EMBO/soda-nel huggingface dataset:
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases (2016)
Figure 7

Figure 7. Overexpression of CSPα in the brain of MPS-IIIA mice re-establishes normal SNARE proteostasis.

(A) Anti-myc immunostaining in different brain regions derived from 10-months-old MPS-IIIA mice intraventricularly injected with AAV2/9 vectors encoding CSPα under Synapsin-1 promoter (MPS-IIIA-myc-CSPα experimental group). As a control immunostaining was performed in brain sections from 10-months-old MPS-IIIA mice injected with empty AAV2/9 vectors (MPS-IIIA-empty experimental groups). To evaluate co-localization between exogenous CSPα and presynaptic compartment brain sections were also co-stained with anti-myc and anti-synapsin1 antibodies (confocal images are also shown).

(B) Anti-myc WB in synaptosomal fractions derived from MPS-IIIA-myc-CSPα mice. As a control WB was performed in samples from 10-months-old WT and MPS-IIIA mice injected with empty AAV2/9 vectors (WT-empty and MPS-IIIA-empty experimental groups). Synaptosomal fractions were also blotted with anti-CSPα.

(C) VAMP2, SNAP-25 and Syntaxin-1 were immunoblotted in synaptosomal samples derived from the three experimental groups of mice. Protein levels were quantified.

(D) The amount of SNARE-complexes was detected in synaptosomal brain samples derived from the three experimental groups of mice by immunoblotting analysis of non-boiled samples with VAMP2, SNAP-25 or Syntaxin-1 antibodies. The amounts of SNARE complexes were quantified.

(E) Total brain lysates derived from the three experimental groups of mice were immunoprecipitated with Syntaxin-1 antibodies and co-immunoprecipitated VAMP2 and SNAP-25 proteins were revealed by WB.

Data are means ± s.e.m. N=3 (biological triplicates) in WB quantitation. *P<0.05, Student’s t-test: Either MPS-IIIA-empty or MPS-IIIA-myc-CSPα vs WT-empty (C, D). Scale bar: 30 µm (epifluorescence images in A), 10 µm (confocal images in A).
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases (2016)
Figure 8

Figure 8. Overexpression of CSPα in the MPS-IIIA mouse brain prevents presynaptic failure, protect against neuropathology and prolonged survival

(A) EM analysis of cortical and hippocampal synapsis derived from the three experimental groups of mice. Synaptic vesicles number per synapse was quantified from ~40 different images (taken from 5 mice for each group), normalized by the length of synaptic cleft and expressed as % of WT. The synaptic density was measured from 20 different images (taken from 5 mice for each group) as the number of synapses/area(#/500μm2) and expressed as % of WT. Arrows indicate the synaptic cleft while asterisks indicate abnormal vacuoles. Values are means ± s.e.m. *P<0.05, Student’s t-test: Either MPS-IIIA-empty or MPS-IIIA-myc-CSPα vs WT-empty. Scale bar: 0.2 µm

(B) Extracellular recordings (fEPSPs) in hippocampal brain slices from WT-empty (n=8), MPS-IIIA-empty (n=5) and MPS-IIIA-myc-CSPα mice (n=5). The upper panel shows representative fEPSP traces. Summary graph shows the fEPSPs slope as a function of the applied stimulus intensity. Data are means ± s.e.m.; *P < 0.05 (WT-empty vs MPS-IIIA-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), Kruskal-Wallis ANOVA followed by Dunn’s test.

(C) Mean distance travelled, maximal speed, immobility time and line crossing during 10 min testing in the open field, divided into 5 minute intervals in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα female mice (n=9, n=9, n=7 respectively). Distance, percentage open entries and open time in the elevated plus maze and latency to fall off the wire (*body weight) in the wire hanging test in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα male mice (n=10, n=8, n=9 respectively). Values are means ± s.e.m; *P < 0.05 (MPS-IIIA-empty vs. WT-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), two-way ANOVA for repeated measures for open field measures and t-test for the plus maze and the wire hanging followed by Duncan post-hoc test. Animals’ average age was 28±2 weeks.

(D) Neuronal cell death was evaluated in the cerebellum (Calbindin immunostaining) and in different layers of cortex (NeuN immunostaining of frontal cortex) of 10 months-old mice belonging to each experimental group of mice. Number of cells was quantified from 20 different images (taken from 5 mice for each group) and expressed as % of WT. Values are means ± s.e.m. *P<0.05, **P<0.001 Student’s t-test. Scale bar: 50 µm.

(E) Kaplan–Meier survival analysis in WT-empty (n=13), MPS-IIIA-empty (n=13) and MPS-IIIA-myc-CSPα (n=13) male mice. The Kaplan–Meier survival curve was analyzed with the Chi-square test. A P value <0.05 was considered to be statistically significant. P= 0.00003 (MPS-IIIA-empty vs WT-empty), P= 0.000776 (MPS-IIIA-myc-CSPα vs MPS-IIIA-empty).
Centriolar distal appendages activate the centrosome-PIDDosome-p53 signaling axis via ANKRD26 (2020)
Figure 1

Figure 1. PIDD1 is a distal appendage protein whose localization relies on ANKRD26.

(A) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies. ­Scale bar: 200 nm.

(B) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/ODF2 fluorescence ratio in the indicated cell lines and genotypes. Mean values (red lines) ± s.e.m. are reported. Data obtained from images as in (C). N ≥ 50 centrosomes were assessed for each condition, a.u. = arbitrary units. Unpaired Mann-Whitney test (****P < 0.0001).

(C) Representative fluorescence micrographs from the indicated cell lines co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.

(D) Co-localization between ODF2 and PIDD1 (n = 11), FBF1 and PIDD1 (n = 21), and ANKRD26 and PIDD1 (n = 17) assessed on images as in (A and E). The value represents the fraction PIDD1 objects touching either ODF2, FBF1 or ANKRD26 objects. Mean values (red lines) ± s.e.m. are reported. Kruskal-Wallis test (***P < 0.001; *P < 0.05; n.s. = non-significant).

(E) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies, scale bar: 200 nm.

(F) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.

(G) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/CEP128 fluorescence ratio in RPE1 of the indicated genotypes. Mean values (red lines) ± s.e.m. are reported. Data obtained from images as in (F). N ≥ 50 centrosomes were assessed from as many individual cells for each condition, a.u. = arbitrary units. Statistical significance was assessed by Kruskal-Wallis test, comparing each sample to the wild type (****P < 0.0001).

(H) Scheme summarizing the epistatic interdependencies between DAPs emerging from work displayed in this Figure.
Centriolar distal appendages activate the centrosome-PIDDosome-p53 signaling axis via ANKRD26 (2020)
Figure 2

Figure 2. ANKRD26 directly interacts with PIDD1 at DAs.

(A) Schematic of the ANKRD26 domain identified as PIDD1 interactor by yeast-two-hybrid screen. PMID = PIDD1 Minimal Interaction Domain; CCDC144C-like = Coiled-Coil Domain similar to CCDC144C; DUF: Domain of Unknown Function.

(B) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.

(C) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).

(D) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).

(E) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.
Centriolar distal appendages activate the centrosome-PIDDosome-p53 signaling axis via ANKRD26 (2020)
Figure 3

Figure 3. PIDD1 localization to DAs is necessary for PIDDosome activation.

(A) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h. A fraction of DHCB treated cells were released into fresh medium for other 24h (release). Samples were subjected to immunoblotting; n = 3 independent experiments.

(B) Fluorescence micrographs of RPE1 cells of the indicated genotypes, either treated for 24h with DHCB or with vehicle alone (DMSO). Blow-up without Hoechst 33342 is magnified 2.5X. Scale bar: 5 μm.

(C) Immunofluorescence micrographs of RPE1 as in (B) were used to visually assess the percentage of cells presenting one or two nuclei. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of binucleated cells between the wild type sample and all the other genotypes was assessed (ANOVA test; *P < 0.05).

(D) Immunofluorescence micrographs of RPE1 as in (B) were used to visual score the number of centrosomes per cell by counting the number of γ-tubulin-positive centrioles. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of cells with > 2 centrosomes between the wild type sample and all the other genotypes was assessed (ANOVA test; **P < 0.01).

(E) Quantitative assessment of the fraction of RPE1 cells undergoing cytokinesis failure upon DHCB treatment, inferred on the basis of the increase of ploidies ≥ 4C (upper panel), and of the fraction of the abovementioned cells undergoing genome reduplication upon release after DHCB treatment (lower panel). Individual values of biological replicates, their mean and standard deviations are reported. ANOVA test, comparing each sample to the wild type (****P < 0.0001; n.s. = non-significant).

(F) RPE1 cells were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated Myc-ANKRD26 constructs. Subsequently, cells were treated either with DMSO or ZM447439 for 24h and subjected to immunoblotting; n = 3 independent experiments.
Centriolar distal appendages activate the centrosome-PIDDosome-p53 signaling axis via ANKRD26 (2020)
Figure 4

Figure 4. Only the PIDD1 non cleaved precursor is capable of localizing to centriole DAs.

(A) Schematic of the PIDD1 domain structure and of the different PIDD1 species generated by autoproteolysis. LRR = Leucin Rich Repeat Domain; ZU5 (N and C) domains = Domain present in ZO-1, Unc5-like netrin receptors and in ankyrins; UPA domain = conserved in UNC5, PIDD, and Ankyrins; DD = death domain.

(B) A549 cells of the indicated genotypes were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated PIDD1-V5 derivatives and subjected to immunoblotting. N = 2 independent experiments.

(C) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced (mock), or transduced with PIDD1-V5 lentiviral vectors as in (B), and co- stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.

(D) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. Data obtained from images as in (C). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001).

(E) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. Data obtained from images as in (F). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).

(F) Representative fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors expressing the PIDD1 non-cleavable derivative (PIDD1S446A-S588A) or truncations thereof. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.
Centriolar distal appendages activate the centrosome-PIDDosome-p53 signaling axis via ANKRD26 (2020)
Figure 5

Figure 5. PIDD1 autoproteolysis is constitutive, occurs independently of DAs localization but is necessary for PIDDosome activation.

(A) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1-V5 in its wild type form. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.

(B,C) A549 cells of the indicated genotypes were either left untransduced (mock), or transduced with lentiviral vectors expressing PIDD1-V5 in its wild type form or carrying the indicated point mutations. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.

(D) Fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors carrying the indicated point mutations, and stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.
Centriolar distal appendages activate the centrosome-PIDDosome-p53 signaling axis via ANKRD26 (2020)
Figure 6

Figure 6. Extra PIDD1-positive centrosomes generate clusters.

(A) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1-GFP. Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm. The centrioles subjected to fluorescence intensity measurement in (B) are labelled with numbers.

(B) Quantification of the PIDD1 average pixel intensity at individual centrioles across the indicated cell cycle phases of RPE1 cells stained as in (A). Mean values ± s.e.m. are reported. N > 50 centrioles were assessed for each phase, a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; **P < 0.01; *P < 0.05; n.s. = non-significant).

(C) Fluorescence micrographs of RPE1 cells stably expressing CETN1-GFP treated either with DMSO or with DHCB for 24h. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.

(D) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in (C). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition, a.u. = arbitrary units. Unpaired Student's t-test, two tails (n.s. = non-significant).

(E) Movie stills of a representative RPE1 cell stably expressing CETN1-GFP treated with DHCB and subjected to time-lapse video microscopy in the presence of SiR-DNA. Time is expressed in minutes, relative to anaphase onset. The dashed line indicates the plasma membrane of the cell of interest, arrowheads indicate the centrosomes' position. Scale bar: 5 μm.

(F) Centrosomal distance over time in RPE1 cells stably expressing CETN1-GFP and treated with DHCB. Time zero corresponds to the frame preceding anaphase onset. Coloured dots (lower panel) summarize the clustering time for each cell, mean ± standard deviation in black. Data calculated from four-dimensional imaging as in (E). N = 10 cells.
Centriolar distal appendages activate the centrosome-PIDDosome-p53 signaling axis via ANKRD26 (2020)
Figure 8

Figure 8. PIDD1 localization to DAs is required for PIDDosome activation in response to DNA damage.

(A) A549 cells of the indicated genotypes were treated for 24h as indicated (CPT = camptothecin, ZM = ZM447439). Samples were subjected to immunoblotting; n = 3 independent experiments.

(B) A549 cells treated as in (A) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ-tubulin-positive centrioles per cell. Mean values ± s.e.m. are reported. N = 3, ≥ 50 cells from each independent experiment. ANOVA test (n.s. = non-significant).

(C) RT-qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. The average fold of induction ± standard deviation is shown. N = 3 biological replicates with two technical replicates each. Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type (WT) cells, ANOVA test (****P < 0.0001; ***P < 0.001; n.s. = non-significant).

(D) Immunoblot analysis of samples treated as in (C). N = 3 independent experiments.

(E) Proposed model for the centrosome-dependent PIDDosome activation upon different stimuli. The centrosome constitutively acts as PIDD1 centralizer. A local increase in PIDD1 concentration (achieved either by centrosome clustering or upon p53 activation) triggers PIDDosome activation. In ANKRD26-deficient cells, the inability of the centrosome to generate a local increase in PIDD1 concentration hinders the activation of the complex in response to both stimuli.

Expanded View Figure legends
The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments (2019)
Figure 1

Figure 1. Rab39a in dendritic cells is required for optimal cross presentation

(A) Relative expression of Rab39a in siRNA-treated cells after 48 hours of knockdown. Rab39a expression in I-Ab or Rab39a siRNA-treated cells was normalized to expression in cells treated with control β2m siRNA. Error bars show SD between three independent experiments. * p < 0.05 using two-way Student's t-test.

(B-D) XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe (B), Ova-latex (C) or unconjugated soluble Ova (D). The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.

(E) DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 Cells were then fed with Ova-Fe and exposed to reporter T cells as in (B). Error bars show SD of duplicate wells. * p < 0.05 using two-way ANOVA. Representative plot of 3 independent experiments.
The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments (2019)
Figure 2

Figure 2. Rab39a functions selectively in XPT

(A) MHC-II presentation of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe. The cells were then exposed to MF2.2D9-Luc Reporter CD4 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control β2m using two-way ANOVA. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m. Representative plot of 3 independent experiments.

(B) DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with Ova-Fe and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m using two-way ANOVA. Representative plot of 3 independent experiments.

(C, D) MHC-I Presentation of SIINFEKL-H2-Kb complexes on siRNA treated DC3.2 NS-Ova (C) or DC3.2 UbS8L (D) cells. 48 hours after siRNA transfection, the indicated amounts of dox was added to the cells to induce Ova expression. After 2 hours at 37oC, RF33.70-Luc CD8 T cells were added along with Brefeldin A (Golgiplug) to a final concentration of 1:1000. The cells were incubated overnight for reporter T cell luciferase expression. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control I-Ab using two-way ANOVA. None of the dox concentrations are p < 0.05 for Rab39a siRNA vs I-Ab. Representative plot of 3 independent experiments.

(E) Flow cytometric analysis of surface H2-Kb and I-Ab levels of siRNA treated DC3.2R cells. Shown are results after 48 hours of knockdown. Representative plot of 3 independent experiments.
The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments (2019)
Figure 3

Figure 3. Rab39a localization and function in endosomes and phagosomes of DCs.

(A) Phagocytosis assay after 48 hours of siRNA knockdown. DC3.2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads. The mixture was then placed in media at 37oC for 1 hour. Cells were harvested and incubated on ice with 1 mg/ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry. As a control, some cells were incubated with 10 µM Cytochalasin D during the feeding period. Shown are percentages of cells that have taken up beads. Representative plot of two independent experiments.

(B) Rab39a was induced in DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) via overnight incubation with 1µg / ml dox. KO and rescued cells were then harvested and assayed as in (A) for phagocytosis. Representative plot of two independent experiments.

(C) DC3.2-Rab39a cells were induced to express Rab39a overnight. Cells on coverslips were washed, fixed and stained according to the listed protocol and antibodies. Green = Rab39a (HA tag), Magenta = Lamp2. Arrows highlight punctate structures with colocalization. Scale bar = 10 µm. Representative image of two independent experiments.

(D) DC3.2 Rab39a KO cells were transduced with dox inducible mcherry-tagged Rab39a. Cells were plated in 35 mm glass bottom dishes and induced with 1 µg/ml dox. The next day, 1 µm latex beads conjugated with Alexa 488-NHS (1 bead/cell) were added. After 2 hours at 37oC, cells were imaged under a Leica TCS SP5 confocal microscope. Green = Alexa 488 labeled beads. Magenta = mcherry-Rab39a. Arrows highlight internalized beads surrounded by Rab39a. Scale bar = 10 µm. Representative image of two independent experiments.

(E) 5x105 of DC3.2-Rab39a cells were induced to express Rab39a overnight with 1 µg/ml dox in a 6 well plate. Cells were fed at 1 bead/cell of biotinylated-6µm magnetic beads for the indicated lengths of time. Magnetic-bead-phagosomes were isolated using the listed protocol. Phagosomes were washed and stained for Rab39a (HA-tag). Representative data for 3 independent experiments.

(F) 1.25 x 105 of DC3.2-Rab39a cells were incubated in a 12 well plate overnight ± dox (1µg/ml) to induce Rab39a. Cells were fed at 4 beads/cell of biotinylated-ova-6µm-magnetic-beads for 3 hours with or without 1:1000 BFA (golgiplug, BD). Phagosomes were isolated using the listed protocol. Isolated phagosomes were washed, permeabilized and stained. Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes. Representative plot of two independent experiments.