EMBO/soda-nel huggingface dataset:
Parkin coordinates mitochondrial lipid remodeling to execute mitophagy (2022)
Figure 1

Figure 1. Parkin stimulates PA and DAG accumulation on depolarized mitochondria. HeLa cells were transfected with a WT or mutant Parkin plasmid (FLAG), shown in blue, along with reporters for either PA or DAG (see Materials and Methods), shown in green. DMSO or 10 µM CCCP was added for the indicated periods of time. Mitochondria were visualized via anti-Tom20 staining (red).

(A) PA accumulated on Parkin-positive mitochondria after 1 hour of 10uM CCCP treatment, with a more dramatic increase by 3hr and 18 hr. Scale bar = 25 µM, and zoom is 9x.

(B) Quantification of imaging experiments shown in A and B. Bars represent mean with SEM from three biological independent experiments; two-way ANOVA analysis was performed for statistical analysis (***P<0.0001).

(C) DAG (diacylglycerol) accumulated on Parkin-positive mitochondria after 5.5 hr of 10uM CCCP treatment. Arrowheads and arrows point to the DAGR concentration at the Golgi and mitochondria, respectively. Scale bar = 25 µM, and zoom is 9x.
Parkin coordinates mitochondrial lipid remodeling to execute mitophagy (2022)
Figure 2

Fig. 2 PLD2 and lipin1 work in tandem to stimulate mitochondrial PA and DAG production.

(A) HeLa cells were transfected with expression plasmids for Parkin (mCherry-Parkin, Red) and YFP-PLD2 (Green) and treated with DMSO or 10 µM CCCP for the indicated time. Mitochondria were visualized via anti-Tom20 staining (blue). Note that YFP-PLD2 accumulated on Parkin-positive mitochondria after CCCP treatment. Scale bar = 25 µM, and zoom is 9x.

(B) HEK-293T cells were transfected with Parkin-FLAG and DAGR-YFP followed by CCCP treatment for indicated times. Mitochondrial and cytosolic fractions were purified and analyzed by Western blots by indicated antibodies. Citrate synthetase and α-tubulin were used as a mitochondrial and cytosolic marker, respectively. Note that PLD2 and Parkin levels were elevated in the mitochondrial fraction in response to CCCP treatment.

(C) Hela cells were co-transfected with mCherry-Parkin and the PA reporter, followed by CCCP treatment alone or with a PLD2 inhibitor VU 0364739 (3 µM) for 5.5 hrs. Line scan analysis (Image J software) corresponding to the line drawn in the images indicate colocalization between the PA reporter (green) and mitochondria (red). Note that VU 0364739 suppressed mitochondrial PA accumulation. Scale bar = 10 µM.

(D) Quantification of the numbers of cells with the PA reporter positive mitochondria shown in (C). Asterisks indicate statistical significance (***P<0.001, Student's t-test) from three biological independent experiments. The bars indicate mean± SEM.

(E) Hela cells were transfected with a lipin1 siRNA, followed by the DAG reporter and mCherry-Parkin, treated with CCCP (10 µM) for 9 hrs and then subject to image analysis. Scale bar = 25 um, and zoom is 4x. Lipin 1 KD reduced mitochondrial DAGR, but not Golgi-DAGR, accumulation (arrowheads)

(F) The numbers of cells with DAG-reporter positive mitochondria shown in (E) and in Lipin-1 knockdown cells transfected with siRNA-resistant wildtype (WT) and catalytically dead (CD; D712E;D714E) mutant cDNA. Note that Lipin1 KD reduced mitochondrial DAG accumulation, which can be significantly restored by the re-expression of WT, but not CD mutant, Lipin-1. The graph shows the means with SEM (error bars) from three biological independent experiments. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001).
Parkin coordinates mitochondrial lipid remodeling to execute mitophagy (2022)
Figure 3

Figure 3. Lipin-1-mediated mitochondrial DAG production is required for mitophagosome production.

(A-B) Hela cells were transfected with a Lipin1 or control (cKD) siRNA followed by a GFP-Parkin expression plasmid. Transfected cells were treated with CCCP at 10 µM for 18 hrs, and subject to immuno-staining. (B) Quantification of Parkin-positive cells in (A) that have lost a majority of mitochondria (marked by TOM20, arrows). Note that mitochondrial clearance is reduced in Lipin-1 knockdown cells (arrowheads in (A)). Scale bar = 25 µM.

(C). Control or lipin1 knockdown Hela cells stably expressing parkin-mCherry were treated with DMSO or CCCP (10 µM for 18 hrs) and Bafilomycin A1 (1μM, lysosomal inhibitor) treatment, followed by immunoblotting with indicated antibodies and quantified by the Image J. software. Note that lipin1 silencing suppressed CCCP-induced mitochondrial protein degradation. Lipin-1-mediated mitochondrial DAG production is required for mitophagosome production.

(D-F). Hela cells were transfected with control or a Lipin1 siRNA followed by the expression plasmids of GFP-Parkin and RFP-DAGR. Transfected cells were treated with DMSO or CCCP at 10 µM for 9 hr, with or without further incubation with 1,2-Dipalmitoyl-sn-glycerol (DPG) at 100 µM, as indicated. Autophagosome formation was assessed by an LC3 antibody (pseudocolored in white in single-channel and blue in the overlay images). Arrows point to DAGR concentration at the Golgi under basal conditions. The number (E) and size (F, arbitrary unit) of LC3 vesicles were quantified. n=4 biological replicates. The bars indicates mean± SEM. Asterisks indicate statistical significance by one-way ANOVA (*P< 0.05, **P<0.01). Scale bar = 25 µM and zoom is 5x. Note that both the number and size of LC3-vesicles in Lipin-1 knockdown cells were much smaller than those in WT cells, and these defects were corrected by DPG treatment.
Parkin coordinates mitochondrial lipid remodeling to execute mitophagy (2022)
Figure 4

Figure 4. Requirement of OPTN, NDP52 for DAG production and exogenous DAG restores mitophagy.

(A) E3 ligase-deficient Parkin mutants (T240R and T415N) have no effect on PA accumulation (green) on depolarized, Parkin-positive mitochondria. The expression of WT and Parkin mutants were determined by Immunoblotting. Scale bar = 25 µM, and zoom is 3x.

(B) DAG accumulation depends upon intact Parkin E3 ligase activity. Scale bar = 25µm, and zoom is 3x.

(C) Quantification of imaging experiments shown in (A) and (B). Bars represent mean with SEM; two-way ANOVA analysis was performed for statistical analysis(***P<0.001). n=3 biological replicates.

(D) Hela cells were transfected with control or optineurin- and NDP52- siRNAs and expression plasmids of FLAG-Parkin and YFP-DAGR. These cells were treated with DMSO or 10 µM CCCP for 5.5 hrs, as indicated. Note that OPTN/NDP52 double knockdown prevented mitochondrial YFP-DAGR accumulation. (Scale bar = 25 µM and zoom is 3x)

(E) Hela OPTN/NDP52 knockdown cells were transfected with a siRNA-resistant wildtype or ubiquitin-binding deficient E478G OPTN mutant, as indicated. The percentage of cells with mitochondrial YFP-DAGR following CCCP treatment was quantified. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001) from three biological independent experiments. The bars indicate mean± SEM. Bottom panel: Expression of OPTN, OPTN E478 and NDP52 was determined by immunoblotting.

(F-G) Hela OPTN/NDP52 knockdown cells were incubated with 1,2-Dipalmitoyl-sn-glycerol (DPG) followed by CCCP treatment for 18 hrs. Mitophagy efficiency was assessed by TOM20 staining and quantified in (G) (n = 2 biological replicates). Note that DPG significantly restored mitophagy in OPTN/NDP52 knockdown cells. Scale bar = 10 µM.
Parkin coordinates mitochondrial lipid remodeling to execute mitophagy (2022)
Figure 5

Figure 5. OPTN and NDP52-depedent EndoB1 mitochondrial recruitment is required for mitochondrial DAG production.

(A) Hela cells were co-transfected with expression plasmids for mCherry-OPTN and GFP-Parkin and treated with DMSO or CCCP for 5.5 hrs. Mitochondria were visualized by immunostaining with a Tom20 antibody and EndoB1 localization was assessed by an EndoB1 antibody. Note that EndoB1 (Blue) translocates to Parkin- and OPTN-tagged mitochondria in response to CCCP treatment. Scale bar = 25 µM, and zoom is 9x.

(B) Mitochondrial and cytosolic fractions obtained from control and CCCP treated cells, as was described and analyzed in Fig 2B, were immunoblotted for EndoB1 and OPTN, as indicated.

(C). Cytosolic and mitochondrial fractions purified from control and OPTN/NDP52 knockdown cells treated with CCCP were immunoblotted with indicated antibodies. Note that mitochondrial EndoB1 levels were reduced in OPTN/NDP52 knockdown cells

(D-E) Hela cells were transfected with an EndoB1 siRNA, followed by the expression plasmids for mCherry-Parkin and YFP-DAGR, and CCCP treatment (10 µM) for 9 hrs. Cells with mitochondrial YFP-DAGR were quantified in (E) (*P<0.05, Student's t-test, n=3 biological replicates). Scale bar = 25 µM and zoom is 5x. Note that knockdown of EndoB1 suppressed mitochondrial DAG production.

Expanded View Figure Legends
Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation (2014)
Figure 1

Comparative profiling of AGO4‐ and AGO6‐dependent methylomes

Heatmap depiction of the 3,731 DNA hypomethylation loci in ago4‐6. Each hypomethylated region corresponds to a colored horizontal bar, and the bars are clustered numerically into a column (y‐axis). Cytosines were examined as CG, CHG, and CHH. The color‐scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions.
Heatmap depiction of the 3,678 DNA hypomethylation loci in ago6‐2.
Overlapping patterns of hypomethylated loci in ago4‐6 and ago6‐2. Upper panel, Venn diagram showing the numbers of the following three types of hypomethylation loci: (1) loci where ago4‐6 shows lower methylation levels than ago6‐2 (green alone); (2) loci where ago4‐6 and ago6‐2 show equal methylation levels (green and red overlap); and (3) loci where ago6‐2 shows lower methylation levels than ago4‐6 (red alone). Lower panel, box plots showing methylation levels at the three groups of loci.
Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation (2014)
Figure 3

AGO4 and AGO6 mediation of canonical and non‐canonical RdDM pathways

Venn diagram showing the overlapping patterns among DNA hypomethylation loci identified in ago4‐6 ago6‐2, nrpd1‐3, and nrpe1‐11.
DNA methylation levels in different mutants. The 2,174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered (y‐axis). Cytosines were examined as CG, CHG, and CHH.
DNA methylation levels at TAS1a, TAS1c, and TAS3a loci. Snapshots from whole‐genome bisulfite sequencing results are shown.
RT–qPCR measurements of transposon RNA levels. Double: ago4‐6 ago6‐2 double mutant. Actin 2 was used as an internal control. RNA levels in the mutants are relative to those in the wild‐type (Col‐0). Means ± SD are shown, n = 3.
Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation (2014)
Figure 4

AGO4 and AGO6 do not co‐localize with each other or with Pol IV

AGO4 and AGO6 were visualized by immunofluorescence using their specific antibodies. Yellow signals would be expected in the merged images if the two proteins co‐localize, as a result of the overlap of red and green channels. DNA (blue) was stained with DAPI.
The largest subunit of Pol IV, NRPD1 (red), was visualized by its specific antibody in cells expressing FLAG‐tagged AGO4 or AGO6 (green).

Data information: The perinucleolar part was marked with dashed white lines, and nucleolar dot was marked with white arrow. The number next to the images showing the total number of nuclei with the presence of both red and green fluorescence signals. The percentage shows the proportion of the nuclei with similar co‐localization patterns. Qualitative images are shown here.
Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation (2014)
Figure 5

AGO4 and AGO6 exhibit different co‐localization patterns with Pol V

The largest subunit of Pol V, NRPE1 (red), was visualized by immunofluorescence using its specific antibody in cells expressing FLAG‐tagged AGO4 (green). AGO4‐NRPE1 co‐localized to nucleolar dot (yellow dot, marked with white arrow, upper panel) but not in nucleoplasmic foci (lower panel).
NRPE1 (red) was visualized by its specific antibody in cells expressing FLAG‐tagged AGO6 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co‐localization. DNA (blue) was stained with DAPI. AGO6‐NRPE1 co‐localized to nucleoplasmic foci (yellow dots, marked with white arrow).

Data information: The perinucleolar part was marked with dashed white lines. The number next to the images showing the total number of nuclei with the presence of both red and green fluorescence signals. The percentage shows the proportion of the nuclei with similar co‐localization patterns. Representative images are shown here.
Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation (2014)
Figure 6

Differential regulation of scaffold RNAs by AGO4 and AGO6

AGO6 dysfunction reduces levels of Pol V‐dependent transcripts. RNA levels were measured by RT–qPCR. Means ± SD are shown, n = 3.
AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6. Anti‐NRPE1 antibody was used for ChIP assays. Means ± SD are shown, n = 3.
Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation (2014)
Figure 7

The effects of AGO4 and AGO6 on siRNAs

Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol IV. Identities of siRNAs were retrieved from Havecker et al (), and siRNAs were grouped into 1,210 AGO4‐bound clusters and 1,486 AGO6‐bound clusters (see Materials and Methods for details), each of which corresponds to a colored horizontal bar; the bars are stacked numerically into a column (y‐axis). Information on siRNA levels in Pol IV mutant and wild‐type plants was retrieved from Zhang et al ().
Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol V.
Quantification of individual 24‐nt siRNAs by TaqMan small RNA assays. Double: ago4‐6 ago6‐2 double mutant. snoR101 was used as an internal control. RNA levels in the mutants were relative to those of the wild‐type (Col‐0). Error bars indicate SD, n ≥ 3.
Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit (2021)
Figure 1

Figure 1- Actin clearance and dynamic re-localization of Aurora B in early anaphase upon centralspindlin depletion.

(A) Stills from time-lapse sequence of representative Hela cells expressing Lifeact-GFP and H2B-mCherry, exiting mitosis under different conditions i.e. Control siRNA and knock-down of centralspindlin protein RACGAP1. Scale bar- 10µm

(B) High magnification view of boxed regions in (A) pseudo-colored to show actin depletion in anaphase. Scale bar -1 µm.

(C) Schematic representation for quantifying actin intensity along the cortex as in D. Segmented line was drawn along the cortex from one equatorial region to the other. Actin intensity across the line is plotted in D.

(D) Quantification of actin intensity profile of representative siControl and siRACGAP1 treated cells shown in (A). While actin levels were uniform across the cortex at metaphase (black line), they reduced from the polar regions (Blue line - segments 2-8) in both siControl and siRACGAP1 treated cells at early anaphase.

(E) Consolidated quantification of actin intensity at the poles of cells at 6 mins post anaphase onset under different conditions as in (A). Despite reduced elongation, actin is cleared from the poles in both Control and RACGAP1 depletion. Data represented as mean±SD. siControl-n=44, 0.98±0.051, siRACGAP1-n=48, 0.99±0.041. Unpaired Welch's t-test comparing siControl and siRACGAP1 shows no significant difference between them, p=0.5199. One sample t test comparing siControl and siRACGAP1 to theoretical mean 1 (implying no change in actin compared to metaphase levels), shows significance for both treatments, with *p=0.0195 for siControl and *p=0.0392 for siRACGAP1. With means lower than 1 (red dashed line) for both treatments, it suggests significant clearance of actin from poles.

(F) Stills from time-lapse sequence of representative Hela cells expressing mNeon-Green Aurora B and Cell Mask to label cell membrane, exiting mitosis, showing the dynamic re-localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment (arrows top). With knock-down of RACGAP1, Aurora B still re-localises from the DNA to the microtubules and furrow in the midzone in early anaphase (arrows bottom), but the microtubule localization is lost at later stages (asterisk bottom). Scale bar - 10 µm.

(G) Maximum projection of 2-z slices of Hela-Cdk1as cells stained with anti-RACGAP1 antibody when treated with DMSO and 2µM ZM447439 (ZM) at metaphase and early anaphase, showing the localization of RACGAP1 at spindle midzone even upon Aurora B inhibition at early anaphase. Scale bar - 10 µm
Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit (2021)
Figure 2

Figure 2 - Aurora B is required for polarization of actomyosin network in the absence of a midzone

(A) Maximum projection of 2 z slices of Hela cells immunostained for Aurora B (top) and actin-membrane cross-linker pERM (bottom), showing the change in localization upon Cdk1 inhibition with RO3306 (exit onset). As DNA moves to one side, Aurora B re-localizes from DNA to cortical region of the side opposite the DNA. In addition to Aurora B, the membrane-actin cross-linker pERM also polarizes in the direction opposite to DNA localization during the course of mitotic exit. Scale bar-10 µm.

(B) Schematic representation of cell, showing the quantification of actin and pERM along the cortex during mitotic exit. Regions 5 and 6 are cortex close to the DNA, while regions 1 and 10 are the furthest away.

(C) Maximum projection of 2 z slices of Hela cells immunostained for pERM following 10 mins of forced mitotic exit in presence of DMSO and 2µM ZM447439. Cells fail to polarize their pERM along the perimeter with Aurora B inhibition, quantified in (D). Scale bar-10 µm.

(D) Quantification of pERM intensity across the perimeter of the cells as shown in (B) where 5 and 6 represent regions closest to DNA, and 1 and 10 furthest. DMSO (n=28 cells) show increase in pERM levels away from the DNA, cells treated with ZM447439 (n=26 cells) during exit, fail to polarize the pERM proteins. Data presented as mean ± SD.

(E) Stills of representative Hela cells expressing Lifeact-GFP and H2B-mCherry at 10 mins following forced mitotic exit with siControl, siRACGAP1, DMSO and 2µM ZM447439 treatment. Control siRNA, RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA, while ZM447439 treated cells fail to clear actin, quantified in (F). Note, failure in actin accumulation in RACGAP1 depleted cell, asterisk. Scale bar - 10 µm

(F) Quantification of actin intensity across the perimeter of the cells based on distance from DNA, with 5 and 6 being closest to DNA and 1 and 10 furthest away, as in cartoon (B). Graph shows decrease in actin intensity closer to the DNA in siControl (n=26), siRACGAP1 (n=37) and Control DMSO treated cells (n=38 cells), seen by levels lower than 1 (dotted red line), while this decrease is absent in ZM447439 treated cells (n=42 cells). Data presented as mean ± SD.
Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit (2021)
Figure 3

Figure 3 - Aurora B inhibition affects DNA-dependent actin clearance during flat monopolar cytokinesis

(A) Schematic of experimental set up. Expression of dominant-negative Rap1 (RapQ63E), prevents adhesion disassembly during mitotic entry and forces cells to remain flat during mitosis. Using Cdk1 inhibitor to induce monopolar cytokinesis in these STLC treated cells, the short cell height forces anaphase DNA into close proximity to the basal actomyosin network, which we measure to determine the ability of DNA dependent cues to relax the actomyosin network by quantifying actin directly beneath the DNA.

(B) Stills from time-lapse sequence of the basal most region of Hela cells expressing Lifeact-GFP and H2B-mCherry forced to undergo flat monopolar mitotic exit. While actin is cleared underneath the DNA in control DMSO treated cells, it fails to be cleared around the DNA when cells are treated with Aurora B inhibitor ZM447439 during mitotic exit. Dotted mask shows position of DNA in actin channel. Scale bar -10µm.

(C) Quantification of clearance of actin underneath the DNA for still shown in (B). While DNA under control cell starts clearing actin within 10 mins, Aurora B inhibited cells fail to do so.

(D) Maximum projection of 2z slices of basal region of Hela cells stained with Phalloidin at 15 mins post anaphase onset, when cells were treated with DMSO or ZM447439 during flat monopolar cytokinesis, showing clearance under DMSO treatment and a failure to clear actin with ZM447439 treatment. Dotted mask shows position of DNA in actin channel. Scale bar - 10 µm.

(E) Quantification of fraction of cells undergoing actin clearance based on Phalloidin staining as in (D). With DMSO treatment, 50% of the cells clear actin completely, and remaining 50% clear partially beneath the DNA (n=34). With ZM447439 treatment, only 23% of cells clear completely, while 73% clear partially and 4% there is no clearance (n=22 cells). Chi-square test comparing distribution of cleared vs partial/no clearance shows significant difference between Control DMSO and ZM treatment, p=0.0413.
Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit (2021)
Figure 4

Figure 4 - Synergistic effect of Aurora B and centralspindlin during anaphase

(A, B) Maximum projection of 2z slices of representative Hela cells stained for Anillin (A) and pMLC- S19 (B), showing reduction of these proteins in cells depleted of centralspindlin protein RACGAP1, but is unaltered upon Aurora B inhibition with ZM. Scale bar - 10 µm

(C) Maximum projection of 2-z slices of representative Hela Cdk1as cells treated with siControl, siRACGAP1, 2µM ZM and siRACGAP1 with 2µM ZM, immunostained with tubulin to visualize spindle midzone at early anaphase and late anaphase. Midzone is specified in siControl and siRACGAP1 treated cells, but fails to be maintained in siRACGAP1 treated cells at later stages. With ZM treatment, a diffuse midzone persists throughout anaphase. No recognizable midzone is seem in the double treatment. Scale bar - 10 µm

(D) Still images of representative Hela cells expressing Lifeact-GFP and H2B-mCherry captured at early anaphase under different conditions i.e. Control siRNA, knock-down of centralspindlin protein RACGAP1, in the presence and absence of Aurora B kinase inhibitor (2µM ZM). Scale bar - 10 µm

(E) Quantification of aspect ratio of cells treated with different conditions as in (D) at 6 mins after anaphase onset, before furrow formation. Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio, while together have a stronger impact during mitotic exit. In combined treatment, mitotic cells hardly undergo any elongation and remain spherical before re-spreading. Ordinary one-way ANOVA with multiple comparison. Each treatment is compared with siControl, ****p<0.0001 for all comparisons. Data represented as box-whisker plot, with box showing 25th-75th percentile values, whiskers-min to max value and line representing median. Mean±SD for different treatments- siControl (n=38) - 1.455±0.19, ZM (n=32) - 1.219±0.16, siRACGAP1 (n=25) -1.144±0.078 and siRACGAP1+ZM (n=16) - 1.013±0.03. Cells for different treatment pooled from n>3 independent experiments.