EMBO/soda-nel huggingface dataset:
OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring (2021)
Figure 3

Figure 3 OCRL deficiency causes failed microtubule nucleation

A Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies. Scale bar, 10 μm.

B Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in A. n = 3 cells per group, three independent experiments.

C WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs derived from the corresponding mice were costained with anti-α-tubulin and anti- γ-tubulin antibodies. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.

D Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in C. n = 3 per group in which 30 cells were counted per condition per experiment.

E Microtubule regrowth assay was performed on NHF, Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice, followed by rinse with PBS, and addition of pre-warmed medium to induce microtubule regrowth. Cells were then immunostained with anti-α-tubulin and anti-γ-tubulin antibodies at the indicated time point after addition of pre-warmed medium. The magnified view shows the cytoplasmic microtubules organization center. Scale bar, 10 μm.

F Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center, MTOC) measured in the experiments shown in E. n = 3 per group in which 30 cells were counted per condition per experiment.

Data information: In (B,D,F), data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.
OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring (2021)
Figure 4

Figure 4 OCRL localizes to the proximal end of centrioles

A Representative images of RPE-1 cells co-stained with anti-OCRL (red) and anti-γ-tubulin (green) antibodies. Insets show the enlarged centrosomal region. Scale bar, 10 μm.

B Representative images of the co-localization of GFP-OCRL-WT (green) with centrosome marker γ-tubulin (red) in RPE-1 cells. Insets show the enlarged centrosomal region. Scale bar, 10 μm.

C The purified centrosome fraction (50%) isolated from RPE-1 cells and the cell lysate (5%) were subjected to immunoblot analysis with anti-OCRL antibody. The centrosomal protein γ-tubulin was probed as control.

D Representative images of RPE-1 cells co-stained with anti-OCRL (red) and anti-γ-tubulin (green) antibodies during mitosis. Insets show the enlarged centrosomal region. Scale bar, 2 μm.

E Super-resolution images of centrosome stained with anti-CEP164 (red, labeling distal appendages of mother centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm.

F Super-resolution images of centrosome stained with anti-CNAP1 (red, labeling proximal ends of centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm.

G Model depicting the localization of the OCRL on mother and daughter centrioles.

H Representative images of the co-localization of OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP with γ-tubulin. Insets, enlargements of the outlined areas. Scale bars, 10 μm.

I Quantitative analysis of cells with and without centrosomal OCRL (centrosomal GFP signal) in the experiments shown in A. n = 3 per group in which 10 cells were counted per condition per experiment. Means ±SEM, ****P < 0.0001, n.s., not significant, calculated by two-way ANOVA, multiple comparisons.
OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring (2021)
Figure 5

Figure 5 OCRL deficiency does not affect centrosomal PI(4,5)P2 pool

A RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5)P2 (green), γ-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm.

B Quantitative analyses of centrosomal PI(4,5)P2 fluorescence intensity in the experiments shown in A. n = 3 per group in which 50 cells were counted per condition per experiment.

C NHF, Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI(4,5)P2 (green), gamma-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm.

D The fluorescence intensity of PI (4,5)P2 on centrosomes in C was measured. n = 3 per group in which 50 cells were counted per condition per experiment.

Data information: In (B,D), data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.
OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring (2021)
Figure 6

Figure 6 OCRL mediates microtubule nucleation through SSX2IP

A SSX2IP interacts with OCRL. Plasmids carrying GFP-OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP were transfected into RPE-1 cells. Immunoprecipitation was performed with GFP-Trap, followed by blotting with the indicated antibodies.

B WT (Ocrl+/y) and OCRL knockout (Ocrl-/-) MEFs were costained with anti-SSX2IP and anti-γ-tubulin antibodies. Nuclei were stained with DAPI (blue). Insets show the enlarged centrosomal region. Scale bar, 5 μm.

C Quantification of centrosomal fluorescence intensity of SSX2IP in the experiments shown in B. n = 3 per group in which 50 cells were counted per condition per experiment.

D NHF, Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP-SSX2IP or GFP-PACT-SSX2IP for 48 h, then immunostained with anti-β-tubulin (red) antibody. Nuclei (blue) were stained with DAPI. The centrosomal microtubule nucleation in GFP-PACT-SSX2IP transfected cells was indicated by white arrowhead. Scale bar, 5 μm.

E Quantitative analyses of cells with and without MTOC. NHF, Lowe 1676 and Lowe 3265 cells were transfected with wild type GFP-SSX2IP or GFP-PACT-SSX2IP for 48 h, then immunostained with anti-γ-tubulin (red) antibody. Nuclei (blue) were stained with DAPI. n = 3 per group in which 10 cells were counted per condition per experiment.

F Western blot analysis of GFP, endogenous phospho-mTOR, total mTOR, in NHF and Lowe 1676 cells. NHF and Lowe 1676 cells were left untransfected, transfected with GFP-SSX2IP-WT or GFP-PACT-SSX2IP, then immunoblotted with indicated antibodies.

G Diagram illustrating that in normal conditions, OCRL recruits SSX2IP to the centrosomes, thus maintaining microtubule nucleation. Nucleated microtubules allow transport of perinuclear lysosomes to the cell periphery to activate mTOR activity. OCRL deficiency in Lowe syndrome causes a significant loss of microtubule nucleation and a marked perinuclear accumulation of lysosomes, which leads to mTOR inactivation.

Data information: In (C), data are presented as mean ± SEM. * ≤ 0.05, calculated by two-tailed Student's t-test. In (E), data are presented as mean ± SEM. ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.

Expanded View legends
How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release (2016)
Figure 2

Figure 2. RIBEYE KO does not impair synaptic organization of the retina

A. Overview of the synaptic organization of the retina in RBEWT/WT and RBEKO/KO mice. Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B-domain (CtBP2; green) and SV2. Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice for PSD95 (green, to label postsynaptic specializations) and mGluR6 (red, to label specifically postsynaptic sites formed by bipolar neurons in photoreceptor synapses). Abbreviations are as in A. Scale bars: 20 µm (left and right overview), 5 µm (left panel magnification), 2 µm (right panel magnification).

C. Same as B, except that retinas were stained for PKCα (green, to label bipolar neurons) and PSD95 (red, to label amacrine cells).
How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release (2016)
Figure 3

Figure 3. RIBEYE KO results in altered Cav1.4 localization

A and B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice, respectively, for PSD95 (green, to label presynaptic photoreceptor terminals) and Cav1.4 (red, to label presynaptic Ca2+-channel). Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (A1 to A4) Magnification of Cav1.4 staining in the outer plexiform layer (OPL) in the retina of RBEWT/WT and (B1 to B4) RBEKO/KO mice. White arrow heads indicate Ca2+-channel distribution at selected synapses. Images represent 2 µm z-stack projections. Scale bar: 2 µm.
Semaphorin-3C signals through Neuropilin-1 and PlexinD1 receptors to inhibit pathological angiogenesis. (2015)
Figure 1

Sema3C inhibits endothelial tube formation and sprouting angiogenesis

Sema3C domain structure with two consensus motifs for furin and furin-like proteases. Sema3CΔ13 and Sema3Cp60 expression vectors were generated by replacing the amino acids in the consensus motifs with stop codons (asterisk).

Conditioned medium was collected from pericytes 48 h after adenoviral transduction. Western blotting verified the secretion of Sema3CΔ13 (81 kDa) and Sema3Cp60 (60 kDa) isoforms.

HUVECs were suspended in conditioned medium with VEGF (25 ng/ml) and plated on Matrigel. Branch points of the capillary-like network were counted after 18 h. n = 4 independent experiments; scale bar, 200 μm. Control vs. Sema3C, *P = 0.0375 and control vs. Sema3Δ13, *P = 0.0162.

HUVEC spheroids in collagen were treated with conditioned medium with or without VEGF (25 ng/ml). After 24 h, the cumulative sprout length per spheroid was quantified. Data are representative of three independent experiments with ten spheroids per group. Scale bar, 100 μm. Control vs. Sema3C (− VEGF), *P = 0.0276; control vs. Sema3CΔ13 (− VEGF), *P = 0.0443; control vs. Sema3C (+ VEGF), *P = 0.028 and control vs. Sema3CΔ13 (+ VEGF), *P = 0.032.

Data information: (C, D) Mean ± s.e.m; n.s., non-significant using paired Student's t-test.
Semaphorin-3C signals through Neuropilin-1 and PlexinD1 receptors to inhibit pathological angiogenesis. (2015)
Figure 2

Sema3C inhibits vessel formation in a xenotransplantation model

Scheme of the experimental procedure. Pericytes were lentivirally transduced with Sema3C or GFP expression vectors. HUVEC/pericyte spheroids (1:1 ratio) were injected s.c. into SCID mice.

Microvessels within the plugs stained against human-specific CD34. Cell nuclei were stained with DAPI. Scale bar, 100 μm.

The area of CD34-positive microvessels was quantified and normalized to the total area of plug. Results represent the means ± s.e.m of 5 plugs (control) and 7 plugs (Sema3C), *P = 0.018.

Vessel coverage was assessed by αSMA staining. Scale bar, 100 μm.

Vessel coverage was quantified as the ratio of endothelial cell-associated mural cells to total amount of vessels (n = 5 (control) and n = 7 (Sema3C)).

Vessel perfusion was evaluated by i.v. injection of FITC-lectin (0.15 mg) 30 min before mice were sacrificed. The data show the percentage of perfused vessels in the plug and normalized to total vessel area (n = 5 (control) and n = 7 (Sema3C)).

Data information: (C, E, F) Mean ± s.e.m; n.s., non-significant using unpaired Student's t-test.
Semaphorin-3C signals through Neuropilin-1 and PlexinD1 receptors to inhibit pathological angiogenesis. (2015)
Figure 3

Intravitreal injection of Sema3C inhibits retinal vessel formation

A C57BL/6N mice pups at P3 received 1 μg of mouse Sema3C-Fc (right eye) or control IgG-Fc (left eye). The retinal vasculature was examined at P5 by whole-mount CD31 immunofluorescent staining. Representative images show the total retinal vasculature and sprouting tips at the vascular front (iii and iv, arrows) and the enlarged intercapillary space (ii and iv, asterisks). Scale bar, 100 μm and 20 μm (iii and iv).

B,C (B) Retinal vessel outgrowth measured as the total area from the central optic disk to the vascular front normalized to control IgG-Fc treatment, ***P = 0.0009. (C) Vessel density was quantified by CD31-stained vessels and normalized to the total vessel area, ***P = 0.0002. (Mean ± s.e.m; n = 23 retinae in each group from four independent experiments; unpaired Student's t-test.)

D,E (D) Number of sprouting tips per retina, *P < 0.0001. (E) Branch points in the capillary plexus represented as the ratio to the control IgG-treated retinae, ***P < 0.0001. (Mean ± s.e.m; n = 18 retinae in each group from three independent experiments; unpaired Student's t-test.)

F Pups received same treatment as described in (A). The astrocyte layer was examined at P5 by GFAP staining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of six retinae per group.

G Six-week-old C57BL/6N male mice were intravitreally injected with Sema3C-Fc (right eye) or control IgG-Fc (left eye), and retinae were collected 2 days later. The retinal vessel network was examined with CD31 immunostaining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of four retinae per group.
Semaphorin-3C signals through Neuropilin-1 and PlexinD1 receptors to inhibit pathological angiogenesis. (2015)
Figure 4

Sema3C inhibits endothelial cell survival and disrupts endothelial cell–cell contacts

HUVECs were treated with Sema3C- or GFP-conditioned medium for 24 h. Cell proliferation was quantified by BrdU incorporation (mean ± s.e.m; n = 3; n.s., non-significant).

Apoptosis of HUVEC was measured by caspase-3 and caspase-7 activities after 3 h of incubation with Sema3C- or control-conditioned medium (mean ± s.e.m; n = 3, *P = 0.018 using unpaired Student's t-test).

Serum-starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF (50 ng/ml) for 15 min. Cell lysates were subjected to SDS–PAGE and immunoblotted with anti-phospho-AKT antibody.

Quantification of phospho-AKT normalized to total AKT levels (mean ± s.e.m; n = 4; control vs. Sema3C (− VEGF), *P = 0.034 and control vs. Sema3C (+ VEGF), *P = 0.024 using unpaired Student's t-test).

HUVECs were adenovirally transduced with VE-cadherin-GFP and plated on ibidi slides (n = 4; control vs. Sema3C (− VEGF) at 20× magnification (Supplementary Movies S1 and S2)). Representative pictures are shown at time points 0 and 1 h. Arrowheads indicate gaps between endothelial cells. Scale bar, 100 μm.

C57BL/6N mice pups at P5 received 1 μg of recombinant Sema3C or IgG-Fc proteins. Endothelial cell junctions were examined by whole-mount VE-cadherin immunofluorescent staining 6 h later. Representative images show VE-cadherin at cell junctions (arrows). The linear structure (i) was disrupted in Sema3C-treated retinae (ii). Scale bar, 100 μm (upper panel) and 20 μm (lower panel).

Whole-mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase-3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG-Fc. Representative images show active caspase-3 signals that co-localize with endothelial cells (arrows). Scale bar, 100 μm.

Quantification of cleaved caspase-3 normalized to area of CD31-stained vessels (mean ± s.e.m; n = 5; **P = 0.007 using paired Student's t-test).

Source data are available online for this figure.
Semaphorin-3C signals through Neuropilin-1 and PlexinD1 receptors to inhibit pathological angiogenesis. (2015)
Figure 5

Sema3C suppresses endothelial cell adhesion and migration by inhibiting FAK and p38MAPK signaling pathways

A HUVECs were transduced with adenoviral vector expressing RFP-conjugated F-actin binding peptide (LifeAct). Time-lapse imaging of F-actin in HUVEC was obtained at 20× magnification (Supplementary Movies S3 and S4). Representative images are shown at 0 and 2 h. Arrowheads indicate detached cells after Sema3C treatment. Scale bar, 100 μm.

B Endothelial cell migration was determined using a scratch wound assay with a co-culture model. A wound was generated in a confluent HUVEC monolayer, and cells were cultured in basal medium with or without VEGF (25 ng/ml) in a 24-well plate. At the same time, the transwells containing Sema3C or GFP-expressing pericytes. Representative pictures show remaining gaps within the HUVEC monolayer. Scale bar, 200 μm.

C Migration distance was measured after 24 h and normalized to control treatment (mean ± s.e.m; n = 3 independent experiments, control vs. Sema3C (− VEGF), *P = 0.021 and control vs. Sema3C (+ VEGF), *P = 0.039 using unpaired Student's t-test).

D,E (D) Serum-starved HUVECs were treated with Sema3C- or control-conditioned medium with or without VEGF (50 ng/ml) for 10 min. Cell lysates were subjected to SDS–PAGE and probed with anti-phosphoT180/Y182 p38MAPK antibody. Quantification of p38MAPK activation was normalized to total p38MAPK and presented as the ratio to the cells with control medium without VEGF treatment. Control vs. Sema3C (− VEGF), *P = 0.046 and control vs. Sema3C (+ VEGF), *P = 0.043. (E) HUVECs were treated as described before and the activation of FAK detected by anti-phosphoY397 FAK antibody and normalized to the total amount of FAK. Control vs. Sema3C (− VEGF), *P = 0.033 and control vs. Sema3C (+ VEGF), *P = 0.047. (Mean ± s.e.m; n = 3 independent experiments; unpaired Student's t-test.)

F Serum-starved HUVECs were treated with conditioned medium for 15 min. Focal adhesion proteins were examined with anti-phosphoY397FAK and anti-vinculin antibodies. F-actin was stained with Alexa Fluor 546-conjugated phalloidin. Merged images show the coupling of stress fibers with focal adhesion complexes in the cells treated with control medium. Inset, phospho-Y397FAK and vinculin formed focal adhesions that were stained in an elongated dot-like structure (i). Lower panel (ii), Sema3C treatment induced F-actin disassembly and membrane ruffling. Phospho-Y397FAK and vinculin were randomly distributed at the peripheral membrane. Scale bars, 10 μm.

Source data are available online for this figure.
Semaphorin-3C signals through Neuropilin-1 and PlexinD1 receptors to inhibit pathological angiogenesis. (2015)
Figure 6

Sema3C, but not Sema3Cp60, acts through Neuropilin-1 and PlexinD1 to suppress endothelial cell adhesion

HUVECs were transfected with siRNA against PlexinD1 or control NS (non-silence) siRNA. qPCR analyses and Western blotting were performed after 48 h (n = 3; paired Student's t-test, NS siRNA vs. PlexinD1 siRNA, ***P = 0.0004). Cell lysates were subjected to SDS–PAGE and probed with anti-PlexinD1.

HUVECs were transfected with siRNA against PlexinD1 and plated on gelatin-coated coverslips. Confluent monolayers were treated with Sema3C-, Sema3Cp60- or control-conditioned medium for 30 min. Cells were fixed and stained with Alexa-Fluor 488-conjugated phalloidin to visualize F-actin. Arrows indicate Sema3C-induced cell membrane ruffling. Scale bar, 20 μm. Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei (n = 5; mean ± s.e.m., paired Student's t-test, **P = 0.001).

Silencing of Nrp-1 by lentiviral particles expressing shRNA. After culturing 72 h in selection medium, RNA was harvested for qPCR analysis. NS shRNA vs. Nrp-1 shRNA: #1, *P = 0.017 and #2, *P = 0.02; n = 3; mean ± s.e.m. Cell lysates were subjected to SDS–PAGE and probed with anti-Nrp-1.

Depletion of Nrp-1 abolished the response to Sema3C. Sema3Cp60 had no effect on HUVEC adhesion. Arrows indicate membrane ruffling. Scale bar, 20 μm. Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei (n = 5; mean ± s.e.m.; paired Student's t-test, ***P = 0.0003).

HUVECs were treated with Sema3C-conditioned medium for 10 min and lysed, and Nrp-1 was immunoprecipitated. Bound Sema3C was detected by Western blotting.

Schematic model of Sema3C signaling through Nrp-1 and PlexinD1 receptors in endothelial cells.

Source data are available online for this figure.
Semaphorin-3C signals through Neuropilin-1 and PlexinD1 receptors to inhibit pathological angiogenesis. (2015)
Figure 7

Sema3C inhibits pathological angiogenesis in an oxygen-induced retinopathy model in mice

Scheme of the experiment. C57BL/6N mice pups at P7 with nursing mothers were subjected to hyperoxic conditions leading to vessel obliteration in the central zone of the retina. At P12, mice pups were returned to room air, which induces vessel regrowth but also pathological neovascularization, characterized by pre-retinal neovascular tufts. At P15, mice pups were injected intravitreally with 1 μg control IgG-Fc (left eye) and Sema3C-Fc (right eye). Retinae were analyzed 2 days later after intravitreal injection.

RNA scope analyses showed Sema3C and PlexinD1 expression in pre-retinal tufts (OIR model, P19). The retinae were subsequently stained against collagen IV. Sema3C mRNA was detected in peri-endothelial cells. Scale bar: 20 μm.

Nrp-1 expression in pre-retinal tufts (OIR model, P17). Abundant expression of Nrp1 in endothelial cells. Pericytes at vascular tufts were immunostained with anti-NG2. Scale bar, 20 μm.

H&E staining shows neovascular tufts (arrows). Representative of five retinae per group in two independent experiments. Scale bar, 50 μm.

Whole-mount CD31 immunostaining. The white line defines the whole retinal area, and red line outlines the vaso-obliterative zone. Inset image shows large numbers of peri-retinal tufts (i, arrow heads) in control IgG-Fc-treated retinae compared to Sema3C-treated retinae (ii). Scale bar, 500 μm (upper panel) and 100 μm (lower panel).

The avascular area is presented as percentage of the whole retinal area (n = 10 retinae in each group; n.s., non-significant using paired Student's t-test).

The formation of pre-retinal tufts was quantified in the superficial layer of the retinal vasculature and normalized to whole retinal area (n = 6 retinae in each group; *P = 0.033 using paired Student's t-test).