EMBO/SourceData huggingface dataset:
Figure 1 (A) Negative stain electron micrograph showing the tendency of S. cerevisiae prApe1 (sc-prApe1) dodecamers to form higher-order assemblies or chains (black arrowheads). (B) Negative stain electron micrograph of S. cerevisiae mApe1 (sc-mApe1) showing monodisperse dodecameric particles (circles) as well as hexameric particles (white arrowheads). (C) Bar plot showing the occurrence of single, double, triple or multiple aggregates of sc-prApe1 in comparison with sc-mApe1. Insets show single, double, triple or multiple dodadecamers. Data information: n=3,186 particles were counted in the case of sc-prApe1 and n=3,107 particles were counted in the case of sc-mApe1. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).  (D) Negative stain electron micrograph of GraFixed sc-mApe1 showing dodecameric particles (circles). (E) Class averages of sc-mApe1 dodecamers including views of two-fold and three-fold symmetry. (F) Fourier shell correlation of the EM reconstruction of the sc-mApe1 dodecamer indicates a resolution of 24 Å. (G) C. thermophilum mApe1 crystal structure fitted into electron microscopy (EM) map of sc-mApe1 dodecamer. Each of the 12 chains is depicted by a different color. A black sphere represents the N-terminus where the propeptide would emanate from the dodecamer. From left to right: two-fold axis view from the edge, three-fold axis view from the facet and three-fold axis view from the vertex.
Figure 2 (A) Electron micrograph of negatively stained Ams1 with particles and higher-order assemblies (arrows). Class averages exhibit two distinct two-fold views consistent with D2 symmetry. Scale bar 10 nm. (B) Electron cryomicrograph of Ams1 with particles highlighted (circles). (C) Selected particles (top row) are shown with corresponding class averages (bottom row). Scale bar 10 nm. (D) Fourier shell correlation indicates a resolution of 6.3 Å according to the 0.143 criterion.  (E) Top view of the cryo-EM structure, four Ams1 molecules were segmented and colored (dark blue, beige, red, light blue).  (F) Molecular architecture of Ams1 monomer. Primary structure, two map segments with fitted pseudo-atomic models are shown. Left. The N-terminal portion was built from distant homology models: a four-helix bundle (17 - 27, 209 - 271) in yellow and a jelly-roll fold (45 - 203) in red. Right. The α-mannosidase core (287 - 1083) with an α/β barrel (287 - 573) in blue, a three-helix bundle (574 - 671) in green, and the β-sandwich domain (672 - 1083) in purple.  (G) Density section through the Ams1 monomer with three-helix bundle in the center.  (H) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo-EM map. Corresponding 3D structure in Movie EV1.  (J) The β-sandwich domain (purple) makes up the transverse interface between chain A and chain B.  (K) The N-terminal four-helix bundle (orange) mediates the longitudinal interface. Asterisk denotes position of W234E mutation between chain A and C/D.  (L) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild-type tetramers. Representative class averages show a two-lobed structure consistent with reprojections of chain A and B (dark blue and beige) dimers depicted in ribbon and surface representation below. Scale bar 10 nm.
Figure 3(A) SEC-MALS profile of Atg19 yields a molecular weight estimate of 142 kDa (measured in triplicate). (B) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay.  (C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.  (D) Size-exclusion chromatography profile (Superose 6 column) of the prApe1/Atg19 complex after anti MBP-Atg19 purification and cleavage of the MBP tag (top). SDS-PAGE of peak fractions of the gel filtration profile (bottom). Samples were run on two separate gels.  (E) Negative stain electron micrograph corresponding to the size-exclusion chromatography peak at 10 ml retention volume of (D). Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets.  (F) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co-purified with Atg19.  (G) Bar plot of occurrence of single, double, triple and multiple dodecamers from the prApe1 and the prApe1/Atg19 sample. Data information: n=3,186 particles were counted in the case of prApe1 and n=3,340 particles were counted in the case of prApe1/Atg19. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).  H) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1-GFP spots. BF represents bright-field images.  I) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1-GFP/Atg19-mCherry/ypt7Δ versus prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells. The intensity of Ape1-GFP was normalized over the average fluorescence intensity of Nuf2-GFP measured in kinetochores as performed in [36]. Data information: a total of 54 prApe1-GFP spots and 80 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19Δ/ypt7Δ cells, whereas 52 prApe1-GFP spots and 98 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19-mCherry/ypt7Δ control cells. Error bars indicate standard error of the mean (SEM). *P = 0.03 (Z test).
Figure 4 Figure 4: Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells. (A) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, Ams1-GFP/Atg19-mCherry/ypt7Δ, Atg19-GFP/Ape1-mCherry/ypt7Δ S. cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots. Only co-localizing GFP and RFP spots were considered. BF represents bright-field images. (B) Bar plot showing the number of molecules of prApe1, Ams1 and Atg19 found in Cvt vesicles in living cells. Data information: data are representative of two independent experiments in which a total of 217 prApe1-GFP, 181 Ams1-GFP and 233 Atg19-GFP spots were analyzed. Error bars indicate standard deviation (SD). ***P < 0.001; *P = 0.0176 (Z test).
Figure 5 (A) Fluorescence microscopy image of a resin section of prApe1-GFP/Atg19-mCherry/ypt7Δ cells, merge of green (prApe1-GFP) and red (Atg19-mcherry) channels. Tetraspeck beads are detected in the blue channel. (B) Electron tomography slice of the boxed area in (A). The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50% and 90% localization accuracy, respectively. Insets show close-up views of a double (top) and single (bottom) membrane. Corresponding tomogram Movie EV2. (C) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation. The average dimensions give rise to spheroids with an average ellipticity of 11 % (shown in dark grey). Both prApe1-GFP and prApe1-GFP/Atg19-mCherry datasets were included. The cases representing autophagosomes were excluded.  (D) Dense Cvt aggregate that is not surrounded by membrane. Corresponding tomogram Movie EV3. (E) Cvt aggregate that is partially enwrapped by double membrane. Corresponding tomogram Movie EV4. (F) Cvt vesicle fully enclosed by double membrane. Bottom: schematic representation. White arrowheads indicate the double membrane. Corresponding tomogram Movie EV5.
Figure 6 Figure 6: Model of the Cvt pathway. (A) prApe1 the major Cvt cargo forms homo-oligomers as dodecamers that (B) assemble into higher-order assemblies mediated by the N-terminal propeptide. (C) These assemblies are capable of binding Atg19 that forms a trimer in solution. (D) Ams1, which forms a homo-oligomer as a tetramer, joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site. (E) prApe1 dodecamers, secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol. (F) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form (G) the Cvt vesicle. Cvt assembly stages in (E), (F) and (G) are drawn respecting the determined average size of the vesicles, the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article. (H) After fusion with the vacuole, prApe1 is subjected to maturation while maintaining the dodecamer organization. (I) Ams1 does not undergo a maturation process in the vacuole.
Figure 1Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (A) CTGF interacts with LRP4 but not with MuSK. Full-length CTGF (CTGF-full-myc) was immunoprecipitated with the ectodomain of LRP4 (LRP4ect-Flag, lane 6) but not with the ectodomain of MuSK (MuSKect-Flag, lane 3). However, when CTGF-full-myc, LRP4ect, and MuSKect-Flag were expressed together, CTGF-full-myc was immunoprecipitated with MuSKect-Flag (lane 5). The CT domain of CTGF (CTGF-CT-myc) was sufficient to immunoprecipitate LRP4ect-Flag (lane 7). Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (B) Deletion of the CT domain of human CTGF (CTGF-ΔCT_ΔTSP1 and CTGF-ΔCT) impairs the interaction between CTGF and LRP4ect-Flag. Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (C) Anti-CTGF antibody immunoprecipitated human LRP4 deleted with the 1st, 2nd, and 4th β-propeller domains (Δ1, Δ2, and Δ4) but not with the 3rd β-propeller domain (∆3).Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (D) LRP4ect-Flag, but not MuSKect-Flag, bound to overlaid CTGF-full-mycAP and CTGF-CT-mycAP. In contrast, LRP4ect-Flag did not bind to CTGF-ΔCT-mycAP. Rspo2-mycAP were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (D) p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (E, upper panel) LRP4ect-Flag bound to overlaid MuSKect-mycAP in a dose-dependent manner of CTGF-full-myc, but not of CTGF-ΔCT-myc. (E, lower panel) LRP4ect-Flag bound to overlaid agrin-mycAP independent of CTGF-full-myc. Rspo2-mycAP and Rspo2-myc were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (E). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.
Figure 2(A) Representative immunoblotting of Lrp4, MuSK, Transferrin Receptor (TfR), CTGF, and β-actin in whole-cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM (1 ng/ml) agrin and/or 250 pM (100 ng/ml) CTGF for 24 h. CTGF increases Lrp4 on the plasma membrane in the presence of agrin.(B) C2C12 myotubes were treated with 1 pM agrin and/or 250 pM CTGF for 6 h. Lrp4 was immunoprecipitated (IP) with an anti-Lrp4 antibody, and phosphorylated Lrp4 was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody.(C) Representative immunoblotting of Lrp4, MuSK, CTGF, and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18.5 of wild-type (WT) and Ctgf-/- mice.(D) Representative immunoblotting of Y755-phosphorylated MuSK, total MuSK, and β-actin in the diaphragm at E18.5 of wild-type (WT), Ctgf-/-, and Ctgf+/- mice.
Figure 3(A) C2C12 myotubes were treated with indicated concentrations of agrin-mycAP, CTGF-mycAP, and/or CTGF-∆CT-mycAP for 3 h. Total MuSK was immunoprecipitated (IP) with an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. CTGF-mycAP, but not CTGF-∆CT-mycAP, enhanced agrin-mediated MuSK phosphorylation.(B) C2C12 myotubes were treated with 5 pM agrin-mycAP, 250 pM CTGF-mycAP, and/or 250 pM CTGF-∆CT-mycAP for 18 h. BSA was added to the control medium. AChRs and the plasma membrane were visualized with Alexa594-conjugated α-bungarotoxin (red) and CellMask (green), respectively. Scale bar = 20 µm. Arrowheads point to the AChR clusters with an axis length of 4 μm or more. CTGF enhances agrin-mediated AChR clustering.(C) C2C12 myotubes were infected with lentivirus expressing shControl, shCtgf-1, or shCtgf-2. Doxycycline was added for 2 days to induce shRNA expression. BSA or agrin was added to the medium at 10 pM. Total MuSK was immunoprecipitated (IP) by an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. Ctgf knockdown suppressed MuSK phosphorylation.(D) Ten pM agrin was added to the medium. AChRs were visualized with Alexa594-conjugated α-bungarotoxin (red signals) in the infected myotube (green GFP signals). Ctgf knockdown decreased the number and length of AChR clusters. Scale bar = 10 µm.
Figure 4(A) Quantitative RT-PCR of Chrne, Musk, Lrp4, and Ctgf in C2C12 myotubes treated with 10 ng/ml BSA or recombinant human CTGF.(B) Quantitative RT-PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng/ml agrin and/or 10 ng/ml neuregulin-1.(C) Localizations of CTGF and AChR in a representative cross section of the lower limb muscle at E18.5. Co-localization of AChR (red) and CTGF (green) is indicated in the merged images. Red arrow points to an AChR cluster. Scale bar = 20 μm.(D) Quantitative RT-PCR of Ctgf and neural Agrn in the spinal cord and the diaphragm in mouse. Mean and SD (n = 3 mice) are indicated. p-value < 0.05 by two-way repeated measures ANOVA. p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.
Figure 5(A) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-peripherin antibody (green) to visualize AChRs and the motor axon, respectively. The endplates of Ctgf-/- diaphragm were smaller (lower left panel) than those of the wild-type diaphragm (upper left panel). In the Ctgf-/- diaphragm, 10.0 ± 3.2% (mean and SD, n = 105 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. In contrast, in the wild-type diaphragm, 2.0 ± 1.4% (mean and SD, n = 102 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. Quantification was performed in a blinded manner. Scale bar = 5 µm.(B) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-synaptophysin antibody (green) to visualize AChRs and the nerve terminal, respectively. Synaptophysin signals mostly overlapped with α-bungarotoxin signals in the wild-type diaphragm, whereas synaptophysin signals extended into the axon in the Ctgf-/- diaphragm. Scale bar = 5 µm.(C-E) Blinded morphometric analysis of AChR signals (C), synaptophysin signals (D), and the ratio of synaptophysin signals not colocalized with AChR signals (E) of confocal images shown in (A) and (B). AChR areas (C), but not synaptophysin-positive areas (D), were decreased in Ctgf-/- mice at E18.5. (E) A large fraction of synaptophysin signalswere not colocalized with AChR signals in Ctgf-/- diaphragm. Mean and SD (n = 40-50 NMJs in 6 left diaphragms) are indicated. p-value < 0.05 by one-way ANOVA for (C and E), but not for (D). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.(F) Representative electron micrographs of the NMJs in the diaphragm of wild-type and Ctgf-/- mice at E18.5. In Ctgf-/- mice, synaptic vesicles are sparser and the number of mitochondria at the nerve terminals is reduced compared to those in wild-type mice. See Table 1 for morphometric analysis. Regions indicated by rectangles are enlarged in the insets. Arrowheads point to synaptic vesicles (SV). Mit, mitochondria. Blinded morphometric measurements are shown in Table 1. Scale bar = 500 nm.
Figure 1(B-C) The phospholipases pla2g4aa and pla2g4ab are highly expressed in neutrophils. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.(D-E-F) The expression of cyclooxygenases cox1, cox2a and cox2b is enriched in macrophages. (G-H) The prostaglandin synthases ptges3a and ptges3b are only expressed by ECs. (I-O) The prostaglandin receptors ptger1a, ptger1b, ptger2a, ptger3, ptger4a are mostly expressed in HSPCs. (P-Q) The prostaglandin transporters slco2b1 and abcc4 are specifically expressed in ECs. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.
Figure 2(A) Schematic indicating the imaging area in the tail at 72hpf, as indicated by the black box; fluorescence imaging of the CHT in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. (B) Quantification of runx1:mcherry positive-cells (C) Quantification of cd45:CFP positive-cells in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. Data information: Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001 ****P<.0001. Scale bar 200μm (A).
Figure 3(A) WISH for cmyb expression at 60hpf in wild type and slco2b1-/- embryos. (B) Quantification of cmyb-expressing cells. Each n represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 500μm (A)(C) Schematic indicating the imaging area in the tail at 60hpf, as indicated by the black box; fluorescence imaging in the CHT of kdrl:mCherry;cmyb:GFP embryos injected with control- and slco2b1-Mos. (D) Quantification of HSPCs associated to ECs. Each n represents the number of yellow spots for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 200μm (C)(E) Experimental outline and quantification of time-lapse live imaging in controls and slco2b1-morphants cmyb:GFP+ cells, using Imaris software. Data information: The statistical analysis was completed using an unpaired two tailed t test. ***P < .001
Figure 4(A) Anti-GFP and pH3 immunostainings of either controls (Ctrl-Mo) or slco2b1-morphants (slco2b1-MO) cmyb:GFP embryos. (B) Quantification of the number of the pH3+ HSCs in controls or slco2b1-morphants. Centre values denote the mean, and error values denote s.e.m, statistical analysis was completed using an unpaired two tailed t test. ***P < .001. Data information: Scale bar 50μm (A)(D) Quantification of PGE2 concentration in control and slco2b1 morphants. The statistical analysis was completed using an unpaired two tailed t test *P < .01. Centre values denote the mean, and error bars denote s.e.m.(E) Fluorescence imaging in the CHT of cmyb:GFP embryos injected with control- and slco2b1-MOs and treated with PGE2. (F) Quantification of GFP positive cells. Statistical analysis: one-way ANOVA, multiple comparison, *P < .01; ****P < .0001. Centre values denote the mean, and error bars denote s.e.m. Data information: Scale bar 200μm (E).
Figure 5(A) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO. (B) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ. (C) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ+PGH2. (D) Quantification of runx1:mcherry positive-cells. Each dot represents the number of red cells for each embryo (biological replicates). Each experiment has been repeated three independent times. (E) Quantification of cd45:CFP positive-cells. Each dot represents the number of blue cells for each embryo (biological replicates). Each experiment has been repeated three independent times.Data information for (D) and (E): Statistical analysis was completed using one-way ANOVA, multiple comparison test. ****P<.0001. Centre values denote the mean, and error bars denote s.e.m Scale bar 200μm (A-C).
Figure 6(A) WISH for cmyb at 60hpf in kdrl:Gal4+ embryos injected with control- and slco2b1- morpholinos and/or co-injected with the Tol2-UAS:slco2b1 vector and tol2 mRNA. The region indicated by the dashed bas been magnified and shows in (B). (C) Quantification and statistical analysis were completed using one-way ANOVA, multiple comparison test. ****P< .0001. Centre values denote the mean, and error bars denote s.e.m. Each dot represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeat three independent times.
Figure 8(A) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and CD31 (endothelial cell marker) at E13 and E16 stages of mouse fetal liver. (B) Distance distribution between cKithigh cells and endothelial cells (n=79 from 3 E13 fetal livers; n=92 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test. *P<.01; **P<.001; ***P<.0001. Centre values denote the mean, and error values denote s.e.m All nuclei marked with DAPI. Scale bar 20μm(C) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and F4/80 (macrophage cell marker) at E13 and E16 stages of mouse fetal liver. (D) Distance distribution between cKithigh cells and macrophages (n=54 from 3 E13 fetal livers; n=64 from 3 E16 fetal livers), binned into 5μm intervals. Statistical analysis was completed using t-test. All nuclei marked with DAPI. Scale bar 20μm(E) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and MBP (neutrophil cell marker) at E13 and E16 stages of mouse fetal liver. (F) Distance distribution between cKithigh cells and neutrophils (n=45 from 3 E13 fetal livers; n=53 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test ***P<.001. All nuclei marked with DAPI. Scale bar 20μm