EMBO/soda-nel huggingface dataset:
C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency (2022)
Figure 3

Figure 3. AID∆C condensate traps wild-type AID in B cells.

(A) Representative immunofluorescence images of endogenous AID in the presence of ectopically-expressed EV, AID, AIDR190X, or AIDcry in cytokine-stimulated CH12F3 cells. EV, empty vector control. An antibody against AID C terminus (185-198 aa) detected both endogenous and ectopically-expressed full-length AID. The nucleolus is indicated by co-nucleofected mCherry-FBL. The white dashed line depicts the shape of the nucleus based on Hoechst staining. Arrows indicate the location of puncta formed by endogenous AID. The images were taken by the N-SIM microscope and processed via N-SIM software. A middle slice of the images was shown. Scale bar, 5 µm.

(B) Co-condensation of AID and AIDR190X in the nucleus of CH12F3 cells in the early G1 phase. Top: experimental layout. GFP-tagged wild-type AID (green) and mCherry-tagged AID variants (red) were co-nucleofected in Aicda-/- CH12F3 cells. The resulting cells were arrested with nocodazole for 12 hours after transfection. Six hours later, nocodazole was removed from the system for 1.5 hours before imaging. Bottom left: representative images showing subcellular localization of indicated AID variants. The white dashed line depicts the shape of the nucleus based on Hoechst staining. The white arrow indicates the location of the co-condensed puncta. Scale bar, 5 µm. Bottom right: plot profiles of the images. Cyto: cytoplasm, Nuc: nucleus.

(C) Quantification of the relative fluorescence intensity of AID-GFP in the diluted phase of cells shown in (B). The fluorescence intensity of AID-GFP in the diluted nuclear phase was normalized by the total intensity from the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell (n = 25), and representative result from three biological replicates were shown. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.

(D) Dynamics of trapping of wild-type AID in optoAIDR190X condensates. Top, representative fluorescence images at different time points. Dashed circles indicate the co-condensed puncta formation of AID-GFP. Scale bar, 5 µm. Bottom, a summary of 12 observed cells is shown as mean ±SD in a bar graph. Percentages of optoAIDR190X puncta with trapped wild-type AID are plotted for the second and third blue-light activations, and the colored dot indicates one cell.
C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency (2022)
Figure 4

Figure 4. Intrinsic sequence features contribute to AID∆C condensation and dominant-negative effect.

(A) Summary of optoDroplet formation driven by indicated AID mutants. Left, AID domain architecture is illustrated based on the published structure and AlphaFold prediction. The orange and black lines indicate predicted low-complexity sequence. Asterisks indicate residues for AID mutations. Right, AID mutants are grouped into three categories based on protein architecture and the optoDroplet formation capacity is summarized at right. "+++": strong capacity with a cutoff of 90% cells showing optoDroplet puncta formation. "++": a lower capacity with 50-90% of cells showing puncta formation. "+": capacity with less than 50% of cells showing puncta formation. "-": empty vector control level.

(B) The optoDroplet formation of indicated AID mutants along with light stimulation. Top, the procedure of optoDroplet assay is illustrated with indicated light stimulation (blue arrow) and image acquisition (black arrow) time points. Bottom, line plots show quantification of cells with optoDroplet formation as mean ± SD at each acquisition time point. The total acquired cell numbers of each genotype are indicated in parentheses. Significance assessment between AIDR190X and AIDR190X-4R was shown at each time point. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.

(C) CSR levels of ectopically-expressed AID variants in AID-proficient CH12F3 cells. Left, representative flow cytometry plots of CSR to IgA. Co-expressed GFP indicates the infected cell fractions. Right, the CSR mean with SD of three biological replicates is shown in the bar plot.

(D) CSR levels of ectopically-expressed AID variants in CSR-activated B cells. Left, representative flow cytometry plots of CSR to IgG1. Right, the bar plot shows the CSR mean with SD of four biological replicates.

Data information: One-way ANOVA followed by Dunnett's multiple comparisons test was performed for (C) and (D). ***, p<0.001; **, p<0.01; *, p<0.05.
C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency (2022)
Figure 5

Figure 5. AID∆C has the tendency to condensation in vitro

(A) Left, representative images of MBP-GFP-AID in the presence of 10% PEG8000. Scale bar, 10 µm and 2 µm, respectively. Right, the schematic protein sequences.

(B) Left, representative images of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry in the presence 10% PEG8000 at the indicated protein concentrations. Scale bar, 10 µm. Right, sizes of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry protein condensates are plotted as box plots with three technical replicates. The box represents values between the lower quartile and the upper quartile, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. All points are listed and mean is indicated by "+". Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.
C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency (2022)
Figure 6

Figure 6. Unbalanced molecular ratio of AID∆C/AID facilitates the dominant-negative effect.

(A) AID deamination assay was performed in vitro. Left, a schematic diagram of deamination activity assay. Right, the deamination reaction with indicated protein combinations was processed and visualized. "+": 0.25 μM of indicated protein; "++": 0.5 μM of indicated protein; "-": not added.

(B) CSR to IgA induced by AID variants in CH12F3 cells. Left, representative flow cytometry plots. Right, the CSR mean with SD of three biological replicates is shown in the bar plot. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ***, p<0.001; **, p<0.01.

(C) Representative images of the co-condensation of MBP-mCherry-AIDcry and MBP-GFP-AID. The protein concentrations and molecular ratios are indicated. Scale bar, 10 µm.

(D) A working model of AID∆C dominant-negative effect. In cells with one C-terminal deletion AICDA allele, large amounts of pathological AID∆C protein accumulates in the nucleus and are frequently enriched in nuclear subcompartments through its condensation tendency, thereby blocking its proper targeting to the physiological IgH substrates. While the wild-type AID dynamically shuttles between cytoplasm and nucleus, the high nuclear AID∆C:AID ratio causes the trapping of wild-type AID into AID∆C-associated condensates, manifesting a dominant-negative effect.
Specialization of actin isoforms derived from the loss of key interactions with regulatory factors (2022)
Figure 4

Figure 4 - In vitro reconstitution of branched- and linear- actin networks assembly from purified actins. Standard conditions include Las17- (branched) and Bni1- (linear) coated beads, 8 µM F-actin assembled from different purified variants and 1% labeled with Alexa-568 rabbit muscle actin, 15 µM profilin, 1 µM capping protein, 500 nM Arp2/3 complex and 600 nM ADF/cofilin. Snapshots of representative actin networks were taken after 30 min. Scale bars: 6 µm.

A (Top) Snapshots of actin networks assembled from three different actins sources: Act_Sc, Act_N2 and Act_Ca. (Bottom left) Quantification of actin fluorescence on beads. Data are presented as mean +/- SD (n = 52 for Act_Sc branched, n = 70 for Act_N2 branched, n = 81 for Act_Ca branched, n = 32 for Act_Sc linear, n = 34 for Act_N2 linear and n = 47 for Act_Ca linear). **P<0.01, ***P<0.001 (Kruskal-Wallis test, with multiple comparisons). (Bottom right) In vitro actin network deviation indexes.

B (Left) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa-488-labeled ADF/cofilin (replacement of unlabeled ADF/cofilin). (Right) Quantification of ADF/cofilin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 34 for Act_Sc branched, n = 28 for Act_N2 branched, n = 43 for Act_Ca branched, n = 24 for Act_Sc linear, n = 15 for Act_N2 linear and n = 27 for Act_Ca linear). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).

C (Left) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488-tropomyosin. (Right) Quantification of tropomyosin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 45 for Act_Sc branched, n = 63 for Act_N2 branched, n = 57 for Act_Ca branched, n = 22 for Act_Sc linear, n = 32 for Act_N2 linear and n = 27 for Act_Ca linear). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).

Data information: For all microscopy images, intensity levels were adapted for images of branched- and linear- actin networks separately as their brightness is different. To obtain a correct representation of the amount of ADF/cofilin and tropomyosin bound to the two networks, the intensity levels of ADF/cofilin and tropomyosin were matched similarly to those of the corresponding actin networks. Abbreviations: ns - non-significant, Act_Sc - purified S. cerevisiae actin, Act_N2 - purified Node 2 actin, Act_Ca - purified C. albicans actin (for more details see Table EV1, Fig 1 and Fig S1 B-C).
Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy (2020)
Figure 1

Figure 1. OFD1 interacts with components of the ULK1 complex.

A. Lysates of HK2 cells cultured in FM or STV(2h) were immunoprecipitated with anti-OFD1 (left) and anti-ATG13 (right) antibodies or IgG and analyzed by WB; ACTIN was used as loading control. n=3 independent experiments. IP=immunoprecipitation, FM=full medium, STV=HBSS starvation.

B. Airyscan confocal analysis of endogenous FIP200/γTUBULIN and ATG13/γTUBULIN colocalization with lentiviral delivered HA-OFD1 in HK2 cells. Cells were cultured in HBSS (90min). Green, FIP200 and ATG13; red, HA-OFD1; Grey, γTUBULIN; blue, Hoechst for nuclei. Insets show magnifications and single-color channels of selected areas. Scale bar=3,5μm.

C. Quantification of HA-OFD1/ATG13 and HA-OFD1/FIP200 colocalization, expressed as % of total OFD1 fluorescence (mean ± SEM). n=3 independent experiments; n=53 cells for ATG13 and n=39 for FIP200 were analyzed.
Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy (2020)
Figure 2

Figure 2. OFD1 controls ULK1 complex stability through autophagy.

A. Top, WB of ULK1-complex components in wt and KO-OFD1 cells assessed in FM (-) or STV (2,4h). Bands at same exposure, noncontiguous on the same gel. Bottom, ACTIN-normalized protein levels are expressed as fold increase compared with wt cells, represented by the dashed line (mean ± SEM); n=5 independent experiments. FM=full medium, STV=HBSS starvation.

B. Top, wild-type and KO-OFD1 cells were incubated with 50µg/ml CHX. Immunoblots were probed with indicated antibodies. Bottom, quantification of βTUBULIN and ACTIN-normalized protein levels versus untreated conditions (-), data are expressed as mean ± SEM; n=4 independent experiments. CHX=Cycloheximide, ns=not significative.

C. Immunofluorescence of ULK1 and ATG13 puncta in wt and KO-OFD1 cells cultured in HBSS (90min). Green, ATG13 and ULK1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) Graphs display quantification of puncta/cell, data are expressed as mean ± SEM; n=5 independent experiments, n≥200 cells/antibody.

D. Western Blot of ULK1 complex components in KO-OFD1 cells transfected with empty vector (Empty) or 3xFLAG-OFD1 (OFD1). Histograms show quantification of band intensities normalized versus ACTIN expressed as mean ± SEM. n=5 independent experiments.

E. Western Blot of ULK1-complex components in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or empty vector (Empty) and treated (+) or not (-) with SAR405 (10μM, 6h). ACTIN is the loading control. On the right, ATG13 levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.

F. Representative confocal images of co-staining of 3xFLAG-OFD1 with ATG13 in KO-OFD1 cells incubated in HBSS, treated (+) or not (-) with Baf-A1(100nM, 90min). Green, ATG13; red, 3xFLAG-OFD1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=3 independent experiments, n≥100 cells.

Data information: Statistical analysis: One tailed Student's t-test was applied in A, B, D and E; the likelihood ratio test for Negative Binomial generalized linear models was applied in C and F. *p≤0.05, **p≤0.01, ***p≤0.001, ns=not significative.
Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy (2020)
Figure 3

Figure 3. OFD1 interacts with LC3B and GABARAP-L1 through a classical LIR motif.

A. Middle. Lysates from 3xFLAG-OFD1 overexpressing HEK293 cells were added to beads with immobilized GST-fused LC3B, LC3BF52A-V53AdG, dNLC3BdG and GST alone. Right. Lysates from 3xFLAG-OFD1(OFD1) and empty vector (Empty) overexpressing HEK293 cells were added to GST-GABARAP-L1 and GST. Left. WB of total lysates (input). All panels display WB for FLAG, ACTIN is the loading control.

B. Representative confocal images of endogenous OFD1 (green) co-staining with LC3B (red), and of 3xFLAG-OFD1 (red) with GABARAP (green) in HK2 cells (HBSS,90min). Hoechst labels nuclei (blue). Insets show magnifications and single-color channels of selected areas. Scale bar=10μm; n≥50 cells/sample.

C. OFD1 domains architecture and alignment of LIR motif (boxed) with orthologous sequences. Residues with full (*), strong (:), weak (.) conservation are indicated. LisH=Lis Homology domain, CC=Coiled coil, LIR=LC3 interacting region.

D. Lysates from 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1∆LIR(OFD1∆LIR) and empty vector (Empty) overexpressing cells were added to GST-GABARAP-L1, GST-LC3B and GST.

E. Representative confocal images of OFD1∆LIR co-staining with LC3B (left) and GABARAP (right) in HK2 cells (HBSS,90min). Green, LC3B and GABARAP; red, 3xFLAG-OFD1∆LIR; blue, Hoechst for nuclei. Scale bar=5μm. n≥50 cells/sample.
Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy (2020)
Figure 4

Figure 4. OFD1 is an autophagy receptor for ATG13.

A. Representative confocal images of 3xFLAG-OFD1 (OFD1) and 3xFLAG-OFD1∆LIR (OFD1∆LIR) co-staining with ATG13 in KO-OFD1 cells (HBSS,90min). Green, ATG13; red, FLAG; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=4 independent experiments, n≥100 cells.

B. Western blot of ATG13 in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) or empty vector (Empty). (Right) ACTIN-normalized ATG13 protein levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.

C. Top. OFD1 fragments (a,b and c). Bottom left. Lysates from empty vector (Empty), 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1a (OFD1a), 3xFLAG-OFD1b (OFD1b), 3xFLAG-OFD1c (OFD1c) overexpressing HEK293 cells were added to GST-ATG13; lysates from 3xFLAG-OFD1(OFD1) overexpressing HEK293 cells were added to GST as control. Right. WB of total lysates (input). All panels display WB for FLAG, HSP90 is the loading control. LIR=LC3 interacting region.

D. Scanning confocal microscopy analysis of wt and KO-OFD1 cells incubated in HBSS (8h), treated (+) or not (-) with Baf-A1(50nM, 8h). Green, LAMP1; red, ATG13; blue, Hoechst for nuclei. Scale bar=6μm. Insets show higher magnification and single colour channels of the boxed area. (Bottom) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1/cell (mean ± SEM). n=30 WT cells, n=28 KO-OFD1 cells counted; three independent experiments.

E. Time course of OFD1 levels in HBSS-starved wt HK2 cells treated with Baf-A1 (100nM) and collected at different time points. LC3B is the control for Baf-A1 treatment. (Right) Graphs show ACTIN-normalized OFD1 levels versus FM condition (-) expressed as mean ± SEM; n=4 independent experiments. STV=starvation, Baf-A1=bafilomycin, Ctrl=control.

F. Western blot of FLAG in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) and treated or not (-) with Baf-A1 (100nM,2h). (Right) Quantification of GAPDH-normalized FLAG levels versus untreated (-) is expressed as mean ± SEM; n=3 independent experiments. Baf-A1=bafilomycin.

Data information: Statistical analysis: One tailed Student's t-test was applied in B, D, E and F; the likelihood ratio test for Negative Binomial generalized linear models was applied in A. *p≤0.05, **p≤0.01, ***p≤0.001.
Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy (2020)
Figure 5

Figure 5. OFD1 regulates autophagy.

A. Representative blots of ATG13 (left) and VPS34 (right) phosphorylation levels in wt and KO-OFD1 cells transiently expressing GFP-ATG13 and FLAG-VPS34 in FM.

B. Wild-type and KO-OFD1 cells were starved in HBSS with/without Baf-A1 (100nM, 2h) and chloroquine (CQ) (50μM, 2h) and total lysates were analyzed by immunoblotting using anti-OFD1, -LC3B (LC3B-I 18 kDa and LC3B-II 16 kDa) and -ACTIN antibodies. (Right) ACTIN-normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of wt cells. n=5 independent experiments.

C. Representative images of LC3B and WIPI2 puncta in wt and KO-OFD1 cells (HBSS, 90min). Green, LC3B; red, WIPI2; blue, Hoechst, nuclei. Scale bar=10μm. (Right) Quantification of LC3B and WIPI2 puncta is shown. ≥100 cells analyzed/sample, n=5 independent experiments.

D. Transmission electron microscopy (left) and quantification (right) of autophagic vacuoles (arrows) in wt and KO-OFD1 cells (HBSS, 90min).

E. Representative images of siOFD1 depleted and control (Ctrl) HeLa cells transiently expressing mRFP-eGFP-LC3B (HBSS, 90min). Insets show higher magnification of colocalization in selected areas. Scale bar=10μm. Bottom, tandem structure of the mRFP-GFP-LC3B construct and quantification of RFP+GFP+ and RFP+GFP- puncta in siOFD1-depleted cells and controls, cultured in full medium (FM) or kept in HBSS for 2h (STV) are depicted. n≥90 cells/sample analyzed, n=5 independent experiments.

F. Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP-OFD1 (HBSS,90min). Green, eGFP-OFD1, red, WIPI2; blue, Hoechst labels nuclei. On the right, quantification of WIPI2 puncta in eGFP-OFD1 transfected cells compared with non-transfected cells (Ctrl) is shown. Scale bar=10μm. n≥90 cells analyzed/sample, n=3 independent experiments.

G. Representative images of WIPI2 and OFD1 co-staining in KO-OFD1 cells transfected with empty vector (Empty), 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) (HBSS,90min). Red, WIPI2; green, OFD1; blue, Hoechst labels nuclei. Scale bars: 10μm. On the right, quantification of WIPI2 puncta. ≥100 cells analyzed/sample, n=4 independent experiments.

Data information: Data are expressed as mean values from the indicated independent experiments. Error bars indicate SEM. One tailed Student's t test (B and D) and the likelihood ratio test for Negative Binomial generalized linear models (C, E, F and G) were applied; *p≤0.05, **p≤0.01 and ***p≤0.001. FM=full medium, STV=starvation, Baf-A1=bafilomycin, Ctrl=control.
Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy (2020)
Figure 6

Figure 6. Autophagy is increased in OFD type I patients.

A. Western blot of LC3B in lymphoblasts of OFD type I patients and controls, either untreated (-) or exposed to Baf-A1 (10nM, 2h)(+). Cells were cultured in complete medium (FM, top) or under serum starvation for 4h (STV, bottom). ACTIN and GAPDH were used as loading controls.

B. (Left) Graphs show LC3B-II levels normalized on ACTIN in patients lymphoblasts and controls cultured in complete medium (FM). (Right) Graphs show LC3B-II levels normalized on GAPDH in patients lymphoblasts and controls in serum starvation for 4h (STV). Normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of control cells, n=4 patients and 3 controls.

C. Western blot of ATG13 and ATG101 proteins in serum starved lymphoblasts from controls and OFD type I patients. (Right) Protein levels relative to ACTIN (loading control) are expressed as fold change versus controls, represented by the dashed line, n=4 patients and 3 controls.

Data information: Data are expressed as mean values, error bars indicate SEM. One tailed Student's t test was applied (B and C); *p≤0.05, **p≤0.01.FM=full medium, STV=starvation, Baf-A1=bafilomycin, ns= not significative.
Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy (2020)
Figure 7

Figure 7. Excessive autophagy contributes to renal cystogenesis in OFD type I syndrome.

A. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1fl/y;creKsp and control mice. (Right) LC3B-II protein levels normalized to Actin (loading control) are expressed as fold increase versus control mice, n=4 mice/group.

B. Representative images of GFP-LC3 puncta (autophagosomes, green) in aquaporin2-positive (AQP2, red) renal distal tubules from P8 Ofd1fl/y;creKsp;GFP-LC3 and Ofd1fl/y;GFP-LC3 mice. Scale bar=10μm. Hoechst labels nuclei. Bar graphs on the left show quantitative analysis of GFP-LC3 puncta; n=4 mice/group for a total of 300 AQP2 positive nuclei/group analyzed.

C. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1-IND and control mice. (Right) LC3B-II protein levels relative to βTubulin (loading control) are expressed as fold increase compared with control mice, n=5 mutant/8 control mice .

D. Western blot of Atg13 in Ofd1fl/y;creKsp and control kidneys at pre-cystic (P8, left) and cystic (P22, right) stages. Gapdh was used as loading control. Atg13 protein levels are expressed as fold change compared with control mice; n=7 mutant/5 control mice at P8, n= 4mice/group at P22.

E. Haematoxylin & eosin staining of kidney sections from Ofd1fl/y;Atg7+/+;creKsp and Ofd1fl/y;Atg7fl/fl;creKsp mice. (Right) Graph shows fold change of the cysts number observed in Ofd1fl/y;Atg7fl/fl;creKsp animals compared with Ofd1fl/y;Atg7+/+;creKsp; n=12 sections/animal were analyzed; n=6 mice/group. Scale bars=1mm.

F. Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl/y;Atg7fl/fl;creKsp, Ofd1fl/y;Atg7+/+;creKsp and control mice at P45. n=8 mice per group.

Data information: Data are expressed as mean values from the indicated independent experiments. Error bars indicate SEM. One tailed Student's t test was applied; *p≤0.05, **p≤0.01 and ***p≤0.001. BUN=blood urea nitrogen.

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