EMBO/soda-nel huggingface dataset:
The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments (2019)
Figure 4

Figure 4. Silencing Rab39a affects the ability of phagosomes to generate peptide-class I complexes

(A-B) XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of C8L peptide (A) or Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.

(C) 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 C8L peptide beads and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. * p < 0.05 between no dox and + dox using two-way ANOVA. Representative plot of 3 independent experiments.

(D) Same as in (A) but cells were fed with 8 µg of A9M peptide bead and exposed to 12.64-Luc Reporter T cells. Experiments were combined by normalizing RLU values to the average of the negative control I-Ab. Error bars show SD of 3 independent experiments. * p < 0.05 for Rab39a or β2m siRNA vs negative control I-Ab using ANOVA.
The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments (2019)
Figure 5

Figure 5. Effect of Rab39a on intraphagosomal MHC-I molecules

(A-B) DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. Representative histograms shown in (B). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2-Ld (dox/no dox) was calculated. Three independent experiments were combined for analysis and error bars show the SD between experiments. Open H2-Ld, but not Closed H2-Ld was statistically significant (p<0.05) between KO and rescued cells using two-tailed ratio-paired Student's t-test.

(C) H2-Ld cell surface levels of KO and induced cells as in (A).

(D) Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains.

(E) Flow cytometry plots of (D).

(F) Analysis of phagosomes shown in (D) and (E). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated. Shown are p-values as computed by two-tailed ratio-paired t-test of 3 independent experiments. Error bars show SD between experiments.

(G) DC3.2 Rab39a-H2-Ld cells were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours in the presence or absence of BFA. Isolated phagosomes were stained for H2-Ld. Representative of two independent experiments.
Photoreceptor nanotubes mediate the in vivo exchange of intracellular material (2021)
Figure 1

Figure 1. The onset of material transfer coincides with the resolution of transplant-induced retinal detachment and can occur between donor photoreceptors located in the subretinal space.

A, Photoreceptor transplantation schematic

B, C, Analysis of GFP transfer after transplantation of Nrl::GFP donor photoreceptors into wildtype C57BL/6J (B) and Nrl-/- (C) recipient retinas after 14 (wt, n=9; Nrl-/-, n=7) and 21 (wt, n=6; Nrl-/-, n=4) and 90 (wt, n=5; Nrl-/-, n=4) days. Tissue is stained for GFP and nuclei (Hoechst). In this and all following figures, donor cells (D) are located above, and acceptor cells with GFP (A) are located below the outer limiting membrane (white dashed line), which delimits the apical border of the outer nuclear layer (ONL). In wildtype recipients, donor cells are typically located above the outer segment region (between the grey dashed lines). In Nrl-/- recipients, donor cells are located in the inner segment (IS) region. Plotted is the ratio of the numbers of GFP+ photoreceptors in the ONL (acceptor photoreceptors) and GFP+ donor photoreceptors in the subretinal space (SRS) over time.

D, Analysis of Cre-recombinase transfer after transplantation of Crx::Cre donor photoreceptors into Nrl-/- recipients after 14 (n=6), 21 (n=5), 42 (n=4), 90 (n=6) and 150 (n=6) days. The number of acceptor photoreceptors in the recipient retina that activated the Cre reporter gene is quantified.

E, Resolution of the transplant-induced retinal detachment in C57BL/6J recipient retinas (n=11), as measured by the maximum detachment length (mm) over time. Fundoscopy (images on the left) was used to locate the region of cell delivery for OCT imaging (green line) shown in the images on the right. Red lines correspond to the callipers used to determine the maximum length of the detachment at each time point. Before injection (B.I.) (n=11), immediately post-injection (I.P.I.) (n=10), 5 days post-injection (5 D.P.I.) (n=11), 9 days post-injection (9 D.P.I.) (n=11), 21 days post-injection (21 D.P.I.) (n=6),

F, Co-transplanted P3-4 Crx::Cre and P3-5 ROSAmT/mG donor photoreceptors into C57BL/6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post-transplantation.

Data information: Abbreviations: A, acceptor cells; D, donor cells; GFP, green fluorescent protein; RFP, red fluorescent protein; NFL, nerve fibre layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; IS, inner segments; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane; OS; outer segments; SRS, subretinal space; RPE, retinal pigmented epithelium. DPT, days post-transplant. BI, before injection; IPI, immediately post-injection; DPI, days post-injection. All data are presented as mean ± SEM; n.s. not statistically significant, ****p ≤ 0.0001, ***p < 0.001, *p < 0.05; one-way ANOVA with Tukey's post-hoc multiple comparisons test. Scale bars: 50 μm.
Photoreceptor nanotubes mediate the in vivo exchange of intracellular material (2021)
Figure 2

Figure 2. Transfer of donor-derived proteins and transcripts to host photoreceptors.

A-C, Combined FISH and IHF to detect GFP (A, B) and GNAT1 (C) 21 days post-transplantation of Nrl::GFP photoreceptors into C57BL/6J (A) and Nrl-/- (B, C) recipients.

D, E, Differences in mean fluorescence intensity of GFP and GNAT1 protein staining (D) and transcripts (E) between donor and acceptor photoreceptors. (GFP: wt recipients, n=9 images from 3 transplanted animals; Nrl-/- recipients n=8 images from 4 transplanted animals. GNAT1: Nrl-/- recipients, n=7 images from 3 transplanted animals).

F-K, FISH and IHF for GFP and GFP transcripts in C57BL/6J recipients transplanted with lentiviral-infected donor cells expressing cytoplasmic GFP (Lenti -GFP) (F, I) and nuclear-localized GFP (Lenti-nls-GFP) (G, J). H, MT index for cytosolic (Lenti-GFP, n=5) and nuclear localized (Lenti-nls-GFP, n=3). K, Quantification of transcripts in donor and acceptor photoreceptors. (Lenti-GFP, n=6 images from 3 transplanted animals; Lenti-nls-GFP, n=8 images from 4 transplanted animals).

Data information: All data are presented as mean ± SEM; n.s. not statistically significant, ****p ≤ 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; one-way ANOVA with Tukey's post-hoc multiple comparisons test. White dashed line delimits the apical border of the outer nuclear layer; grey dashed lines indicate the outer segment (OS) region. IS, inner segments. Scale bars: A-C, I, J: 10 μm. F, G: 50 μm.
Photoreceptor nanotubes mediate the in vivo exchange of intracellular material (2021)
Figure 3

Figure 3. Mitochondria transfer to photoreceptors in vivo.

A, Detection of GFP, Mito-DsRed, mitochondria marker (ATP5B, blue) and nuclei (Hoechst, white) in wildtype C57BL/6J recipient retinas 21 days after transplantation with Mito-DsRed-expressing Nrl::GFP donor photoreceptors.

B-D'', Higher magnification of regions in A (indicated by yellow dashed boxes) showing colocalization of ATP5B and Mito-DsRed-labelled mitochondria in the outer segments (B), soma (C), and the axon (D) of acceptor photoreceptors.

Data information: White dashed line delimits the apical border of the outer nuclear layer; grey dashed lines delimit the inner segments and/or outer segments of recipient photoreceptors. Scale bar: A: 50 μm. B-D'': 5 μm.
Photoreceptor nanotubes mediate the in vivo exchange of intracellular material (2021)
Figure 5

Figure 5. GFP-labelled acceptor photoreceptors are connected to transplanted donor photoreceptors via cell protrusions.

A, Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl-/- recipient retina 21 days after transplantation with Nrl::GFP donor photoreceptors, showing protrusions (pink arrows) connecting donor and acceptor photoreceptors.

B, C, 3D reconstruction of confocal microscopy images of whole-mounted retinas from Nrl-/- (B) and C57BL6/J (C) recipients 21 days after transplantation with Nrl::GFP donor photoreceptors (top layer) shows that donor and GFP+ acceptor photoreceptors appear to be attached via protrusions. Higher magnification 3D reconstruction (C) showing protrusions (pink arrows) and GFP labelling in acceptor cells.

D, E, Left: 3D reconstruction of two-photon (D) and light sheet (E) microscopy images of intact cleared ROSAmT/mG eye showing that GFP+-donor and -acceptor photoreceptors are in the same area. D, Right: A high magnification image showing GFP+-donor and -acceptor photoreceptors connected by protrusions (pink arrows). E, Right: In detail, donor and acceptor photoreceptors also appear connected through cell protrusions (pink arrow).

F, Percentage of GFP+-acceptor photoreceptors connected via a donor cell protrusion across different imaging modalities (n=3 animals per imaging technique).

Data information: Abbreviations: RGCsL, retina ganglion cells layer. All data presented as mean ± SEM; n.s. not statistically significant, one-way ANOVA with Tukey's post-hoc multiple comparisons test. White dashed lines delimit the apical side of the ONL in the recipient retina, grey dashed lines delimit the apical border of recipient photoreceptor inner segments. Scale bars: A, B, D: 50 μm. C: 20 μm. E: 1000 μm.
Photoreceptor nanotubes mediate the in vivo exchange of intracellular material (2021)
Figure 6

Figure 6. Characterization of protrusions that connect donor and acceptor photoreceptors in transplanted retinas.

A, Confocal image shows a Lenti-GFP infected donor photoreceptor (yellow arrow) connected to an acceptor photoreceptor. The donor protrusion (pink arrow) co-localizes with α-tubulin but not the outer segment marker GNAT1.

B, Nrl::GFP donor photoreceptors transplanted in a Prphr2-/- recipient retina exhibit polarized Prphr2 expression (yellow arrow) on the apical side of the donor photoreceptor. Basal protrusions (pink arrows) indicate donor protrusions that contact the host retina.

C, D, Expression of β-tubulin (C), SV2 (D) and GFP in the protrusions connecting Lenti-GFP-infected C57BL/6J donor photoreceptors with acceptor photoreceptors. Pink arrows point to the protrusions connecting donor and acceptor cells.

Data information: White dashed lines delimit the apical side of the ONL of the recipient retina, grey dashed lines delimit the apical border of the inner segments in the recipient retina. Scale bars: A, C: 50 μm. B, D: 10 μm.
Photoreceptor nanotubes mediate the in vivo exchange of intracellular material (2021)
Figure 7

Figure 7. Analysis of photoreceptor protrusions and GFP transfer in vitro.

A, B, Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin+ (SirActin) and GFP+ (A) and tubulin+ (ViaFluor) and MitoTracker Green+ (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy.

C, Quantification of photoreceptor protrusions in Nrl::GFP retinal cultures (culture wells, n=8, images, n=24, protrusions, n= 868). Data is presented as mean ± SEM;

D, Images from FRAP experiments carried out on protrusion connected and isolated Nrl::GFP cultures pre- and post-photobleaching. Area of photobleaching (white dashed circles). Analysed regions (yellow, orange and red dashed circles). Photoreceptor FRAP results shown from multiple experiments (isolated cells, n=14; connected to recipient, n=21; connected donor, n= 21). All data presented as percentage, mean ± SEM and fitted to the curve in a non-linear regression with two phase association.

E, Time lapse imaging showing movement of MitoTracker Green+ puncta in photoreceptors connected by a protrusion. White arrows point MTG+ puncta inside of the protrusion during the time lapse imaging.

Data information: n.s. not statistically significant, ****p ≤ 0.0001, ***p < 0.001; Unpaired T-test and extra-sum-of-squares F-test (Y0 constrained to 0.0). Scale bars: 5 μm.
Photoreceptor nanotubes mediate the in vivo exchange of intracellular material (2021)
Figure 8

Figure 8. RhoA-Rac1 plays a role in the extension of photoreceptor protrusions and MT.

A, B, Flow cytometric analysis showing enhancement of material transfer, of protein (biological replicate n=12; technical replicate (n=3 per biological replicate) (A) and mitochondria (biological replicate n=3; technical replicate (n=3 per biological replicate) (B), in co-cultures treated with ROCK inhibitor Y27632. ME, material exchange.

C, Confocal microscopy image of cryostat sections of C57BL/6J recipient retinas 21 days after transplant of C57BL/J6 photoreceptors infected with empty vector (n=5), RHOA overexpression (RHOA OE) (n=7) or RAC1 dominant negative (RAC1 DN) (n=7) lentiviruses. Quantification of MT in retinas transplanted with control, RHOA and RAC1 DN lentiviruses.

D, GFP staining of whole-mounted retinas after transplantation with control, RHOA and RAC1 DN lentivirus infected donor cells (n=4 per group). Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control.

Data information: All data presented as mean ± SEM; n.s. not statistically significant, ****p ≤ 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; one-way ANOVA with Tukey's post-hoc multiple comparisons test and unpaired T-test. White dashed lines delimit the apical side of the ONL of the recipient retina. Scale bars: 50 μm.

Expanded View Figure Legends
Enzyme promiscuity shapes adaptation to novel growth substrates (2019)
Figure 2

Figure 2: Evaluation of enzymatic activity for the wild-type and mutated promiscuous enzyme, YihS. A) YihS V314L + R315S mutant enzyme activity on D-mannose and D-lyxose. LC-MS was used to analyze YihS activity at saturating substrate concentrations to compare turnover rates on each substrate. Product formation was followed over time at a constant enzyme concentration. Turnover rates were calculated using linear regression (n=3 replicates for each enzyme, Dataset EV4). The error bars represent standard deviation (n=3) of the peak area. B) Turnover ratios of substrate conversion of D-lyxose / D-mannose are shown for the wild type YihS and mutant YihS enzymes. A ratio < 1 indicates a higher turnover rate on D-mannose compared to D-lyxose. Error bars represent standard error (n=3) calculated from the linear regression analysis.
Enzyme promiscuity shapes adaptation to novel growth substrates (2019)
Figure 3

Figure 3: Optimization mutation analysis for D-arabinose evolution experiments. A) Structural mutations observed in sequencing data of Experiments (Exp.) 1 and 2 (green) as well as residues previously identified as important for binding L-arabinose (blue) are highlighted on one chain of the AraC homodimer protein structure. The six base pair deletion observed in Exp. 1 appears to be most clearly linked to affecting substrate binding. B) Expression data (RNAseq) for significantly differentially expressed genes (q-value < 0.05, FDR-adjusted p-value, n=2 biological replicates for each condition). Scatter plot shows log2(fold change) of gene expression data comparing endpoint to initial populations for Exp. 1 and Exp. 2 (grey dots) with the location of the gene in the reference genome as the x-axis. Those genes that are associated with AraC transcription units are highlighted (red dots for Exp. 1 and blue dots for Exp. 2). Above the plot, the transcription units are labeled green if AraC activates expression (in the presence of arabinose) or red if AraC represses expression of those genes. C) The proposed two pathways for metabolizing D-arabinose. The pink pathway is enabled by the optimizing mutations observed in araC. D) Growth rate analysis of various weaned (starting point of static phase) and optimized (endpoint of static phase) strains with or without fucK or araB genes knocked out. Strains were grown in triplicate (n=3) on M9 minimal media with D-arabinose as the sole carbon source. The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation. The p-values reported were calculated using a two-sided Welch's t-test.
Homozygous NOTCH3 null mutation and impaired NOTCH3 signaling in recessive early-onset arteriopathy and cavitating leukoencephalopathy. (2015)
Figure 2

NOTCH3 and NOTCH3 targets expression profile

Gene expression of NOTCH3, HES1, HEY1, HEYL and the 17 recently identified targets were established by a real-time PCR panel assay in skeletal muscle of controls (n = 6), proband (II:1), parents (mother I:1, father I:2) and CADASIL patients (n = 3). Graph shows gene expression fold changes relative to controls and normalized on GAPDH (reference gene), expressed as mean of two experiments ± SEM. Among the 17 novel assayed target genes, RCAN2, ANGPT4, HP and SORBS were not detected in any sample and were therefore not reported. Green and red lines indicate 2.0 and 0.5 fold changes, respectively. Statistical significances and P-values are reported in Supplementary Table S1.

Direct sequencing of NOTCH3 cDNA from skeletal muscle of controls, proband, parents and two CADASIL patients. In the proband, residual mutant cDNA is amplified and the sequence shows only the mutant allele (A allele, arrow). Predominance of the wild-type allele (C allele, arrow) in the parents documents mRNA-mediated decay of the mutant allele (A allele), in contrast to what was observed for a canonical CADASIL mutation where there is balanced composition of mutant and wild-type alleles (arrows).
Homozygous NOTCH3 null mutation and impaired NOTCH3 signaling in recessive early-onset arteriopathy and cavitating leukoencephalopathy. (2015)
Figure 3

Morphologic analysis of vessels in skin and skeletal muscle of the proband as compared to control and CADASIL patient

A–C Histopathological examination of skin (A) and skeletal muscle biopsies (B, C) of control (left), proband (middle) and CADASIL patient (right). (A) Collagen IV staining (in green) in skin vessels: Collagen wall appears compact in the control, while it is disorganized in the skin vessels of the proband and of the CADASIL patient. Derangement of the collagen wall in single collagen fibers is more evident in the proband than in the CADASIL patient, where collagen wall is quite compact. Vessel's endothelium is delineated by ULEX staining (in red). Collagen IV staining in skeletal muscle vessels (B) recapitulates the skin picture. Smooth muscle actin immunostaining of skeletal muscle biopsies (C) shows attenuated SMCs in the tunica media of both the proband and the CADASIL patient, with foci characterized by a complete SMCs loss (particularly in the CADASIL patient) and thinning of the vessel wall (arrows).

D, E Ultrastructural analysis of skin biopsies. (D) Skin biopsy of the proband: (left panel) At low magnification, the tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal membrane with a multilayered aspect (arrows); SMCs are identified by the presence of “focal adhesions” (square boxes); (middle panel) collagen fibrils appear organized in a parallel pathway along SMC (arrow), note the “focal adhesions” (square box); (right panel) collagen fibrils (“CF”) are more represented in dense bundles than elastic ones (“E”). No GOM is detected, in contrast to CADASIL patient (E), showing deposits of granular osmiophilic material (arrows) located between SMC plasmalemma and basal lamina.

F KCNA5 immunostaining of skeletal muscle biopsies of control (left), proband (middle) and CADASIL patient (right). A global decrease in reactivity is evident in the vessels of both the proband and the CADASIL patient. Note multiple areas in which KCNA5 is barely detectable (arrows).

Data information: Scale bars: (A) 50 μm; (B) 30 μm; (C, F) 10 μm; (D, left panel) 7 μm; (D, middle and right panel) 1 μm.
Mitochondrial-nuclear co-evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins (2016)
Figure 1

Figure 1. CCM1 is involved in the incompatibility between S. bayanus chromosome 7 and the S. cerevisiae mitochondrial genome

(A) Chromosome 7 of S. bayanus is incompatible with the S. cerevisiae genome. The chromosome 7 replacement stain (Sc+Sb-chr7) exhibited serious growth defects when grown on the non-fermentable carbon source (glycerol). The respiration defects could be partially rescued when a whole set of S. bayanus chromosomes were provided (Sc+Sb-chr7 x Sb-ρ⁰), or fully rescued with the addition of S. bayanus mtDNA (Sc+Sb-chr7-ρ⁰ x Sb). Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. Sc, S. cerevisiae. Sb, S. bayanus. ρ⁰, mtDNA-less strains. (B) Ectopically-expressing Sc-CCM1 rescues the respiration defect of Sc+Sb-chr7. Spot assays for the wild-type S. cerevisiae (Sc) and the chromosome 7 replacement stain (Sc+Sb-chr7) carrying an empty plasmid (+ vector) or the plasmid with the S. cerevisiae CCM1 gene (+ Sc-CCM1). (C) Sb-CCM1 incompatibility could only be rescued in the presence of S. bayanus mtDNA. The native CCM1 ORF in S. cerevisiae was replaced by either Sb-CCM1 (Sc+Sb-CCM1) or Sc-CCM1 (Sc+Sc-CCM1, as a control) coding regions. Crossing the Sc+Sb-CCM1 strain with the mtDNA-less Sb-ρ⁰ strain (Sc+Sb-CCM1 x Sb-ρ⁰) could not rescue the growth defect. (D) CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb+Sc-CCM1) or Sb-CCM1 (Sb+Sb-CCM1, as a control) coding regions. Only the Sb+Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb+Sc-CCM1 x Sc-ρ⁰). (E) CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates.