EMBO/soda-nel huggingface dataset:
Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders (2021)
Figure 1

Figure 1: AAV2.GL and AAV2.NN achieve panretinal transduction in mice using ubiquitous promoter.

Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice.

(A) In vivo cSLO examinations of the mouse retina at 1- and 2-weeks post injection (WPI) of 2E9 total vg (in 1 µl) of sc-CMV-eGFP packaged with parental serotype AAV2, state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.

(B) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI. Acquisition settings were kept constant for all samples. Scale bar: 100 μm.

(C) Confocal tile-scans of retinal cross sections immunolabelled for eGFP and Hoechst. Scale bar: 500 μm

(D-E) Immunolabelled cross sections of mouse retina infected with AAV2.GL and AAV2.NN respectively, with focus on the ONL and INL, stained for eGFP, recoverin and DAPI. Scale bar: 50 μm

(F) qRT-PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2.7m8, AAV2.GL and AAV2.NN compared to AAV2. Biological replicates n ≥ 3. Bars show mean ± SEM. One-way ANOVA was performed with post hoc Holm-Sidak multiple comparisons test. * p≤0.05, ** p≤0.01

Data information: GFP=eGFP; ONL, outer nuclear layer; INL, inner nuclear layer. (F) Detailed statistical analysis in Appendix Table S1.
Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders (2021)
Figure 2

Figure 2: Novel capsids outperform AAV2 and AAV2.7m8 in mice using photoreceptor-specific promoter.

Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice.

(A) In vivo cSLO examinations of the mouse retina at 1-, 2- and 3- weeks post injection (WPI) of 8E9 total vg of ss-hRho-eGFP packaged with parental serotype AAV2, state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.

(B) qRT-PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants. Biological replicates n ≥ 3. Bars show mean ± SEM. One-way ANOVA was performed with post hoc Holm-Sidak multiple comparisons test. * p≤0.05, ** p≤0.01, *** p≤0.001.

(C) Confocal scans of retinal cross sections, with focus on the ONL, immunolabelled for DAPI, eGFP and recoverin. The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images (noted with asterisk *) with merged DAPI, eGFP and recoverin signal. Acquisition settings were kept constant for all samples. Scale bar: 50 μm.

Data information: GFP=eGFP; ONL, outer nuclear layer. (B) Detailed statistical analysis in Appendix Table S1.
Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders (2021)
Figure 3

Figure 3: AAV2.GL and AAV2.NN transduce the dog retina more efficiently than AAV2.

Vector mediated eGFP expression after intravitreal injection in 10-month-old dog eyes. (A) In vivo cSLO examinations of the dog retina at four, five and six weeks post intravitreal injection of 2E11 total vg (in 200 µl) of sc-CMV-eGFP packaged with wildtype AAV2 (right eye), AAV2.GL (right eye) and AAV2.NN (left eye) capsid variants.

(B-C) Confocal scans of immunolabelled retinal cross sections (14 μm) stained for GFP and DAPI at 6 WPI time point.

(D-E) Confocal scans of immunolabelled retinal cross sections (14 μm) at 6 WPI time point, focusing on the photoreceptors, using antibodies against eGFP, Rhodopsin (Rho) and M-opsin (MO).

Data information: DAPI was used a nuclear marker. Scale bar panel (B): 200 μm, (C-E): 50 μm. Acquisition settings were kept constant for all samples. GFP=eGFP; RPE, retinal pigmented epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders (2021)
Figure 6

Figure 6: Intravitreal gene supplementation with AAV2.GL restores cone function in Cnga3-/- mice.

Restoration of cone function following intravitreal delivery of 1E10 vg AAV2.GL/mSWS-mCNGA3 therapeutic vector.

(A) Representative photopic ERG flicker series at 1, 5 and 10 Hz with 3 cd.s/m2 illumination at 10 WPI. Arrowheads indicate light stimulus.

(B) Representative photopic single flash ERG signal with 1, 3 and 10 cd.s/m2 illumination. The vertical dotted line indicates light stimulus.

(C) Quantification of photopic B-wave amplitude from single flash responses to 1, 3 and 10 cd.s/m2 illumination of wildtype C57BL6/J x 129/sv eyes (WT, n = 3) compared to treated eyes (IVT/GL, n ≥ 3) and untreated eyes (UN, n ≥ 3) at 10 weeks post injection. Symbols show mean ± SEM. Ordinary two-way ANOVA with α = 0.05 revealed significant difference (*** p < 0.0001) between samples. Additional analysis with multiple t-tests using the Holm-Sidak method, α = 0.05, assuming sample populations have the same scatter also revealed significant difference between samples at individual illuminations.

(D) Graphical representation of mouse fundus (d=dorsal, n=nasal, v=ventral, t=temporal) divided into quadrants. Q2 indicates locus of intravitreal injection.

(E-F) Confocal images of sections proximal to the injection site in Q2 (E) and distal to the injection site in Q4 (F), showing Cnga3 expression in PNA-positive cone outer segments. (G-I) Confocal images of Cnga3-/- treated (G), wildtype (H) and Cnga3-/- untreated (I) retinal sections stained for Cnga3. Note some non-specific signal detected in INL and adjacent synaptic layer. Scale bar: 50 μm.

Data information: OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer. (C) Detailed statistical analysis in Appendix Table S1.
Fungal phytochrome chromophore biosynthesis at mitochondria (2021)
Figure 4

Fig. 4: Localization of A. alternata HoxA and HoxB in A. nidulans.

A, Strains were grown over night at 28°C. Mitochondria were stained for 30 min with MitoTracker Red CMOXRos. Microscopy was done with a LSM 900 Airyscan 2 (Zeiss). Pictures were taken in the GFP and the RFP channel and overlaid (merge).

B, HoxA-GFP and CitA-GFP (ca. 170 amino acids from the N-terminus of citrate synthase) were expressed over night at 30°C in minimal medium with 2% threonine and 0.2% glucose. Mitochondria were incubated with 100 μg/ml proteinase K (PK) for 20 min on ice. Crude extract (CE), supernatant 1 (S1) containing mitochondria, pellet 1 (P1), supernatant 2 (S2) and pellet 2 (P2) with mitochondria were analyzed by Western Blot using anti GFP antibodies. CitA-GFPc = cytoplasmic form; CitA-GFPi = imported version.

C, D Role of the C-terminal anchor in HoxA. Microscopy was done as in A using a fluorescent microscope without the Airyscan technology. C, Expression of GFP-HoxA lacking the C-terminal anchor (∆CTA) in A. alternata. D, Expression of GFP fused to the CTA motif.
Fungal phytochrome chromophore biosynthesis at mitochondria (2021)
Figure 5

Fig. 5: Characterization of the HO-phytochrome protein complex.

A, BiFC (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2% threonine and 0.2% glucose. MitoTracker Red CMOXRos was used for mitochondrial staining. The mitotracker signal was observed in the RFP and the split-YFP signal in the YFP channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast (DIC). The scale bar represents 10 µm.

B, HoxA was expressed in E. coli 20 h at 15°C. Purification was performed with the Strep-tag system. HoxA was analyzed by size exclusion chromatography.

C, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot using the Anti-His antibody (lower panel).
Fungal phytochrome chromophore biosynthesis at mitochondria (2021)
Figure 6

Fig. 6: Analysis of the interaction of HO and the phytochrome photosensory domain in vitro.

A, B, Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiments.
Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy and Collagen quality control (2021)
Figure 1

Figure 1. Structure and dynamics of human FAM134 family members.

A) Schematic representation of FAM134 protein structures and sequence of the LC3 interacting region (LIR) domain of human (h) and mouse (m) proteins (red square).

B) Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm.

C) Structural models of reticulon homology domains (RHDs) of FAM134 proteins were built and coarse-grained before embedding in POPC bilayers and simulated using MD simulations. The overall shape of the RHD along with its membrane footprint was monitored by measuring the radius of gyration Rg of the protein (grey: TM segments; yellow: AH segments; red, green, blue: cytosolic loop of FAM134A, FAM134B, FAM134C).

D) Sequence alignment of human FAM134 proteins based on hydrophobicity profiles computed using AlignME. The helical structural elements characteristic of the RHD are denoted in red below along with a consensus sequence.

E) Probability density of Rg for FAM134-RHDs, indicating compact (small Rg value) or widespread (high Rg value) conformations.

F) Distinct conformational states of the RHD of FAM134 proteins were obtained from conformational sampling (total n=10.000) using coarse-grained molecular dynamic simulations (10 μs) and subsequent clustering. Representative conformations corresponding to the top 3 populated clusters are shown for each system. Conformation states in light colours represent the most populated states in the reported simulation.

G) Snapshots showing the curvature of bicelles containing DMPC (gray) and short-chain DHPC lipids (red) together with FAM134-RHDs over time.

H) Curvature time-traces (smoothed running averages over 11 ns widows) from individual replicates (n=20) quantify the bicelle shape transformation process during simulations. Black lines (mean value) and shaded region (s.e.m) denote the average behaviour of the system. From the 20 replicates, data on waiting times (t) were collected to compute rates and acceleration factors (Table EV3). Vesicle formation is marked by bilayer-disc curvature, |H| = 0.14 nm−1.

I) Cumulative distribution functions (CDF) of recorded waiting times for formation of vesicle (filled circles: simulations; lines: Single Poisson process with additional lag time).
Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy and Collagen quality control (2021)
Figure 2

Figure 2. FAM134 members are ER resident proteins harboring a functional LIR domain.

A-C) Lysates of HEK293T cells transiently transfected with HA-tagged wild-type (WT, Figure 2A-2C) and wild-type or ∆LIR mutant (Figure 2B) FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) using A, B) purified GST, wild type GST-LC3s, GST-GABARAPs, GST-Ubiquitin (Ub), GST-Tetra-Ubiquitin, or C) GST-LC3B and GST- LC3B F52A-V53A as baits. A representative Ponceau staining is shown.

D) Immunofluorescence images of U2OS expressing wild-type or ∆LIR mutant FLAG-HA-FAM134 proteins after 24hrs doxycycline induction under different conditions (basal=DMSO, Baf.A1=2h 200nM Bafilomycin A1, EBSS=2h starvation in EBSS) Scale bar: 10μm; staining against FAM134 (HA; green) and endogenous LC3B (red).

E) Automatic quantification of HA positive dot-like structures. Each data point represents the average of dots per cell of one view (representative image see Fig.EV2B). Total number of cells analyzed for basal: FAM134A/B/C/A∆LIR/B∆LIR/C∆LIR=592/472/358/280/233/300; EBSS: FAM134A/B/C/A∆LIR/B∆LIR/C∆LIR=364/720/142/364/720/674; error bars indicate s.e.m.; biological replicates WT/∆LIR=3/1.

F) Automated quantification of ER branching of in an automated manner (Valente et al, 2017). Each datapoint represents one view (representative image see Fig.EV2B). same cells as in (E); error bars indicate s.e.m..
Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy and Collagen quality control (2021)
Figure 3

Figure 3. FAM134s overexpression promotes the degradation of ER proteins.

A) Schematic representation of the experimental procedure to obtain the proteomes of U2OS control cells (-Dox) or cells inducible expressing FLAG-HA-FAM134 proteins upon 24hrs doxycycline induction (+Dox) by mass spectrometry (MS). Cells were grown in basal condition or starved with EBSS for 8hrs prior to lysis.

B) Principal Component Analysis of the MS data after indicated treatments.

C) Hierarchical clustering of label-free quantitation (LFQ) intensities of significantly changed proteins (ANOVA, FDR 0.01). Cluster framed in green includes proteins with a "endoplasmic reticulum" enriched GO.

D) Profile-plot of protein levels (LFQ intensities) of the cluster enriched for "endoplasmic reticulum" upon indicated treatments.

E) List and frequency of GOCC terms related to the protein cluster in (D).

F) CANX, REEP5 and SEC22B as sample proteins extracted from (D).

G) Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8.

H) ER-phagy flux, represented by the RFP/GFP ratio (total fluorescent intensities), upon basal (DMSO) or autophagy induced (Torin1) conditions, in U2OS control cells and cells overexpressing FAM134 proteins upon 24hrs Dox induction.

I) Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3

J) Comparison of DMSO or Thapsigargin (Tg) treated U2OS ssRFP-GFP-KDEL cell lines overexpressing indicated FAM134 family members upon 24hrs Dox induction.

Data information: all ER-phagy flux data are representative of three biological replicates and presents averaged data obtained from three individual wells (technical replicates) via the IncuCyte® S3, each view containing >150 cells.
Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy and Collagen quality control (2021)
Figure 4

Figure 4. FAM134 family members induce ER-phagy flux.

A) Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts (MEFs).

B) Western blot for Fam134 proteins in indicated MEFs, expressing the ER-phagy reporter ssRFP-GFP-KDEL and being reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein.

C-E) ER-phagy flux in ssRFP-GFP-KDEL Fam134 knockout and reconstituted MEFs, represented by the RFP/GFP ratio (total fluorescent intensities). Data is representative for three biological replicates and presents averaged data obtained from three individual wells (technical replicates) via the IncuCyte® S3, each view containing >150 cells. C) ER-phagy flux in basal (DMSO) or autophagy induced (Torin1) conditions. D,E) ER-phagy flux in Thapsigargin (Tg) treated MEFs.
Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy and Collagen quality control (2021)
Figure 5

Figure 5. Global proteome analysis of Fam134s knockout MEFs.

A). Schematic representation of the experimental procedure to obtain the proteomes of wild-type and Fam134s knockout MEFs by mass spectrometry (MS).

B) Principal component analysis (PCA) of analyzed proteomes.

C) Scatter plot showing proteins responsible for driving sample segregation along component 1 and component 2. Proteins driving segregation are significantly enriched (FDR<0.05) for GO cellular component (GOCC) terms associated with endoplasmic reticulum (ER) (blue dots).

D) Unsupervised clustering of ANOVA-significant proteins by GOCC terms. Inset showing the profile plot and the GO-term enrichment (FDR<0.05) of 197 proteins belonging to the highlighted cluster.

E) Physical interaction network of proteins significantly upregulated in Fam134 knockout MEFs. The network was generated using the STRING plugin in the Cytoscape environment with a confidence score set to 0.4. Nodes are protein entities while lines represent physical interactions Red, orange, green: Collagen, stress fiber, ER associated.

F) Insets of (E) showing the Collagen and the ER cluster, respectively.

(G,H) Profile plots showing the Log2 LFQ intensity (G) and representative immunofluorescence (H) of Climp63 (green) in wild-type and Fam134 knockout MEFs. (I,J) Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.
Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy and Collagen quality control (2021)
Figure 6

Figure 6. FAM134 absence causes ER expansion.

A) Electron microscopy of wild-type and Fam134 single knockout MEFs (ER=endoplasmic reticulum). Scale bar: 500nm.

B) Immunofluorescence images stained for endogenous Calnexin (Canx) or Climp63 in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. Scale bar: 10μm. Inset scale bar: 5μm.

C) Representative Western blots of total cell lysate derived from MEFs used in (B) and detected for endogenous Fam134 and Actin, respectively. D) Quantification of (B). At least n=10 independent cells across three independent experiments each condition. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.
Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy and Collagen quality control (2021)
Figure 7

Figure 7. Absence of Fam134 impacts Collagen I homeostasis.

A) Percentage of cells that are positive for pro-Collagen I in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. n=150 cells each condition.

(B, C) Representative Western Blot (B) of total cell lysate and the corresponding densitometric analysis (C) of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.

(D,E) Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein.

(F,G) Representative Western Blot (F) and the relative bar plot (G) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c.

(H,I) Representative Western Blot (H) and the relative bar plot (I) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b.

(J,K) Representative Western Blot (J) and the relative bar plot (K) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b.

(L,M) Representative Western Blot (L) and the relative bar plot (M) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c.

Data information: vinculin has been used as reference for ratio calculation. The statistical significance was estimated by ordinary one-way ANOVA. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.

Expanded View Figure Legends
Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases (2020)
Figure 1

Figure 1:

The type II (Pru) Toxoplasma strain is more susceptible to IFNγ-mediated growth inhibition in primary human foreskin fibroblasts (HFFs) than the type I RH strain due to presence of the polymorphic effector GRA15.

A) HFFs were pre-stimulated with IFNγ (10U/mL) for 24 h. Plaque assays were performed for each strain and each condition. Plaque number and area loss were calculated 4 days p.i. for RH and 6 days p.i. for Pru. Assays were performed with RH (n=4), Pru (n=4).

B) Relative parasite growth was measured 24 h p.i. in IFNγ-stimulated and unstimulated HFFs by luciferase assay. Growth of each strain in IFNγ-stimulated HFFs is expressed relative to growth in unstimulated HFFs. Experiments were performed with RH (n=4), Pru (n=4).

C) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U/mL IFNγ for 24 h. Following stimulation, HFFs were infected with either RH (MOI=1) or Pru (MOI=3) for another 24 h. Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei. The number of PVs in 5-6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field. Experiments were performed with RH (n=3), Pru (n=3). Representative images of percentage infected cells for each strain and condition are provided. Scale bar is 100 µm.

D) Parasites per vacuole were determined 24 h p.i. with similar conditions and staining as described in (C) except MOIs used were 0.5 and 1 for RH (n=3) and Pru (n=3), respectively.

E) Plaque assays were performed similarly as described for (a) with Pru WT (n=20 for plaque number and n=7 for plaque area), PruΔgra15 (n=20 for plaque number and n=7 for plaque area), PruΔgra15+GRA15 complemented (n=12 for plaque number and n=7 for plaque area).

F) Plaque assays were performed similarly as described for (A) with RH (n=5 for plaque number and n=3 for plaque area) and RH+GRA15II (n=5 for plaque number and n=3 for plaque area).

G) Relative parasite growth was measured as described in (B) with Pru WT (n=12), PruΔgra15 (n=12), PruΔgra15+GRA15 complemented (n=8).

H) Number of PVs was calculated as described in (C) with Pru WT (n=3), PruΔgra15 (n=3), RH (n=3) and RH+GRA15II (n=3) and normalized with the number of host cells in each field.

Data information: Statistical analysis was done by Two-way ANOVA followed by Tukey's multiple comparison test. Data are represented as mean ± standard error of mean (SEM).