EMBO/soda-nel huggingface dataset:
Structural basis of semaphorin-plexin cis interaction (2020)
Figure 2

Figure 2. Binding of Sema1b to PlexA in cis perturbs the PlexA ring-like conformation

A. FRET-FLIM in live COS-7 cells shows cell surface PlexA-Sema1b cis interaction. Sema1b mutants reveal that both head-to-head and side-on orientations of the PlexA-Sema1b complex are involved in the cis interaction. The cis interaction between Sema1a and PlexA is not observed. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). *P<0.05, ***P<0.001

B. Representative negative stain class averages of PlexAecto correlate well with the ring-like conformation of mouse PlxnA1. The ring-like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue (N terminus) to red (C terminus). Scale bar, 10 nm. All class averages of PlexAecto are shown in Fig EV3H.

C. Superposition of the side-on orientation derived from the 2:2 complex with the mouse PlexinA1 (pdb 5l56) ring structure (green) based on the Drosophila and mouse PlexinA1 sema domains.

D. Design of PlexAecto-FRET Biosensor for monitoring conformational changes. In the ring-like conformation, the fluorescent protein mClover and mRuby2 at N- and C- termini, respectively, are in close proximity generating FRET. Opening the ring-like conformation results in a FRET decrease.

E. In vitro FRET-FLIM measurement of the PlexA-FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). ***P<0.001.

F. Relative change of COS-7 cells surface area upon treatment with Sema1a-Fc. Cells expressing PlexAFL-mClover or PlexAFL-mCover and Sema1b-mRuby2 wild-type or mutants were treated with purified Sema1a-Fc at a final concentration of 5.8 µM. Images were acquired every minute for 30 minutes. Cell surface area was calculated using ImageJ before and after stimulation. Data are presented as means ± sem. *P<0.05, ***P<0.001, p-value calculated by one-way analysis of variance (ANOVA).
Structural basis of semaphorin-plexin cis interaction (2020)
Figure 3

Figure 3. Mouse Sema6A utilises the same molecular mechanism to inhibit plexin function as that observed for Drosophila Sema1b

A. Cultured dorsal root ganglion (DRG) neurons from E12.5 Sema6A knockout (KO) embryos were transfected with EGFP, EGFP-tagged wild-type (WT) Sema6A (Sema6A-WT) or different EGFP-tagged Sema6A mutants (Sema6A-mutA, Sema6A-mutB, and Sema6A-mutA+B) at 1 day in vitro (DIV1) and treated with purified Sema6A-Fc at a concentration of 75 nM for 1 hour at 37°C at DIV4. Transfection of EGFP was used as a control (full collapse). DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments. Transfection of Sema6A-WT, Sema6A-mutA and Sema6A-mutB but not of EGFP or Sema6A-mutantA+B prevented Sema6A-mediated growth cone collapse (Fisher's exact test p<0.0001). Quantification of growth cone collapse was performed using a growth cone morphology matrix (lower part panel A). Data is presented as percentage of morphologically distinct growth cones, divided (represented by the dotted line) in two categories, from uncollapsed (1-4) to fully collapsed (5-8). Totally, 20-40 growth cones were analyzed for each condition per experiment (n=3 experiments). Scale bar 20 µm.

B. Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A-Fc and visualized with immunofluorescent staining to detect GFP (green) and phalloidin (red). Average intensity images for all different constructs are shown in grey color. Scale bar 20 µm.
Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non- human primates by intracerebral lentiviral gene therapy (2016)
Figure 2

Figure 2. LV transduction and transgene overexpression in neurons and glial cells at the injection sites. (A) Representative confocal images showing GFP+ cells (green) expressing markers of neurons (NeuN), astrocytes (GFAP) and oligodendrocytes (CNPase) close to the posterior injection site of NHP P2. Arrows indicate co-localization of IF signals. (B) Representative confocal images showing neurons (NeuN) overexpressing ARSA (green) close to the posterior injection site of NHP S2.2. Arrows indicate co-localization of IF signals. Note the granular perinuclear ARSA staining, likely representing protein localized in the Golgi/lysosomal vesicles. (C) Confocal z-stack images showing the presence of ARSA-positive cells (green) co-stained with oligodendrocyte markers CNPase and APC (arrows). (D) Undetectable ARSA immunoreactivity in tissues of untreated animals. In all pictures nuclei are counterstained with ToPro (blue). Scale bars: A, 80 µm; B, D 60µm, C, 80µm
Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non- human primates by intracerebral lentiviral gene therapy (2016)
Figure 3

Figure 3. Integrated LV genome and transgene overexpression in LV.hARSA-injected NHPs. Vector copy number (VCN) cartography shows integrated LV genome (assessed by qPCR) along the rostro-caudal axis (slices 1-10) in LV.hARSA-injected S1.1 and S2.2 NHP in a side-by-side comparison with ARSA mRNA expression (assessed by qPCR analyses using probe and primers annealing to sequences conserved in both the human and Macaca fascicularis and expressed as fold increase to region-matched blocks of the contralateral hemisphere). Grading of colors for VCN ranged from white (VCN<0.001; corresponding to CT>37) to dark orange (VCN=1-3). The highest VCN is found in close correspondence to the injection sites, as confirmed by comparison with post-surgery MR images (yellow and orange circles indicate viral suspension close to the injection sites). Grading of colors for ARSA expression ranges from white (<1.5 fold the physiological level) to dark purple (100-fold the physiological level).
Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non- human primates by intracerebral lentiviral gene therapy (2016)
Figure 4

Figure 4. Supraphysiological ARSA protein expression in LV.hARSA-injected NHPs. (A) ARSA overexpression detected by IHC in cortical and subcortical regions of the injected and contralateral hemispheres of NHP S2.3. (B) Physiological ARSA expression in cortical and subcortical regions of the injected hemisphere in LV.GFP-injected NHP P1. Scale bar, 100 µm. Insets show ARSA-expressing cells at higher magnification. Note the perinuclear accumulation of ARSA protein. (C) Quantification of immunopositive signal in brain slices 3 to 6 (n= 8 blocks/slice). Data are expressed as fold increase to mean immunopositive signal measured in matched brain regions of NHP P1 (physiological). Two-way ANOVA and Bonferroni post-tests, *p<0.05, **p<0.01, ***p<0.001 vs physiological.
Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non- human primates by intracerebral lentiviral gene therapy (2016)
Figure 5

Figure 5. Supraphysiological ARSA activity in LV.hARSA-injected NHPs. (A) Schematic representation of ARSA activity in the brain of NHP S1.1 and S2.2 along the rostro-caudal axis (slices 2-9). Data are expressed as fold increase to ARSA activity detected in matched blocks of UT and LV.GFP-injected NHPs (physiological levels). Grading of colors ranges from light yellow (<1.2 fold) to dark green (≥ 2 fold). (B-C) Enzymatic activity measured in single brain slices of NHPs of study group 1 (B) and study group 2 (C) (filled grey bars, injected hemisphere; striped grey bars, contralateral hemisphere), and in UT and LV.GFP-injected NHPs (white bars; physiological levels). Arrows on x axis indicate the injection sites. n=2-12 tissue blocks/slice. Number of tissue blocks analyzed per animal: 51 (UT/LV.GFP), 98-122 (S1.1 and S2.2), 12-22 (S1.2, S1.3, S2.2, S2.3). Two-way ANOVA followed by Bonferroni’s multiple comparison tests, *p<0.05; **p<0.01; ***p<0.001 vs matched slices of UT/LV.GFP. (D) Summary of ARSA activity in the injected and contralateral hemispheres of NHPs in study group 1 and study group 2. Number of tissue blocks analysed: 51 (UT/LV.GFP), 105 (study group 1, injected hemisphere), 52 (study group 1, contralateral hemisphere); 103 (study group 2, injected hemisphere), 72 (study group 2, contralateral hemisphere). One-way ANOVA and Tukey’s multiple comparison tests, *** p< 0.001 versus UT/LV.GFP, §p<0.05 versus study group 1. (E) ARSA activity in the spinal cord of individual LV.GFP- (n=4 blocks/animal, 2-3 replicates) and LV.hARSA-injected NHPs (n=2-4 blocks/animal; n=10 and n=11 total blocks for study group 1 and study group 2, respectively). Graph on the right represent the summary of ARSA activity in each treatment group. One-way ANOVA and Dunnet’s multiple comparison test. * p<0.05 versus pilot group. (F) ARSA activity in the sciatic nerve of LV.GFP- (n=2) and LV.hARSA-injected NHPs (n=3). In B-F data are expressed as floating bars (line at mean). (G) ARSA activity in the cerebrospinal fluid (CSF) of UT/LV.GFP (n=3) and LV.hARSA-injected NHP (n=5). Data are expressed as mean±SEM. Student t-test, *p=0.035.
Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non- human primates by intracerebral lentiviral gene therapy (2016)
Figure 6

Figure 6. Efficient transduction of Krabbe CNS tissue by LV.hGALC. (A) Vector copy number (VCN) indicating the distribution of integrated LV genome along the rostro-caudal axis (slices 7-19) in LV.hGALC-injected NHPs (JT02 and JV02) assessed by qPCR. Each dot represent the VCN measured in one block within the slice. Lower threshold (dotted line): VCN<0.001, corresponding to CT>37. (B) GALC mRNA expression along the rostro-caudal axis of JT02 (slices 7-13) is expressed as fold increase to region-matched blocks of the contralateral hemisphere (dotted line, y=1). Data are expressed as floating bars (min to max, line at mean; n=4-8 blocks/slice). Arrows on x axis in A and B indicate the injection sites. (C) Confocal images showing GALC+ cells expressing markers of neurons (NeuN) and astrocytes (S100β) close to the injection site (slice 10) in JT02. Arrows indicate co-localization of IF signals. (D-E) Confocal images showing GALC-expressing astrocytes (S100β; D) and neurons (NeuN; E) in the contralateral hemispheres, anterior (slice 8) and posterior (slice 12) to the injection site. Arrows indicate co-localization of IF signals. Scale bars: C, 70 µm (upper panel) and 40µm (lower panel); D and E, 40µm.
Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non- human primates by intracerebral lentiviral gene therapy (2016)
Figure 7

Figure 7. Gene therapy rescues GALC activity in CNS tissues of Krabbe-affected NHP. (A) Specific GALC enzymatic activity in single brain slices of WT untreated (UT; white bars, physiological levels), WT LV.hGALC-treated (JV02) and Krabbe-affected LV.hGALC-treated (JV02) animals (filled grey bars, injected hemisphere; striped grey bars, contralateral hemisphere). Two-way ANOVA followed by Bonferroni’s multiple comparison tests; *p<0.05, **p<0.01 vs matched slices of WT UT. (B-C) Summary of GALC activity in brain (B) and spinal cord (C; n=3 blocks/animal) samples from JT02 and JV02, as well as from WT and Krabbe UT controls. Data in A-C are represented as floating bars (min to max, line at mean; n=2-6 blocks/slice). Number of blocks analysed/animal: 16 (WT UT), 24 (JT02), 22 (JV02). GALC activity in Krabbe UT samples is < 0.001 nmol/h*mg; n=3 blocks. Kruskal-Wallis test and Dunn’s multiple comparison test; *p<0.05, ***p<0.001 vs WT UT. (D) Chromatographic profile of GALC in selected brain slices and spinal cord (SC) tissue of untreated (UT) WT and LV.hGALC-treated Krabbe NHP (JT02). Extracts were run through a Sephadex S-300 gel filtration column. Fractions (0.2 ml) were assayed for enzyme activity using MUGAL substrate in the presence () or absence of 11µM AgNO3 (●). (E) GALC activity in the CSF of JT02 (pre- and post-treatment) and JV02 (post-treatment). Dots represent technical replicates. (F) Beginning at two months (m) of age, animals were evaluated monthly for motor performance using a modified Bayley’s scale for infant development. For the study animals (JV02 and JT02), scores are reported as pre-surgery and post-surgery. Historical data for normal and affected animals are presented as means plus one standard deviation. Affected animals score significantly lower than normal animals at any age considered. JT02’s motor scores increase over time post LV.GALC injection, being close to the mean observed in normal juveniles at 5 months. Historical data are analyzed by two-way ANOVA and Bonferroni posttests, p<0.05 at 6m, p<0.001 at all other ages. n=5-20 animals/group, except for normal animals at 6 months (n=1).
Connexins modulate autophagosome biogenesis (2014)
figf1

(a) Immunoblot for the indicated Cx of homogenates (Hom) and autophagic vacuoles (AV) isolated from fed or 6 h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a. (b) Immunofluorescence for LC3 and Atg16 in normal rat kidney (NRK) cells expressing GFP–Cx43 maintained in the presence or absence of serum for 4 h. Single channels are shown in reverse black and white, and magnification of single and merged images are shown in colour. Arrows: co-localization of Atg16/Cx43 (yellow) or Atg16/Cx43/LC3 (white). Quantification is shown in Supplementary Fig. 1c. (c) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4 h. Full fields are shown in Supplementary Fig. 1d. (d) Maximum projection confocal microscopy 3D reconstruction (left) followed by a single-plane time-lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2–Cx43 (pseudocoloured in red) and sfGFP–ATG16. (e) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP–Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white, and merged images in colour. Inset shows higher magnification. Bottom: Quantification of co-localization between Atg16 and Cx43 (n = 3 wells, 4 independent experiments, >20 cells per experiment). Values are mean + s.e.m. * P 0.05. (f,g) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence (f,g) or absence of serum (4 h; g). I: input; FT: flow through. (h) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 from untreated WT MEFs, on incubation with 3-methyladenine (3MA) or in MEFs from Atg5-null mice (Atg5−/−). (i) Immunoblot for Flag and Cx43 of immunoprecipitates (IP) of endogenous Cx43 in WT MEFs transfected with the indicated Flag-tagged constructs of truncated Atg16. GAPDH, LC3 and Atg5 are shown as negative controls for immunoprecipitation. Nuclei are highlighted with DAPI. Scale bars, 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
Connexins modulate autophagosome biogenesis (2014)
figf2

(a) Immunostaining for LC3 and GABARAP in MOBs from WT or Cx43-null mice (Cx43−/−) grown in serum-supplemented media. Reverse black and white channels (top) and colour images (bottom). Right: Average number of LC3 and GABARAP puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment). (b) Electron micrographs of WT and Cx43−/− MOBs grown in serum-supplemented media. Yellow arrows: autophagic vacuoles. Insets: autophagic vacuoles (AV) at higher magnification. Right: Percentage of cytoplasm area occupied by AV (top) and number of AV per area (bottom; n = 3 mice, >10 micrographs per mice). (c) Immunoblot for LC3 of the same cells grown in serum-supplemented media untreated or treated with lysosomal protease inhibitors (PI) for 2 or 4 h. Top: representative immunoblot. GAPDH is shown as loading control. Bottom: LC3 flux (left) and increase in LC3-II during lysosomal inhibition (right; n = 3 independent experiments). (d) Representative images of the same cells transfected with the mCherry–GFP–LC3 tandem reporter and grown in serum-supplemented media. Single-channel images (inverted black and white) and merged images (colour). Bottom: Number of vesicles positive for GFP and mCherry (autophagosomes; APG) and only for mCherry (autophagolysosomes; APL; n = 4 wells, 4 independent experiments, >30 cells per experiment). (e) Electron micrographs of livers from WT and Cx43+/− mice. Arrows indicate AV. Right: Percentage of cytosolic area occupied by AVs (n = 3 mice, >10 micrographs per mice). (f) Immunofluorescence for LC3 in Cx43−/− MOBs expressing GFP-tagged full-length Cx43. Single channels (inverted black and white) and merged channels (colour). Asterisks: transfected (green) and untransfected (red) cells. (g) LC3-positive puncta per cell in the indicated cells (n = 3 wells, 3 independent experiments, >50 cells per experiment). All values are mean + s.e.m. and differences from WT were significant for *P 0.01. Nuclei are highlighted with DAPI. Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of b and e. Uncropped images of blots are shown in Supplementary Fig. 9.
Connexins modulate autophagosome biogenesis (2014)
figf3

(a) Immunoblot for LC3 in MOBs from WT or Cx43-null mice (Cx43−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA). Glyceraldehyde-3-dehydrogenae (GPD) is shown as the loading control. Levels of LC3-II (left) and LC3-II flux (right; n = 3 independent experiments). (b) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid. Where indicated, chloroquine (CQ) was added to block lysosomal degradation. Bottom: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (c) Immunofluorescence for LC3 in WT or Cx43−/− MOBs treated with the indicated kinase inhibitors for 2 h. Insets: Higher magnification images in inverted black and white. (d) Endogenous LC3 puncta in the same cells shown in c (n = 3 wells, 3 independent experiments, >50 cells per experiment). *, differences from WT; §, differences from untreated. (e) LC3 puncta in Cx43−/− MOBs transfected with constructs coding for different forms of Cx43 (full length, CT, ΔCT258 or ΔCT245) tagged as indicated in the scheme. Left: Representative images. Merged fields (left) and LC3 channel in reverse black and white (right) are shown (images of GFP–Cx43 are shown in Fig. 2f). Right: Quantification of the average number of LC3 puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment). (f,g) Immunoblot for the indicated proteins of the immunoprecipitates (IP) of Atg16 from MEFs transfected with GFP-tagged full-length (FL-) Cx43 or Flag-tagged C terminus (CT-) Cx43 (f) or ΔCT258-Cx43 (g) I: input; FT: flow through. GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control. All values are mean + s.e.m. Differences from the control were significant for (*, §)P 0.01 and (**, §§)P 0.001. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
Connexins modulate autophagosome biogenesis (2014)
figf4

(a) Immunoblots for the indicated proteins in MEFs treated with tamoxifen (Tx) or lindane (Lind) in the absence or presence of lysosomal protease inhibitors (PI). Right: Changes in LC3-II levels relative to those detected in untreated cells (n = 4 independent experiments). (b) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine (CQ) for 3 h. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (c) Time course of changes in the average LC3-positive puncta per cell after addition of lindane to control NRK cells. (d) Fluorescence images of WT or Cx43−/− MOBs transfected with mCherry–GFP–LC3 and incubated or not with tamoxifen or lindane. Bottom: Quantification of autophagolysosomes (n = 3 wells, 3 independent experiments, >50 cells per experiment). (e) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds. Representative images as merged channels are shown. Right: quantification of the average of cytosolic Atg16-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (f) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP–Cx43 treated with lindane and untreated. Single channel images in inverse black and white and merged colour images are shown. Outlined areas are shown at higher magnification. Arrows: double (yellow) and triple (white) co-localization. (g,h) Immunostaining for Atg16 in Cx43 knocked down (KD) (g) or knocked out (−/−) (h) cells. Right: quantification of the average of cytosolic Atg16 positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment). (i) Immunoblot for Atg16 and Atg14 in immunoprecipitates (IP) of endogenous Cx43 from WT MEFs maintained in the absence of serum (4 h) or incubated in the presence of tamoxifen or lindane. I: input; FT: flow through. All values are mean + s.e.m and differences from untreated cells are significant for *P 0.01. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
Connexins modulate autophagosome biogenesis (2014)
figf5

(a–c) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP–Cx43 maintained in the presence of serum (a), absence of serum (b) or treated with lindane (c). Single black and white inverted channels, merged channels and higher magnification insets are shown. (d) Immunoblots for indicated proteins in isolated fractions from starved mouse liver. Hom, homogenate; LE, late endosomes; Cyt, cytosol; APG, autophagosomes; APL, autophagolysosomes. (e) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice. (f–h) Fluorescence images of NRK cells expressing GFP–Cx43 maintained in the presence of serum (f), absence of serum (g) or treated with lindane (h) and incubated with Alexa594–transferrin for 15 min before fixation. Top: single and merged channels and outlined area at higher magnification. Bottom: 3D reconstructions and higher magnification details of the outlined areas. Nuclei are highlighted with DAPI. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.