EMBO/soda-nel huggingface dataset:
ARHGAP45 controls naïve T and B cell entry into lymph nodes and T-cell progenitor thymus-seeding (2021)
Figure 5

Figure 5. ARGHAP45 deficiency has no effect on ICAM-1-mediated adhesion and motility of activated T cell but affects speed of naive T cells.

A Analysis of migration patterns of WT and Arghap45−/− activated T cells on 2D surface coated with ICAM-1. Each track represents the migratory path of individual WT and Arghap45−/− activated T cells recorded over 466 s in a single field of view, Trajectories were plotted to a common starting point, and > 200 T cells were recorded per plot using time-lapse microscopy at 10 x magnification.

B Individual cell speed in an experiment involving 188 WT (black dots) and 144 Arghap45−/− (red dots) activated T cells, respectively. Each dot corresponds to the mean speed of a given cell. Bars represent the mean and SD. Three replicates are shown in panel D.

C Percentage of WT (black dots) and Arghap45−/− (red dots) activated T cells remaining adherent after application of a shear stress of 1 dyne/cm2 for 120 s. Three independent experiments were performed involving more than 200 cells and each dot correspond to the mean percentage of remaining adherent cells in one given experiment.

D Average cell speed of WT (black dots) and Arghap45−/− (red dots) activated T cells. Three independent experiments were performed involving more than 200 cells and each dot correspond to the mean of the speed in one given experiment. Average cell speed was evaluated by dividing the cumulative displacement of cells tracked over 10 seconds by the total time of displacement.

E Analysis of migration patterns of WT and Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. Same conditions as in (A).

F Individual cell speed in an experiment involving 283 WT (black dots) and 305 Arghap45−/− (red dots) naïve T cells, respectively. Same conditions as in (B). Four replicates shown in panel H.

G Percentage of WT (black dots) and Arghap45−/− naive T cells (red dots) remaining adherent cells after application of a shear stress of 1 dyne/cm2 for 120 s. Same conditions as in (C).

H Average cell speed of WT cell (black) and Arghap45−/− naive T cells (red). Same conditions as in (D).

In B-D and F-G mean and SD are shown. *P ≤ 0.033 (*), ** P ≤ 0.002, *** P ≤ 0.001, **** P ≤ 0.0001, unpaired T test.
ARHGAP45 controls naïve T and B cell entry into lymph nodes and T-cell progenitor thymus-seeding (2021)
Figure 6

Figure 6. ARHGAP45 deficiency stabilizes the polarization and decreases the speed of naïve T cells.

A, B Representative kymographs of WT (A) and Arghap45−/− (B) naive CD4+ T cells on 2D surface coated with ICAM-1 and CCL21.

C, D Top, representative sequences of cell contours during migration for 466 s of WT (C) and Arghap45−/− (D) naive T cells on 2D surface coated with ICAM-1 and CCL21. The color scale highlights each time-resolved shape. Rectangle length, 112,6 μm (WT) and 51,6 μm (Arghap45−/−). Bar, 10 µm. Botttom, three representative time-resolved shapes and their corresponding calculated contours are shown. Bar, 10 µm.

E, F Histograms of instant eccentricities (left) and 2D plots of adherent surface versus speed (right) of WT naive T cells on 2D surface coated with ICAM-1 and CCL21. In the 2D plot, the 3 populations discussed in the results have been highlighted: a, hardly motile (instant speed <5 µm/min) cell population with low (projected adherent area < 20%,) adhesion, b, hardly motile (instant speed <5 µm/min) cell population with high (projected adherent area > 40 %) adhesion, and c, highly adherent (projected adherent area >20 %) cell population with elevated speed (ranging between 15 and 30 µm/min).

G, H Histograms of instant eccentricities (left) and 2D plots of adherent surface versus speed (right) of Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. In the 2D plot, the single adherent (projected adherent area > 25 %) and slow (instant speed < 15 µm/min) cell population is highlighted.

Data information: In (E) and (F), histograms of instant eccentricities correspond to > 60 cells and > 2000 contours, and fitted by a double Gaussian for WT naive T cells (means = 0.54, 0.8 and SD = 0.18, 0.06) and a single Gaussian for Arghap45−/− naïve T cells (Mean=0.58, SD =0.18). 2D plots correspond to > 60 cells and > 2000 events. In (F) and (H), events which combined null motility and adherence over the recorded time have been excluded.
ARHGAP45 controls naïve T and B cell entry into lymph nodes and T-cell progenitor thymus-seeding (2021)
Figure 7

Figure 7. Normal TCR-dependent activation of Arhgap45−/− naïve T cells.

A Irradiated H-2 Kb- positive spleen cells isolated from T cell-deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8+ T cells purified from WT OT-I (WT) or Arhgap45−/− OT-I (Arhgap45−/−) mice. Cell proliferation was measured by luminescence after 48 h. Data are representative of 2 independent experiments with 3 mice per genotype (mean and SEM of triplicate are shown).

B WT mice that received CTV-labelled WT or Arhgap45−/− OT-I T cells were immunized using laser-assisted, dermal delivery of vaccibodies that deliver OVA to XCR1+ DC. Four days after antigen delivery, the extent of OT-I T cell proliferation was determined by CTV dilution in ear-draining, auricular LNs, in LNs that do not drain the ear, and in the spleen. Data are representative of two experiments with 4 mice per group.

C On the basis of the data shown in (B), the index of OT-I proliferation was calculated using FloJo software. Each dot represents an individual mouse. Mean and SD are shown. ns, non-significant.
Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes (2014)
f1

(a) Expression of Ulk1 and Atg5–12 in the indicated erythroid cells. (b–e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). ‘NS’ indicates not significant (ANOVA).
Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes (2014)
f2

(a) Induction of alternative macroautophagy in WT and Atg5−/−, but not Ulk1gt/gt and DKO, erythroid cells. Erythroblasts and reticulocytes from the liver of embryonic mice (E18.5) were incubated with or without STS (1 μM) for 24 h, followed by staining with anti-Lamp2 (red), anti-Ter119 (green) and DAPI (blue). DAPI-positive erythroblasts and DAPI-negative reticulocytes are shown. Lamp2 image and merged image (DAPI, Ter119 and Lamp2) are shown. Scale bar, 1 μm. Large dots for Lamp2 are observed in STS-treated WT and Atg5−/− cells, but not STS-treated Ulk1gt/gt or DKO cells. (b) Representative electron micrographs of EC incubated with or without STS. Erythroid cells were harvested from the liver of embryonic mice (E18.5), incubated with or without STS (1 μM) for 24 h, and analysed by electron microscopy (EM). Scale bar, 1 μm. Insets of WT and Atg5−/− cells show mitophagy (Scale bar, 0.5 μm). Inset of a Ulk1gt/gt cell showing mitochondria that have not been engulfed (Scale bar, 0.5 μm). Arrows point to non-engulfed mitochondria and the arrowheads indicate engulfed mitochondria. (c–e) Quantitative analysis of mitophagy after STS treatment, calculated from EM photos. Population of reticulocytes with mitophagy (c), number of mitochondria per reticulocytes (d) and population of reticulocytes showing macroautophagy (e) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); ‘NS’ indicates not significant versus value of WT (ANOVA).
Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes (2014)
f3

(a) Three-colour flow cytometry of erythroid cells from WT embryonic liver (E14.5) stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red. (Left upper panel) Ter119 versus CD71 fluorescence. (Other panels) FSC versus Mitotracker fluorescence of the R1 (right upper panel), R2 (left lower panel) and R3 (right lower panel) fractions. (b,c) Percentage of cells with a low number of mitochondria (mitochondrialow cells) among CD71−Ter119+ EC. Erythroid cells from the liver (E14.5 and E18.5) and peripheral blood (E18.5) of indicated mice were stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red. (b) Representative histograms of mitochondrial content in CD71−Ter119+ cells. Numbers indicate the population of mitochondrialow cells. (c) The percentage of CD71−Ter119+ cells without mitochondria was determined by gating the mitochondrialow fraction, as shown in (b) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); ‘NS’ indicates not significant versus value of WT (ANOVA). (d) Percent mitochondrialow cells in the CD71−Ter119+ EC of Atg7F/FCre embryo. Erythroid cells from the liver and peripheral blood of WT and Atg7F/FCre embryo (E18.5) were stained with anti-Ter119, anti-CD71, and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test). (e) Percentage of mitochondrialow cells in Syto16low cells. Liver erythroid cells (E18.5) were stained with Syto16 (DNA) and Mitotracker Deep Red. Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig. 5, the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction (mean±s.d., n=6). *P0.05 versus value of WT (ANOVA); #P0.05 versus value of Atg5−/− (ANOVA); ‘NS’ indicates not significant versus value of WT (ANOVA). (f) Immunofluorescent analysis of mitochondrial and lysosomal proteins. Liver Ter119+ cells (E18.5) of the indicated mice were stained with anti-Tom20 (mitochondrial marker) and anti-Lamp2 (lysosomal marker) antibodies, and observed by fluorescent microscopy. Scale bar, 2 μm. Green, red, white, and blue indicate Tom20, Lamp2, Ter119 and DAPI (DNA), respectively. Mitochondrial and lysosomal markers have separate distributions in Ulk1gt/gt cells and DKO cells.
Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes (2014)
f4

(a–f) Representative EM of WT (a), ATG5−/− (b), Ulk1gt/gt (c,d) and DKO (e,f) reticulocytes from embryonic liver (E14.5). (a,b) Mitophagy was detected in WT and ATG5−/− cells (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm). (c,e) In Ulk1gt/gt and DKO cells, numerous mitochondria were in contact with membrane structures (scale bar, 1 μm). Enlarged images are shown on the right side (scale bar, 0.2 μm). (d,f) Some Ulk1gt/gt and DKO cells showed plasma membrane blebs containing mitochondria (scale bar, 1 μm). (g,h) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18.5. Population of reticulocytes with mitophagy (g), and number of mitochondria per reticulocytes (h) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); ‘NS’ indicates not significant versus value of WT (ANOVA).
Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes (2014)
f5

(a) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated embryos (E18.5), and the expression of each protein was examined by western blot. The asterisk indicates a nonspecific band. Uncropped images are shown in Supplementary Figs 11 and 12. (b) Semi-quantitative analysis of the expression level of each protein and the LC3-II/LC3-I ratio (mean±s.d., n=3). *P0.05 versus value of WT EC (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− EC (ANOVA); ‘NS’ indicates not significant versus value of WT EC (ANOVA). (c) Modification of Ulk1 and Atg13 during erythrocyte maturation. Cell lysates were treated with λ-phosphatase (400 U) for 45 min at 30 °C, and band shift was examined by anti-Ulk1 and anti-Atg13 antibodies. Asterisk indicates non-specific band. Uncropped images are shown in Supplementary Fig. 12.
Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes (2014)
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(a) Morphology of cultured erythroid cells on day 0, 1 and 3 of differentiation. Erythroblasts were purified from embryonic liver (E14.5) and incubated for the indicated times in erythrocyte differentiation medium. The cells were stained with a Diff-quick kit. (b) Percentage of cells without nuclei (Syto16low cells) on the indicated days of differentiation (mean±s.d., n=6). Loss of nuclei occurred normally in all mice. (c,d) Percentage of Syto16low cells without mitochondria. Differentiated cells were stained with Syto16 and Mitotracker Deep Red. Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in (c). Numbers indicate the population of mitochondrialow cells. Representative dot plots are also available in Supplementary Fig. 13. (d) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in (c) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); ‘NS’ indicates not significant versus value of WT (ANOVA). (e–h) Impact of various drugs on mitochondrial clearance during in vitro differentiation. Differentiated cells from WT embryos were incubated with or without wortmannin (e), 3-methyladenine (3-MA) (f), rapamycin (g) or compound C (h) at the indicated concentrations and time periods. Percentage of mitochondrialow cells/Syto16low cells was measured by flow cytometry (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA). (i) Similar experiments were performed with cells from Atg5−/− embryos exposed to wortmannin or 3-MA (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).
Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes (2014)
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(a) Number of EC and haemoglobin content in the blood of the indicated mice (10–20 weeks old; mean±s.d., n=6). *P0.05 versus value of WT (Student's t-test). (b) Analysis of early erythrocyte development. Bone marrow cells were harvested from the indicated mice (10–20 weeks old) and stained with anti-cKit and anti-Sca1 antibodies. The population of each cell type was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)). (c) Percentage of mitochondria-containing Ter119+ blood cells. Cells in peripheral blood from the indicated mice (10–20 weeks old) were stained with anti-Ter119 antibody together with Mitotracker Deep Red. The population of mitochondriahigh cells among Ter119-positive cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA). (d) Percentage of mitochondria-containing Syto16low blood cells. Cells in peripheral blood from the indicated mice (10–20 weeks old) were stained with Syto16 and Mitotracker Deep Red. The population of mitochondriahigh cells among Syto16-negative cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA). (e) Representative electron micrographs of bone marrow reticulocytes in 10 week-old WT and Ulk1gt/gt mice (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm). (f,g) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated mice (10–20 weeks old), and the expression of each protein was examined by western blot. Uncropped images are shown in Supplementary Fig. 14. (g) Semi-quantitative analysis of the expression level of each protein (mean±s.d., n=3). ‘NS’ indicates not significant versus value of WT EC (ANOVA). (h,i) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis. (h) Representative images of erythrocyte in PHZ-treated WT and Ulk1gt/gt mice (14 weeks old). Blood cells were stained with anti-Ter119 (erythroid cell marker) and anti-Tom20 (mitochondrial marker) antibodies and observed by fluorescence microscopy. Green and red indicate Ter119 and Tom20, respectively. Scale bar, 10 μm. (i) Percentage of mitochondrialow cells among Syto16low cells. Erythroid cells were stained with Syto16 (DNA) and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student’s t-test). Mitochondrial retention was observed in Ulk1gt/gt mice.
Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features (2020)
Figure 1

Figure 1. Netrin-1 is highly expressed in the substantia nigra and colocalizes with SNpc DA neurons

(A) Netrin-1 (NTN1) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas.

(B) Reporter gene expression in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA)) of netrin-1+/lacZ+ adult (P60) mice incubated with S-Gal. (Scale bar, 2000 µm). Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel.

(C) Netrin-1 protein localization in the SN of adult rat. (Scale bar, 200 µm). Upper panel, confocal image of the SN. Higher magnification of the SNpc (bottoms panels). Netrin-1 (red), Tyrosine hydroxylase (TH) (green).

(D) Localization of netrin-1 protein in the adult rat striatum. (Scale bar, 600µm). Left panel, confocal image of the rat striatum. Netrin-1 (red), Dopamine transporter (DAT) (green). Right panels, higher magnification.

Data information: All images are made from coronal sections, images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.
Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features (2020)
Figure 2

Figure 2. Silencing netrin-1 in the SN elicits TH loss and motor impairment.

(A) Representative images of dopamine neurons (TH-positive) in the substantia nigra (SN) (left panel) and in the SN and striatum (right panel top, and bottom respectively) by immunofluorescence, six weeks after the intranigral injection of control (CTL) (AAV6-GFP) or Cre (AAV6-GFP Cre) adenovirus vectors in Netrin-1fl/fl mice (left panel: green-GFP, red-TH; right panel: Cy-5-TH) (Scale bar, 1000 μm). Filled arrows indicate SN TH dopamine neurons.

(B) Representative images of TH and netrin-1 stainings by immunofluorescence (scale bar, 2000 μm) six weeks after the intranigral injection of CTL adenovirus vector (left panel) or Cre adenovirus vector (right panel), in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA) of Netrin-1fl/fl mice.

(C) Quantification of fluorescence intensity of TH-positive neurons, in the substantia nigra (SN) (left), and fibres, in the striatum (right), six weeks after adenovirus vector injection, in Netrin-1fl/fl mice. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; **P< 0.01. N=3 each group.

(D) Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) reactivity in the SN after Cre and control adenovirus vector injection, in Netrin-1fl/fl mice (Scale bar, 50μm). The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. **P< 0.01. N=3 each group.

(E) Motor behavior assays at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05

(F) Immunoblot of SN lysates from netrin-1 wild type (WT) and netrin-1 conditional KO mice (left upper panel). Densitometry band quantification of cleaved receptor over full length receptor ratio (lower right panel). We randomly selected 2 mice from each group for immunoblot analysis. N=3 independent experiments. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.
Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features (2020)
Figure 3

Figure 3. Unbound DCC participates to netrin-1 depletion-induced dopamine cell loss.

(A) Representative images of TH+ dopamine neurons and fibers staining by immunofluorescence, in the SN and the striatum (Str) 6 weeks after CTL (AAV6-GFP); Cre (AAV6-GFP Cre); sh DCC (AAV6-GFP Cre + Sh-DCC) and shUNC5B (AAV6 GFP Cre + Sh-UNC5B) injection into the SN of Netrin-1fl/fl mice. (Scale bar, 2000 µm).

(B) Quantification of TH fluorescent intensity in the SN (upper bar graph) and striatum (Str) of Netrin-1fl/fl mice (lower bar graph). Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 3 each group. Tukey post hoc analysis after one-way ANOVA *P < 0.05, **P <0.01, N.S., not significant.

(C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01.

(D) Immunoblot of SN lysates (upper panel) and corresponding quantification (bottom panel). Quantification of band intensity (bottom). Mouse N=2. N=3 independent immunoblot analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.

(E) Motor behaviour tests at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; N.S., not significant.