of mitochondrial ATP (10 µg/mL oligomycin) and/or glycolytic ATP (10 mM 2-DG) production for 4 h. N = 3 replicates on at least two occasions. ( I ) Flow cytometry measurements of PINK1-YFP (left graph) and mitochondrial membrane potential with TMRE 20 nM (right graph), following drug treatment for 4 h. Graphs are representative of N = 3 independent experiments. ( J ) Immunoblot of HeLa dCas9-BFP-ZIM3 cells treated with escalating doses of CCCP with or without F 1 F O -ATP synthase inhibition (oligomycin 10 µg/mL) for 4 h, probing PINK1 stabilization and activation. Import block of ATP5A is monitored through the accumulation of uncleaved ATP5A (ATP5A-p) relative to mature ATP5A (ATP5A-m). N = 3 independent experiments. .

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Flow Cytometry, Western Blot, Activity Assay, Blocking Assay, Inhibition, Membrane, Activation Assay

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Heatmaps show log 2 fold changes for the top 20 mitochondrial PINK1–Parkin activators in each of the six screens described in (Fig. ). ( B ) Schematic summarizing the methods used to characterize the top PINK1–Parkin activators. ( C ) Representative TEM micrographs show abnormalities in mitochondrial ultrastructure induced by knockdown of the top PINK1–Parkin activators in HeLa dCas9-BFP-ZIM3 cells transduced with vector co-expressing sgRNA and APEX-ER. Transduced cells were readily identified by dark staining of the osmophilic polymer in the ER lumen produced by the APEX-DAB reaction. In some samples, cupped mitochondria (closed yellow arrowheads), indicative of membrane potential collapse (Ding et al, ; Miyazono et al, ), were found adjacent to intact mitochondria (open arrowheads). A fluffy aggregate in the matrix of mitochondria (arrow) was observed in DNAJA3 sgRNA cells. The percentage of cells with abnormal mitochondria out of ten or more cells imaged per sample is indicated in the upper right of each image. The text below the images describes the predominant abnormal features of the cristae and cup-shaped or small mitochondrial morphology if observed. Control cells from two transductions were analyzed, while single transductions were performed and analyzed for non-control cells. Scale bar = 1 µm. ( D ) CLEM images from a NDUFAB1 KD HeLa PINK1-YFP+MTS-mCh cell. PINK1-YFP selectively accumulates on cupped mitochondria (closed arrowheads) while sparing adjacent intact mitochondria (open arrowheads). Serial sections of two boxed areas (1 and 2) are shown at magnification. Scale bars = 2 µm. ( E ) Representative confocal image of live HeLa PINK1-YFP+MTS-mCh cells transduced with a guide targeting NDUFAB1, showing PINK1-YFP accumulation on a subset of mitochondria that have not accumulated the MMP-dependent dye MitoLite NIR (arrowheads). Scale bar = 10 µm. ( F ) Graph of cells in ( E ) comparing PINK1-YFP intensity on mitochondria with and without MMP within the same cell, measured from confocal images as in ( E ). **** P ≤ 0.0001 (exact P value, P = 7e-15) by two-tailed Wilcoxon matched-pairs signed rank test. N = 48 cells were analyzed in total from five wells and two independent transductions. ( G ) Graph of cells in ( E ) comparing PINK1-YFP co-localization to MTS-mCh and the MMP dye MitoLite NIR. The mean Pearson coefficient for 20 pixel-randomized images (Rrand) above the background for each image. **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Mann–Whitney test. N = 50 cells were analyzed in total from five wells and two independent transductions. ( H ) Volcano plots show changes to whole-cell protein abundance following transduction with the indicated guide vs. a non-targeting guide in HeLa cells without Parkin (HeLa dCas9-BFP-ZIM3 ). Boxes show the top MitoCarta3.0 mitochondrial pathways identified in enrichment analysis of significantly down-regulated proteins. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating an FDR with the Benjamini–Hochberg procedure. Proteins were annotated as significant if they had an FDR < 0.05 and an absolute log 2 fold change of >1 (black outline). N = 4 replicates/sgRNA on one occasion. .

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Knockdown, Transduction, Plasmid Preparation, Expressing, Staining, Polymer, Produced, Membrane, Control, Two Tailed Test, MANN-WHITNEY, Quantitative Proteomics

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Transmitted light image of HeLa dCas9-BFP-ZIM3 cells shows the brown DAB reaction product after ~12 min of development time in cells transduced with APEX-ER (solid arrowheads) compared to cells expressing low or no APEX-ER (open arrowheads). Scale bar = 100 µm. ( B ) TEM image of a cell with low or no APEX-ER staining in the ER lumen (open arrowheads) next to a cell with darkly stained ER lumen (closed arrowheads) indicating a high level of APEX-ER expression. Scale bar = 2 µm. ( C ) Examples of abnormal ultrastructural features of mitochondria expressing sgRNAs for the protein indicated in the lower left corner of each image. Yellow arrows indicate the abnormal cristae feature listed at the top of each image; yellow asterisks indicate areas with sparse cristae. Red arrows indicate the fluffy aggregate observed in the matrix of cells transduced with sgRNA DNAJA3. Blue asterisks indicate cytosol enclosed cup-shaped mitochondria. In some cases, examples shown were cropped from the same cell. Scale bar = 500 nm.

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Transduction, Expressing, Staining

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Flow cytometry measurements of MFN2-Halo degradation (HeLa MFN2-Halo+mCh-Parkin cells). N = 6 biological replicates measured from two independent transductions (separate transductions denoted by open or closed circles). Error bars mean +/− SD. ( B ) Flow cytometry measurements of PINK1-YFP (top) and MMP with dye MitoLite NIR (bottom) obtained from the same cells. (HeLa PINK1-YFP cells) N = 6 biological replicates measured from 2 independent transductions (separate transductions denoted by open or closed circles). Error bars mean +/− SD. ( C ) Graph of experiment in ( B ) directly comparing PINK1-YFP and MMP for each gene perturbation. ( D ) Flow cytometry measurements of mitophagy using HeLa mt-Keima cells without Parkin (left) or with exogenous YFP-Parkin expression (right). N = 6 biological replicates measured from 2 independent transductions (separate transductions denoted by open or closed circles). Error bars mean +/− SD. ( E ) Dot plots comparing z-scores from the MFN2-Halo CRISPRi screen in not treated HeLa cells expressing mCh-Parkin (Fig. , middle) with previously published CRISPRi screens of respiratory ATP (left) and mitochondrial superoxide (right) (Bennett et al, , ). ( F ) Representative 2D kernel density plots comparing single-cell PINK1-YFP intensity and intensity of the MMP-sensitive dye MitoLite NIR from the same experiments as shown in ( B , C ). The arrow indicates a distinct population of cells, high in both MMP and PINK1-YFP, that was observed following TIMM23 KD. ( G ) Quantification of the proportion of cells in each of the quadrant 2D kernel density plots, such as those seen in (Figs. and ). ( H ) Representative confocal image of live HeLa PINK1-YFP+MTS-mCh cells transduced with a guide targeting PMPCB (top) or TIMM23 (bottom). Rectangle images are zoomed in the area of the white rectangle outlined in the square images above. PMPCB KD zoomed in area—arrowheads point to cells that have high PINK1-YFP expression, lost MMP, and import is not blocked. TIMM23 KD—Cells labeled 1— population with preserved MMP, no PINK1-YFP expression, and import is not blocked. Cells labeled 2—population with import block, high PINK1-YFP expression, and preserved MMP (examples denoted by white arrowheads in TIMM23 KD zoomed in area). Cells labeled 3—population with high PINK1-YFP expression, have lost MMP, and import is blocked. Scale bar = 10 µm. ( I ) The graph on the left is as described in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Mann–Whitney test. N = 54 cells were analyzed in total from four wells and two separate transductions. The graph on the right is as described in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Wilcoxon matched-pairs signed rank test. N = 54 cells were analyzed in total from four wells and two independent transductions. ( J ) The graph on the left is as described in (Fig. ). Exact P value, ns P = 0.6838 by two-tailed Mann–Whitney test. N = 65 cells were analyzed in total from six wells and two separate transductions. Graph on the right compares PINK1-YFP intensity on mitochondria following CTRL or TIMM23 KD. Only cells with high MMP were analyzed from both groups, and only cells with mitochondrial block (based on MTS-mCh signal) were analyzed for the TIMM23 KD group. **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Mann–Whitney test. N = 65 TIMM23 KD cells and 82 CTRL KD cells were analyzed in total from six wells and two independent transductions. .

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Flow Cytometry, Expressing, Transduction, Labeling, Blocking Assay, Two Tailed Test, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Volcano plot shows interactors of PINK1-YFP (red) identified by AP-MS following transduction with CTRL or PINK1 guides in HeLa PINK1-YFP cells. Cells in both groups were treated with 10 µM CCCP overnight. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. ( B ) Flow cytometry measurements of PINK1-YFP intensity (top) and intensity of MTS-mCh (bottom) from the same HeLa PINK1-YFP+MTS-mCh cells. N = 6 biological replicates measured from two independent transductions. All statistical comparisons are to the vehicle-treated CTRL guide group. Exact P values, ns P = 0.2179, **** P ≤ 0.0001 (exact P values P = 1.2e-14 for all comparisons) by two-way ANOVA with Dunnett’s multiple comparison test. Error bars mean +/− SD. ( C ) Representative confocal Airyscan images shown as z-projections in HeLa PINK1-YFP cells immunostained for TOMM70 to mark mitochondria. The arrow indicates PINK1-YFP accumulated around lipid droplets; the arrowhead indicates PINK1 co-localizing with TOMM70. Scale bar = 10 µm. ( D ) CLEM images demonstrating that the PINK1-YFP accumulates around lipid droplets (LD) following loss of the TOM translocase (HeLa PINK1-YFP cells). ER (yellow arrowheads) and small vesicles (red arrows) were adjacent to lipid droplet collections and accumulated PINK1-YFP. Scale bars: green = 20 µm; yellow = 5 µm; black = 1 µm; white = 500 nm. ( E ) Representative confocal images of TIMM23 KO pools in HeLa PINK1-YFP cells showing PINK1-YFP accumulates on mitochondria in cells lacking TIMM23 by immunostaining. Scale bar = 10 µm. ( F ) Line scan of dotted line in ( E ). ( G ) Quantification of ( E ). **** P ≤ 0.0001 (exact P value P = 9.0037e-05) via two-tailed unpaired t test with Welch’s correction. N = 4 wells analyzed, plated on two separate occasions from the same KO pool. Cells were analyzed 7 or 8 days after electroporation, and cells without TIMM23 were scored for PINK1-YFP accumulated on TOMM40-positive mitochondria. Error bars mean +/− SD. ( H ) Representative immunoblots from HeLa cells with exogenous PINK1-YFP (left blot) or endogenous PINK1 (right blot) +/− 10 µM CCCP treatment for 4 h, probing for PINK1 activation and stabilization. N ≥ 3 independent experiments. ( I ) Volcano plot shows PINK1-YFP (red) interactors, following CTRL vs. TIMM23 KD in HeLa PINK1-YFP cells. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. ( J ) In gel fluorescence of PINK1-YFP complexes separated by CN-PAGE after mixing lysates with the indicated antibodies. The specific interaction of the antibody with PINK1-YFP-containing complex increases the molecular weight of the complex, causing it to shift up in the gel. 10 µM CCCP treatment in the indicated lanes was for 3 h. N = 2 independent experiments with TOMM20 gel shift, one of which also tested TOMM22 and TOMM40 antibodies. (HeLa PINK-YFP cells). .

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Protein-Protein interactions, Transduction, Flow Cytometry, Comparison, Immunostaining, Two Tailed Test, Electroporation, Western Blot, Activation Assay, Fluorescence, Clear Native PAGE, Molecular Weight, Gel Shift

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Representative confocal images of TOMM22, TOMM40, TIMM23 KO pools in HeLa PINK1-YFP+MTS-mCh cells showing PINK1-YFP accumulates in the same pattern as observed by CRISPRi. Images were obtained 7 or 8 days after electroporation with Cas9 ribonucleoprotein complexes. Scale bar 10 = µm. ( B ) Total protein measured via SimplyBlue SafeStain of same gel as in (Fig. ), demonstrating equal loading. ( C ) Flow cytometry data of HeLa MFN2-Halo cells + BFP-Parkin transfected with ATP5MG-mCherry-sfGFP and treated +/− 10 µM CCCP for 4 h, illustrating differences in MFN2-Halo levels in the presence of the clogger. ns P = 0.5976, **** P ≤ 0.0001 (exact P value P < 1e-15) by two-way ANOVA with Tukey’s multiple comparisons test. Error bars mean +/− SD. N = 3 independent experiments, 9 replicates. ( D ) Flow cytometry data of HeLa MFN2-Halo cells transfected with ATP5MG-mCherry-sfGFP and treated +/− 10 µM CCCP for 4 h, illustrating differences in MFN2-Halo levels in the presence of the clogger. ** P = 0.0086, **** P ≤ 0.0001 (exact P value P = 7.4e-09) by two-way ANOVA with Tukey’s multiple comparisons test. Error bars mean +/− SD. N = 3 independent experiments, 10 replicates. ( E ) Flow cytometry data of HeLa PINK1-YFP cells expressing TOMM70 endogenously tagged with HaloTag and IMMT-DHFR clogger. Cells were treated +/− 20 µM CCCP for 4 h, demonstrating differences in PINK1-YFP stabilization. ns P = 0.4962, *** P = 0.0004 by two-way ANOVA with Šídák’s multiple comparisons test. Error bars mean +/− SD. N = 6 replicates run on two different occasions (separate occasions denoted by open or closed circles). ( F ) Flow cytometry of HeLa PINK1-YFP cells. *** P = 0.0003, **** P ≤ 0.0001 (exact P values - CTRL vs PAM16, P = 1.1e-05; CTRL vs TIMM23, P = 9.3e-07; CTRL vs NDUFAB1, P = 9e-06; CTRL vs PMPCB, P = 3.6e-07; TIMM44 vs TIMM23, P = 1.2e-06; PAM16 vs TIMM23, 1.3e-08). Error bars mean +/− SD. N = 6 replicates (5 for TIMM23) from 2 independent transductions. ( G ) Representative 2D kernel density plots comparing single-cell PINK1-YFP intensity and intensity of the MMP sensitive dye MitoLite NIR as in (Fig. ).

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Electroporation, Flow Cytometry, Transfection, Expressing

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Representative immunoblot comparing endogenous PINK1 activation to import block of ATP5A as described for (Fig. ) in HeLa dCas9-BFP-ZIM3 cells. N = 3 independent experiments. ( B ) Schematic showing the dual functions of HSPA9 in the PAM import motor and folding of newly imported proteins. HSPA9 performs the latter with its co-chaperone DNAJA3. ( C ) Representative confocal images of PINK1-YFP and MTS-mCh in TIMM23 and TIMM44 KO pools 7 days after electroporation. Chronic import block through the TIM23 translocase is identified by accumulation of MTS-mCh in the cytosol (cells with white outlines). Scale bar = 10 µm. (HeLa PINK1-YFP+MTS-mCh cells). ( D ) Quantification of the experiment in ( C ). Mitochondrial PINK1-YFP intensity was measured in the subset of cells with import block (left) and correlation of PINK1-YFP with import block of MTS-mCh for all cells (right). **** P ≤ 0.0001 (exact P value P < 1e-15) by Mann–Whitney test. 89 or more cells per condition were analyzed from N = 4 wells, plated on two separate days, from the same KO pool. Cells were analyzed 7 days after electroporation. ( E ) Immunoblot of 1% Triton X100 soluble and insoluble fractions from whole-cell lysates, probing for OXA1L and NDUFA9. N = 3 replicates on at least two occasions in HeLa dCas9-BFP-ZIM3 cells. ( F ) LFQ proteomics depicting protein abundance from the 1% Triton X100 insoluble and soluble heavy membrane fractions, normalized to the median value of non-mitochondrial proteins in the sample. Left ns P = 0.9827, right ns P = 0.9930, * P = 0.0489, **** P ≤ 0.0001 (Insoluble exact P values from left to right, P = 7.05e-05, P = 1.42e-09, P < 1e-15; Soluble exact P values from left to right, P < 1e-15, p < 1e-15) by Brown–Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons test. N = 4 replicates/sgRNA on one occasion. (HeLa dCas9-BFP-ZIM3 cells). ( G ) Volcano plot showing PINK1-YFP (red) interactors, following CTRL vs. DNAJA3 KD in HeLa PINK1-YFP cells. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. ( H ) The left graph quantification of cells in (Fig. ) was performed as in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Mann–Whitney test. N = 86 cells from six wells and two separate transductions. The right graph quantification of cells in (Fig. ) was performed as in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Wilcoxon matched-pairs signed rank test. N = 91 cells from six wells and two separate transductions. ( I ) CLEM images from a LONP1 KD HeLa PINK1-YFP+MTS-mCh cell demonstrating that mitochondrial aggregates (yellow arrowheads) were observed both in mitochondria that retained MMP (white box 1 in image) and mitochondria that had lost MMP (white box 1 in image) via TEM, but PINK1-YFP preferentially accumulated on aggregate-containing mitochondria that had lost MMP (white box 2 in image). Scale bars white = 10 µm, black = 1 µm, yellow = 500 nm. ( J ) Left graph quantification of cells in (Fig. ) was performed as in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Mann–Whiteney test. N = 80 cells from 4 wells and 2 separate transductions. Right graph quantification of cells in (Fig. ) was performed as in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by Wilcoxon matched-pairs signed rank test. N = 81 cells from four wells and two separate transductions. .

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Western Blot, Activation Assay, Blocking Assay, Electroporation, MANN-WHITNEY, Quantitative Proteomics, Membrane, Two Tailed Test

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Representative confocal image of live HeLa PINK1-YFP+MTS-mCh cells transduced with a guide targeting DNAJA3. Rectangle images are zoomed in area of white rectangle outlined in square image above. Arrowheads point to cells that have high PINK1-YFP expression on mitochondria that have lost MMP and import is not blocked. Scale bar = 10 µm. ( B ) Flow cytometry of HeLa PINK1-YFP cells transduce with the indicated sgRNA, illustrating PINK1-YFP levels. ns P = 0.3001, ** P = 0.0013, **** P ≤ 0.0001 (exact P values - CTRL vs TIMM23, P < 1e-15; CTRL vs HSPE1, p = 1e-15; CTRL vs LONP1, P = 8.1e-07), by ordinary one-way ANOVA with Šídák’s multiple comparisons test. Error bars mean +/− SD. N = at least 7 replicates from 2 independent transductions (separate transductions denoted by open or closed circles). ( C ) Representative 2D kernel density plots comparing single-cell PINK1-YFP intensity and intensity of the MMP sensitive dye MitoLite NIR from the same experiment as shown in Fig. EV5B. ( D ) Representative immunoblots of HeLa dCas9-BFP-ZIM3 cells transduced with indicated sgRNAs, illustrating PINK1 stabilization and activation. N = 3 independent experiments. ( E ) Representative confocal image of live HeLa PINK1-YFP+MTS-mCh cells transduced with a guide targeting LONP1. PINK1-YFP accumulated preferentially on mitochondria with low MMP. Scale bar = 10 µm. ( F ) Representative confocal image of live HeLa PINK1-YFP+MTS-mCh cells transduced with a guide targeting HSPD1. PINK1-YFP accumulated preferentially on mitochondria with low MMP. Scale bar = 10 µm. ( G ) Left graph quantification of cells in (Fig. EV5F) was performed as in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by two-tailed Mann–Whiteney test. N = 56 cells from 4 wells and 2 separate transductions. Right graph quantification of cells in (Fig. EV5F) was performed as in (Fig. ). **** P ≤ 0.0001 (exact P value P < 1e-15) by Wilcoxon matched-pairs signed rank test. N = 56 cells from 4 wells and 2 separate transductions. ( H ) Representative 2D kernel density plots comparing single-cell PINK1-YFP intensity and intensity of the MMP sensitive dye MitoLite NIR in HeLa PINK1-YFP + TOMM70-Halo cells +/− transient transfection of ΔOTC. ( I ) Total protein measured via SimplyBlue SafeStain of same gel in (Fig. ), demonstrating equal loading.

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Transduction, Expressing, Flow Cytometry, Western Blot, Activation Assay, Two Tailed Test, Transfection

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Flow cytometry measurements performed as in (Fig. ). Left graph ns P = 0.0988, Right graph from left to right ns P = 0.5132, 0.2695, **** P ≤ 0.0001 (left graph exact P values from left to right P = 7e-15, 1.7e-14, 7e-15, 7e-15; right graph exact P values from left to right 4.62e-11, 7e-15, 7e-15) by two-way ANOVA with Dunnett’s multiple comparisons test. Error bars mean +/− SD. N = 6 independent experiments from two separate transductions (separate transductions denoted by open or closed circles). Error bars mean +/− SD. ( B ) Representative immunoblots of PINK1 stabilization and activity in HeLa dCas9-BFP-ZIM3 cells with indicated sgRNA +/− 10 µM CCCP for 4 h. N ≥ 3 independent experiments. ( C ) Volcano plots of PINK1-YFP (red) interactors measured by AP-MS as in (5I). CCCP treatment, where indicated, was overnight. Other samples were untreated. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. The untreated control guide group was the same as used in (Fig. ). ( D ) CN-PAGE separated PINK1-YFP complexes visualized by in-gel fluorescence as in (Fig. ). N = 2 independent experiments with TOMM40 KD, one of which was with antibody gel shift. ( E ) Flow cytometry in HeLa PINK-YFP cells with doxycycline inducible expression of wild-type or C-terminal truncated TOMM5. Demonstrating rescue with TOMM5 WT but not TOMM5 ΔC. N = 6 replicates on at least two occasions. ns P = 0.4357, **** P ≤ 0.0001 (exact P values P < 1e-15 for all comparisons) by ordinary one-way ANOVA with Šídák’s multiple comparisons test. ( F ) Crystal structure yeast TOM complex (PDB: 6JNF (Araiso et al, )) demonstrating the location of TOMM5 and TOMM7 subunits. ( G ) LFQ proteomics of HeLa PINK-YFP whole lysates following the indicated knockdown/treatment. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating an FDR with the Benjamini–Hochberg procedure. Proteins were annotated as significant if they had an FDR < 0.05 and an absolute log 2 fold change of >0.5 (black outline). N = 4 replicates/sgRNA on one occasion. .

Article Snippet: Dual sgRNA CRISPRi Library 1–2 , Addgene , 187246.

Techniques: Flow Cytometry, Western Blot, Activity Assay, Protein-Protein interactions, Control, Clear Native PAGE, Fluorescence, Gel Shift, Expressing, Knockdown

Journal: mBio

Article Title: Genome-wide CRISPRi screen and proteomic profiling identify key genes related to ferulic acid’s antifungal activity

doi: 10.1128/mbio.01909-25

Figure Lengend Snippet: Comprehensive genome-wide CRISPRi analysis of S. cerevisiae response to ferulic acid. ( A ) Schematic representation of the CRISPRi library screening and competitive growth. S. cerevisiae cells expressing a genome-wide CRISPRi library were subjected to competitive growth in the presence of FA or DMSO (control). The dCas9-Mxi1 system, guided by gRNAs, repressed target gene transcription upon induction with ATc. Pooled cultures underwent serial dilutions (1:100), followed by plasmid extraction and sequencing to assess gRNA abundance. Enrichment analysis identified genes whose repression either increased sensitivity (depleted gRNAs) or conferred resistance (enriched gRNAs) to FA, as visualized in the volcano plot. ( B ) Correlation matrix showing Pearson correlation coefficients between biological replicates of FA-treated (FA), DMSO control (DM), and initial library samples (Li). The color scale (blue to red) indicates the strength of correlation. ( C ) Volcano plot illustrates differential gRNA abundance between FA-treated and DMSO control samples. Genes significantly depleted (blue) or enriched (orange) are highlighted, with thresholds of log2 fold change >2 or <−2 and P value <0.05. ( D ) Top-ranked genes sensitive to FA treatment. The top 10 genes with the highest RRA scores in negative selection. ( E ) Top-ranked genes contributing to FA resistance. The top 10 genes with the highest RRA scores in positive selection. ( F ) STRING network analysis of 194 genes depleted upon FA treatment. The network is divided into three distinct clusters: blue, green, and red. The blue cluster primarily contains genes involved in ribosomal biogenesis and RNA metabolism. The green cluster includes genes related to mitochondrial function, protein degradation, and stress responses. The red cluster comprises genes associated with mRNA processing, splicing, and transport.

Article Snippet: S. cerevisiae inducible CRISPRi Library (Addgene #161829) was transformed into DH5α Escherichia coli, generating more than 4 M colonies.

Techniques: Genome Wide, Library Screening, Expressing, Control, Plasmid Preparation, Extraction, Sequencing, Selection

Journal: mBio

Article Title: Genome-wide CRISPRi screen and proteomic profiling identify key genes related to ferulic acid’s antifungal activity

doi: 10.1128/mbio.01909-25

Figure Lengend Snippet: Effect of ERG9 silencing on FA resistance and upstream enzyme expression. ( A ) Lollipop plot of ergosterol biosynthesis genes. Log2 fold change (FA-DMSO) obtained from MAGeCK test analysis. Each gene response is colored based on the direction of fold change, with red indicating a negative log-fold change and blue indicating a positive log-fold change. Asterisks denote statistical significance levels: * <0.05, ** <0.01, and *** <0.001. ( B ) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) confirmation of ERG9 silencing. mRNAs were quantified by RT-qPCR analysis from three independent biological repeats, each with three technical repeats, normalized to ACT1 mRNA levels. ( C ) Yeast growth assay comparing the FA resistance of the ERG9 CRISPRi strain to the empty vector (E.V.) control. Growth was monitored in the presence of DMSO and 1.5 mM FA over 48 h. ( D ) Quantitative analysis of Yeast drop assay. Log2 fold-change (FA-DMSO) in the ERG9 CRISPRi strain compared to the empty vector. Error bars represent the standard error of the mean (SEM) from three independent biological repeats, and the P value was calculated by the dependent samples one-tailed t -test. ( E ) Expression levels of nine upstream enzymes in the ergosterol biosynthesis pathway following ERG9 silencing. mRNAs were quantified by RT-qPCR analysis and normalized to ACT1 mRNA levels and empty vector expression levels. The histogram presents the quantification of three independent biological repeats, each with three technical repeats. P values were calculated by the dependent samples one-tailed t -test.

Article Snippet: S. cerevisiae inducible CRISPRi Library (Addgene #161829) was transformed into DH5α Escherichia coli, generating more than 4 M colonies.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Growth Assay, Plasmid Preparation, Control, One-tailed Test

Journal: mBio

Article Title: Genome-wide CRISPRi screen and proteomic profiling identify key genes related to ferulic acid’s antifungal activity

doi: 10.1128/mbio.01909-25

Figure Lengend Snippet: Comprehensive genome-wide CRISPRi analysis of S. cerevisiae response to ferulic acid. ( A ) Schematic representation of the CRISPRi library screening and competitive growth. S. cerevisiae cells expressing a genome-wide CRISPRi library were subjected to competitive growth in the presence of FA or DMSO (control). The dCas9-Mxi1 system, guided by gRNAs, repressed target gene transcription upon induction with ATc. Pooled cultures underwent serial dilutions (1:100), followed by plasmid extraction and sequencing to assess gRNA abundance. Enrichment analysis identified genes whose repression either increased sensitivity (depleted gRNAs) or conferred resistance (enriched gRNAs) to FA, as visualized in the volcano plot. ( B ) Correlation matrix showing Pearson correlation coefficients between biological replicates of FA-treated (FA), DMSO control (DM), and initial library samples (Li). The color scale (blue to red) indicates the strength of correlation. ( C ) Volcano plot illustrates differential gRNA abundance between FA-treated and DMSO control samples. Genes significantly depleted (blue) or enriched (orange) are highlighted, with thresholds of log2 fold change >2 or <−2 and P value <0.05. ( D ) Top-ranked genes sensitive to FA treatment. The top 10 genes with the highest RRA scores in negative selection. ( E ) Top-ranked genes contributing to FA resistance. The top 10 genes with the highest RRA scores in positive selection. ( F ) STRING network analysis of 194 genes depleted upon FA treatment. The network is divided into three distinct clusters: blue, green, and red. The blue cluster primarily contains genes involved in ribosomal biogenesis and RNA metabolism. The green cluster includes genes related to mitochondrial function, protein degradation, and stress responses. The red cluster comprises genes associated with mRNA processing, splicing, and transport.

Article Snippet: To comprehensively identify and characterize molecular pathways and resistance mechanisms associated with FA treatment, we utilized a previously established genome-wide CRISPRi library (Addgene #161829) ( ).

Techniques: Genome Wide, Library Screening, Expressing, Control, Plasmid Preparation, Extraction, Sequencing, Selection