EMBO/soda-nel huggingface dataset:
Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation (2022)
Figure 1

Figure 1. Novel associations of microtubule-associated protein tau with synaptic vesicle cycle in proximity labelled interactomes

(A) Principle of proximity ligation catalysed by promiscuous biotin ligase BioID2 fused with human tau (441aa) (BioID2-tau) to map protein-protein interactions. Biotin supplementation enhances target biotinylation in BioID2-tau-expressing neurons. Labelled proteins are identified by LC-MS/MS after enrichment of biotinylated peptides.

(B) Adeno-associated virus (AAV) constructs for neuron-specific expression of BioID2-tau or BioID2 control. Syn-1, synapsin-1 minimal promoter; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element

(C) Schematic of primary neuron transduction with AAVs expressing BioID2-tau or BioID2 control at day 12 in vitro (DIV12) followed by biotin-enriched media treatment (DIV19-20) and cell harvesting for proteomic analysis (DIV21).

(D) Immunofluorescence of neurons expressing BioID2-tau or BioID2 (myc) or non-transduced control cells. Cells were incubated in biotin medium (50 μM) or vehicle medium for 30 h prior to fixation and probed for biotin (streptavidin, SA) and cell nuclei (DAPI) (n = 3 biological replicates). BioID2-tau and incorporated biotin co-localise in axons and somata. Scale bar, 25 μm.

(E) Venn diagram of overlapping and unique interacting proteins identified by biotinylation in cultured cortical mouse neurons (DIV21) (n = 3 biological replicates). Protein ontology (PO) categories are: 1 biological processes, 2 cell component, 3 molecular function, 4 Kyoto encyclopaedia of genes and genomes (KEGG) pathway, 5 Reactome pathway, 6 PFAM protein domain. Note most enriched functional annotation clusters: 3 cytoskeletal binding and 4 synaptic vesicle cycle. FDR, false discovery rate

(F) Tau interactome network generated by BioID2 in cultured neurons (DIV21) (n = 3 biological replicates). Network of significantly abundant and exclusive interactors in BioID2-tau with network properties and PPI enrichment score (STRING v11.5).

(G) Functional ontology clusters for molecular function: cytoskeletal binding, cell component: synapse and KEGG: synaptic vesicle cycle mapped onto neuronal tau interactome network in (F). Additional associations with synaptic localization/function and synaptic vesicle regulation described in literature are mapped, respectively.
Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation (2022)
Figure 2

Figure 2. Proximity labelling links tau with synaptic vesicle regulation in mouse brain in vivo

(A) Experimental schematic of post-natal interactome labelling of neuronal tau in vivo.

(B) Immunoblot of combined brain lysates from mice (P35) expressing BioID2-tau or BioID2 control (n = 4 biological replicates) probed for myc-tagged BioID2 and BioID2-tau. Controls: lysates from (1) BioID2- or (2) BioID2-tau-expressing and (3) non-transduced mouse brain. Loading, glyceraldehyde-3-phosphate dehydrogenase (Gapdh). *, unspecific band. Relative quantification confirmed similar expression levels of BioID2 and BioID2-tau. Means ± S.E.M. ns, not significant (Student's t-test)

(C) Immunofluorescence of P35 mouse brain expressing BioID2-tau. Hippocampal Cornu ammonis 1 (CA1) region was probed for BioID2-tau (myc), for incorporated biotin (streptavidin, SA) and for cell nuclei (DAPI) (n = 3-5 biological replicates). Scale bar, 50 μm.

(D) Tau interactome in mouse brain (P35) transduced at P0 to express BioID2-tau or BioID2 control (n = 12 biological replicates). Venn diagram shows overlapping and unique interacting proteins identified by biotinylation. Protein ontology (PO) enrichment (false discovery rate (FDR)) for highest ranking categories in 1 biological processes, 2 cell component, 3 molecular function, 4 KEGG pathway, 5 Reactome pathway, 6 PFAM protein domain are shown. Note most enriched functional annotation clusters: 2 synapse, 3 cytoskeletal binding, and 4 regulation of vesicle cycle.

(E) Tau interactome network generated by BioID2 in mouse brain (P35) (n = 12 biological replicates). Network of significantly abundant and exclusive interactors in BioID2-tau with network properties and PPI enrichment score (STRING v11.5).

(F) Functional ontology clusters mapped onto neuronal tau interactome network in (E) for cell component: synapse and for KEGG: synaptic vesicle cycle. Additional associations with synaptic localization/function and synaptic vesicle regulation described in literature are mapped, respectively.
Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation (2022)
Figure 4

Figure 4. Direct interaction with tau regulates activity of essential synaptic vesicular ATPase NSF

(A) Confocal immunofluorescence for endogenous tau and NSF in primary murine hippocampal neurons (DIV15). Colocalization of tau and NSF is apparent in perinuclear compartments (arrows) and neurites (insets i-iii). Scale bar, 25 μm. Inset of magnified neurite with partial overlap of tau and NSF signals (arrowheads). Scale bar, 3 μm. Co-distribution (R Ch1-Ch2) are indicated.

(B-C) Co-immunoprecipitation (Co-IP) of tau and NSF from mouse brain. (B) Endogenous and transgenic tau were immunoprecipitated from mouse brain lysates of tau-/-, ALZ17tg or ALZ17non-tg mice using anti-tau antibody followed by immunoblot probed for NSF, tau and α-actin. Endogenous NSF was co-immunoprecipitated with transgenic and endogenous tau from ALZ17tg or ALZ17non-tg but not from tau-/- with. GAPDH, input loading control (n = 3 biological replicates). Recombinant tau and NSF served as controls. (C) Densitometric quantification of co-IP results in (B).

(D-E) Co-IP of HA-tagged tau with NSF from 293T cells. (D) Schematic of tau deletion and truncation constructs used for expression in 293T cells. (E) Co-IP of NSF with haemagglutinin (HA)-tagged tau variants from cultured cells. HA-tagged tau variants and NSF were expressed in 293T cells followed by immunoprecipitation using HA antibody and immunoblot probed for NSf, tau and GAPDH. (n = 3 biological replicates).

(F) Microscale thermophoretic (MST) measurement of direct tau and NSF interaction. MST binding curve of recombinant hexameric NT-647-NHS-labelled NSF (27 pM) and recombinant tau (18 mM - 0.275 nM). Dissociation constant: KD = 146 ± 70 nM (sigmoidal curve fit, R2 = 0.844). (n = 5 technical replicates).

(G) Inhibition of NSF ATPase activity with increasing concentrations of tau. NSF activity (70 nM) was assessed alone or in the presence of NSF-inhibiting peptide TAT-NSF or control TAT-Scramble peptides (140 nM); or co-incubated with recombinant tau protein (8 nM, 16 nM, 24 nM), or heat-denatured tau (dTau, 8 nM) or albumin (Alb, 16 nM). Data were fitted to Michaelis-Menten model to determine rate constants (R2 = 0.904 - 0.994 for all conditions; n = 3 technical replicates).

(H) Rate constants derived from kinetic data in i. (n = 3 technical replicates) ATP hydrolysis is similar in NSF and NSF incubated with control peptide TAT-Scr, albumin, or denatured tau, yet lower with increasing concentrations of tau or after addition of NSF-inhibiting peptide TAT-NSF.

Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.
Alteration of microbiota antibody-mediated immune selection contributes to dysbiosis in IBD (2022)
Figure 1

Figure 1. Purified fecal IgA1 and IgA2 from IBD patients have altered functionality related to Dectin-1 binding. ELISA assay of fecal IgA1 (A), IgA2 (B), total IgA (C), and secretory component (D) levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool. Data were further separated according to disease activity for IgA1 (E) and IgA2 (F). In vitro assay of purified IgA1 (G) and IgA2 (H) reverse-transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co-culture. (I) ELISA assay of IgA1-Dectin-1 binding, at a rate of one receptor per 10 IgA; (J) ELISA assay of IgA2-Dectin-1 binding, at a rate of one receptor per 10 IgA. Percent of IgA1 (K, L) and IgA2 (M, N) reverse-transcytosis for weak (K, M) and strong (L, N) Dectin-1 binding. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (A)** : p= 0,0098; (B) *: p=0,0406; (C) Non-IBD v. UC *: p=0,0479, CD vs. UC *: p=0,0166; (F) *: p=0,0167; (G) *: p=0,0441; (H) *: p=0,423 ; (J) Non-IBD vs. UC *: p=0,0199 , CD vs. UC *: p= 0,0243; (K) *: p=0,0221 ; (L) *: p=0,0486 . (G-N) For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.
Alteration of microbiota antibody-mediated immune selection contributes to dysbiosis in IBD (2022)
Figure 3

Figure 3. CD and UC IgA have distinct anti-glycan reactivity profiles. Heatmaps displaying standardized log2-transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non-IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance. (C-F) Volcano plots of log2-transformed fold changes for IgA1 anti-glycan reactivity in CD (C) and UC (D) groups, and IgA2 anti-glycan reactivity for CD (E) and UC (F) compared to non-IBD IgA1 and IgA2. For CD, p-values of differentially targeted glycan motifs are liste in Appendix Table S8 and those of UC in Appendix Table S9, S10. Significance threshold was placed at p-value<0,05 (indicated by the dotted line in C to F); For IgA1, Non-IBD: n= 4; CD: n= 4; UC: n= 4. For IgA2, n= 6; CD: n= 6; UC: n= 6. IgA1 and IgA2 were taken matched for the same patient. All patient samples (biological replicates) have been tested in technical duplicates. A list of the glycans from each cluster is provided as supplementary material (Appendix Table S11, S12).
Alteration of microbiota antibody-mediated immune selection contributes to dysbiosis in IBD (2022)
Figure 4

Figure 4. CD IgA1 have biased selection towards the microbiota. Flow cytometry analysis of total IgA1-bound (A) and IgA2-bound (B) fecal microbiota. (C) Flow cytometry analysis of stool IgA1+ IgA2- bacteria (grey dots), IgA1- IgA2+ bacteria (black dots) and IgA1+ IgA2+ bacteria (empty dots). (A-C) Data were analysed with a Kruskall-Wallis multiple comparison test. In (C) *: p= 0,0226. Non-IBD: n= 7, CD: n=18, and UC: n=12. (D) Shannon and Chao1 diversity indices of IgA1-bound microbiota in non-IBD, CD and UC. (E) PCA plot based on the Jaccard distance between samples. (F) Phylum-level composition of IgA1-bound microbiota in non-IBD, CD and UC. (G) Boxplots of OTUs for which the abundance was significantly different between non-IBD and CD. (H) Shannon and Chao1 diversity indices of IgA2-bound microbiota in non-IBD, CD and UC. (I) PCA plot based on the Jaccard distance between samples. (J) Phylum-level composition of IgA2-bound microbiota in non-IBD, CD and UC. (K) Boxplots of OTUs whose abundance was significantly different between CD and UC, and between non-IBD and UC, respectively. OTUs present in less than 25% of samples or with read count lower than 50 were filtered out. Differential abundance was tested using negative binomial model implemented in DESeq2 and p-values corrected with False Discovery Rate (FDR) procedure. Non-IBD: n = 4; CD: n = 4; UC: n = 4 (2 patients excluded for abundance in both IgA1 and IgA2 analyses). All patient samples have been tested in technical duplicates.
Alteration of microbiota antibody-mediated immune selection contributes to dysbiosis in IBD (2022)
Figure 6

Figure 6. CD IgA2 display biased selectivity in active CD patients. (A) Combined FISH-IF staining for each probe. (B) Total fluorescence detected per probe and per biopsy. Erec482 - *: p=0,00233; Ato291 - *: p=0,0268; Bac303 - *: p=0,0196. (C) Percent of co-occurred IgA2 and FISH-bound particles. Erec482 - *: p=0, 0073.Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test. Non-IBD: n = 3; Active CD: n= 6; Inactive CD: n=4. All patient samples (biological replicates) have been tested in technical duplicates.
Plasma cells require autophagy for sustainable immunoglobulin production (2013)
figf1

(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red. (b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm. (c) Immunoblot analysis of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in LPS-stimulated B cells with (+NH4Cl) or without treatment with NH4Cl (left). Actin serves as a loading control throughout. Right, quantification of LC3-II band intensity in the absence of NH4Cl, presented relative to that of actin. (d) Quantitative RT-PCR analysis of transcripts encoding autophagy-related molecules in ASCs 3 d after stimulation with LPS, presented relative to that in unstimulated B cells. (e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c). (f) Live-cell fluorescence microscopy of CD138+ PCs purified from the spleen of GFP-LC3 mice 5 d after the injection of NP-Ficoll (PCs) and of splenic B cells (B cells) and bone marrow CD138+ PCs (BM PCs) purified from unstimulated GFP-LC3 mice (right) and automated quantification of spots (left). Scale bar (right), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of three or more independent experiments (a,c–e) or one experiment or more with >40 cells (b,f; average and s.e.m.).
Plasma cells require autophagy for sustainable immunoglobulin production (2013)
figf2

(a) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE. (b) Size of B cells purified as in a and activated for various times (horizontal axis) with LPS, assessed by the intensity of forward scatter (FSC) by flow cytometry. (c) CD138 expression of B cells purified and activated as in a. APC, allophycocyanin; PE, phycoerythrin. (d) Immunoblot analysis of ER proteins in lysates of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left), and quantification of band intensity (right; as in Fig. 1c). IgM-H, IgM heavy chain. NS, not significant; *P 0.05 and **P 0.01 (Student's t-test). Data are from one experiment (a) or three independent experiments (b,c; average and s.e.m. of three or more mice in a,c), three independent experiments with three mice per genotype in each (d, left) or four independent experiments (d, right; average and s.e.m. of seven or more mice).
Plasma cells require autophagy for sustainable immunoglobulin production (2013)
figf3

(a) Electron microscopy of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left and middle), and quantification of the ER by stereology (right). Scale bars (left and middle), 1 μm. (b) G6Pase electron-microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS (left), and quantification of G6Pase+ (ER) area, relative to total cell area (right). Scale bars (left), 1 μm. (c) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1 (sXBP-1), total XBP-1 (tXBP-1) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS, presented relative to that of unstimulated Atg5f/f B cells at day 3. (d) Quantitative RT-PCR analysis of CHOP mRNA in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS (left and middle), and in Atg5f/f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin (Atg5f/f 24 h tun; 0.5 μg/ml; positive control), presented relative to that in Atg5f/f B cells stimulated for 3 d with LPS. (e) Immunoblot analysis (left) of ER proteins in Atg5f/f B cells left untreated (UT) or treated with lysosomal protease inhibitors (10 μM leupeptin and E-64d) for the final 8 h of the third day of LPS-induced differentiation (Leu + E-64d (8 h)). Right, quantification of band intensity (presented as in Fig. 1c). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are from two or more independent experiments (a–d and e, right) with ≥29 (a) or ≥67 (b) total cells (average and s.e.m.) or three or more mice in each (c,d and e, right; error bars, s.e.m.) or one experiment with three separate mice (e, left).
Plasma cells require autophagy for sustainable immunoglobulin production (2013)
figf4

(a) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells. (b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units. (d) Quantitative RT-PCR analysis of mRNA encoding Blimp-1 and the secreted μ-chain in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, presented relative to that of Atg5f/f cells. (e) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1, BiP, Blimp-1 and secreted μ-chain in Atg5f/f B cells stimulated for 3 d with LPS and given no further treatment (−tun) or treated for the final 24 h with tunicamycin (tun 24 h; 0.5 μg/ml), presented relative to results obtained with LPS-stimulated cells given no further treatment. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of two or more independent experiments with two or more mice in each (average and s.e.m.).
Plasma cells require autophagy for sustainable immunoglobulin production (2013)
figf5

(a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0). (b) Death of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for various times (horizontal axis) with LPS, quantified by flow cytometry by propidium iodide (PI) staining. (c) Quantitative PCR analysis of genomic DNA in differentiating B cells from Atg5f/f and Atg5f/fCD19-Cre mice, assessing nonrecombinant (Undeleted; left) and recombinant (Deleted; right) loxP-flanked Atg5 alleles; results are presented relative to those of unstimulated Atg5f/fCD19-Cre B cells. (d) Quantitative RT-PCR analysis of Atg5 mRNA in differentiating Atg5f/f and Atg5f/fCD19-Cre B cells at 0 and 3 d after stimulation with LPS, presented relative to that in untreated Atg5f/fCD19-Cre B cells. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are from one experiment representative of three with three mice per genotype (a) or are from three independent experiments (b–d; average and s.e.m.).
Plasma cells require autophagy for sustainable immunoglobulin production (2013)
figf6

(a) ELISA of anti-pneumovax IgM titers in diluted serum 1 day before and 4, 10 and 14 d after intraperitoneal immunization of Atg5f/f and Atg5f/fCD19-Cre mice with 1 μg pneumovax. (b) ELISA of anti-pneumovax IgG3 titers in serum 14 d after injection as in a. Results are presented as absorbance (A). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of one experiment with six mice per genotype (error bars, s.e.m.).
Plasma cells require autophagy for sustainable immunoglobulin production (2013)
figf7

(a) Frequency of CD138hiB220lo cells (numbers above outlined areas, bottom) among the IRF4hi population (numbers above outlined areas, top), assessed by flow cytometry (left). 488, Alexa Fluor 488. Right, quantification of bone marrow PCs (each symbol represents an individual mouse; small horizontal lines indicate mean (and s.e.m.). (b) Quantitative RT-PCR analysis of mRNA encoding IRF4 and Blimp-1 in CD138hiB220lo bone marrow PCs and B220hiCD138lo spleen B lymphocytes (control) sorted from Atg5f/f and Atg5f/fCD19-Cre mice, to confirm PC phenotype; results are presented relative to that of histone H3 mRNA (loading control). (c) Quantitative PCR analysis of genomic DNA assessing undeleted and deleted loxP-flanked Atg5 alleles in sorted B cells and PCs (as in b; presented as in Fig. 5c). (d) Immunofluorescence of endogenous LC3 (left, top row) and immunoglobulin content (left, bottom row) in bone marrow PCs sorted from Atg5f/f and Atg5f/fCD19-Cre mice, and quantitative RT-PCR analysis of Atg5 mRNA in bone marrow PCs and spleen B cell populations (right) sorted from Atg5f/f and Atg5f/f CD19-Cre mice. Nuclei (left) are stained blue with Hoechst. Scale bar (left), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of one experiment with four 9- to 12-month-old mice per genotype (a) or are from two independent experiments with two Atg5f/f mice and four Atg5f/f CD19-Cre mice (b–d; average and s.e.m.).
Structural basis of semaphorin-plexin cis interaction (2020)
Figure 1

Figure 1. Sema1b binds PlexA at two independent sites, A and B, indicating two independent Sema1b-PlexA orientations: head-to-head and side-on

A. Schematic domain organization of Drosophila PlexA and Sema1b (SS, signal sequence; TM, transmembrane region, red symbols represent the position of N-linked glycosylation sites)

B, C. Ribbon representation of the PlexA1-4-Sema1b1-2 1:1 (C) and 2:2 (D) complexes. N-glycans are shown in stick representation.

D. Ribbon representation of the side-on orientation derived from the 2:2 complex.

E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.