EMBO/soda-nel huggingface dataset:
A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease (2011)
figf2

(a) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn's disease (CD) (n = 67) without or with active inflammation and labeled for miR-196A. (L, lumen; LP, lamina propria). Scale bars in the upper panel, 25 μm; scale bars in the lower panel, 10 μm. (b) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls (n = 8) or individuals with Crohn's disease with no inflammation (n = 16), quiescent (defined as low-grade inflammation) (n = 8) or acute inflammation (n = 8) using laser capture microdissection. After RNA extraction, miR-196A (black bars) and miR-196B (white bars) relative expression was analyzed using RNU19, 44 and U6. To overcome possible inter-individual bias, the lamina propria fraction value was used for relative quantification. Due to high differences in expression between healthy and inflamed tissues, the results are presented as a log2-fold ratio. Error bars indicate the s.d. of the ΔΔCt values. (c) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn's disease with a defined genotype in a healthy non-inflamed or an acute inflamed phase. Scale bars, 32 μm. (d) Mean (black line), s.e.m. (white box) and 95% CI of the mean of the IRGM expression level for 40 healthy subjects (32 individuals with C/C and 8 individuals with C/T) and 67 individuals with Crohn's disease (45 with C/C and 22 with C/T) with quiescent or inflamed colon mucosa. We performed statistical analysis using ANOVA (P = 0.0015) and an unpaired Student's t-test (the one tail P value is indicated on the figure).
A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease (2011)
figf3

(a) The basal flux of autophagy is affected by IRGM expression level. HEK293 cells transfected with an IRGM-expressing plasmid, miR-196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti-LC3B. (b) Quantification of LC3-II relative to actin (mean of three independent experiments ± s.d.). (c) Downregulation of IRGM expression by miR-196 abrogates AIEC-mediated autophagy in cells treated with autophagic inhibitors (Inh) or transfected with miR-196B and infected for 4 h with AIEC LF82 (mean of three independent experiments ± s.d.). (d) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3-associated (red) LF82 bacteria (green) in miR-196 transfected cells compared to control cells (mean ± s.d.). (e) miR-196 transfection leads to increased intracellular LF82 replication. Results are expressed as a fold increase ± s.e.m. of intracellular bacteria. (f) IRGM overexpression did not inhibit autophagic flux and it increased LC3-II accumulation slightly in response to AIEC infection. HEK293 cells were transfected with IRGM-expressing plasmid, treated with autophagic inhibitors and infected with AIEC bacteria for 4 h (mean of three independent experiments ± s.d.). (g) Confocal microscopic examination showed an increased percentage of LC3-associated AIEC bacteria in IRGM cells compared to control cells (mean ± s.d.). (h) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria. (i) Most of the bacteria reside in non-acidic vacuoles, as shown with lysotracker at 8 h post infection (means of three independent experiments ± s.d.).
A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. (2015)
Figure 1

Novel G-insertion mutation identified in male with FXS symptoms

Predicted effects of the G-insertion mutation on FMRP are schematized. The insertion site corresponds to the beginning of the RNA-binding RGG Box, where it alters the open reading frame and thus disrupts the domain entirely. The frameshift creates a novel peptide sequence [RKRTRRKRTWRRLQRKRRSLPNR] followed by a premature stop codon, causing the truncation of the FMRP C-terminus.

Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.

FMR1 mRNA levels were analyzed via RT–qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.

Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.
A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. (2015)
Figure 2

Patient FMRP aggregates in the nucleolus

A–C HEK293 (A) and SH-Sy5y (B) cells, as well as primary neurons from FMR1 KO mice (C), were transfected with vectors encoding the GFP-tagged wild-type FMRP (GFP-FMR1WT) or the GFP-tagged patient FMRP (GFP-FMR1G-insertion) under the control of a β-actin promoter. Wild-type FMRP localizes predominantly in the cytoplasm, whereas the patient FMRP forms nuclear and nucleolar aggregates that colocalize with nucleophosmin. Scale bars in (A, B) represent 10 μm. Scale bar in (C) represents 5 μm.
A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. (2015)
Figure 3

Patient FMRP contains a novel nuclear localization signal in the C-terminus

Transfected HEK293 cells expressing a GFP-tagged FMRP in which the C-terminus is truncated following the G-ins. mutation site (GFP-FMR1ΔCt). GFP-FMR1ΔCt protein is exclusively cytoplasmic, suggesting that the truncation of the C-terminus alone is not sufficient to explain the localization change observed for the patient FMRP.

Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP (GFP-FMR1G-ins. [NLSmutated]). The novel amino acid sequence in the C-terminus of patient FMRP [RKRTRRKRTWRRLQRKRRSLPNR] contains stretches of arginines and lysines predicted to function as a nuclear localization signal (based on Expasy protein motif scanner, ScanProsite). Mutating adjacent R and K residues into alanines [RARTRAARTWARLQRAARSLPNR] abolishes the nucleolar localization of the patient FMRP, strongly suggesting that the novel amino acid sequence present in the patient FMRP C-terminus is a functional nuclear localization signal (NLS).

Data information: Scale bar represents 10 μm.
A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. (2015)
Figure 4

An intact C-terminus facilitates nuclear export of patient FMRP

Transfected HEK293 cells expressing GFP-tagged wild-type FMRP fused to the patient NLS motif (GFP-FMR1wt+NLS). Unlike the patient FMRP, GFP-FMR1wt+NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus. However, treatment of HEK293 cells expressing GFP-FMR1wt+NLS with leptomycin B—an inhibitor of nuclear export—resulted in the appearance of nucleolar inclusions. This suggests that the presence of a full-length C-terminus facilitates the nuclear export of FMRP in this context.

Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP, in which the truncation of the C-terminus is reverted by restoring the open reading frame (GFP-FMR1G-ins.[Ct restored]). The patient protein is not detected in the nucleus when the C-terminus is intact; however, nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells. In line with results from (A), this suggests that an intact C-terminus enables nuclear export of the FMR1 protein.

Data information: Scale bar represents 10 μm.
A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. (2015)
Figure 5

sLNV neurons are sensitive to changes in Dfmrp function

The morphology of small lateral ventral neurons (sLNvs) is sensitive to dfmr1 activity. Overexpression of wild-type dfmr1 in the sLNv neurons results in a consistent “axonal collapse” phenotype, where the branching of axonal termini of sLNvs is reduced. Scale bars represent 50 and 10 μm (magnified images).

Transgenic fly lines were created bearing UAS-dfmr1 variants, in order to assay in vivo functional changes associated with the effects of the patient mutation: the truncation of the C-terminus (dfmr1ΔCt), presence of the patient NLS sequence (dfmr1wt+NLS), or both (dfmr1ΔCt+NLS).
A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. (2015)
Figure 6

Compound effects of the patient mutation confer a neomorphic function for Dfmrp in sLNV neurons

Overexpression of wild-type dfmr1 (UAS-dfmr1wt) using a LNv neuron-specific driver line (Pdf-Gal4) causes reduction of branching area—“axonal collapse”—in the sLNv axonal termini. dfmr1ΔCt and dfmr1wt+NLS overexpression phenocopies overexpression of dfmr1wt, suggesting that the truncation of the C-terminus or the presence of the patient NLS alone does not impair or alter the functional efficacy of dfmr1 in this context. However, overexpression of dfmr1ΔCt+NLS fails to cause collapse of sLNv termini, indicating a significant loss or change of Dfmrp protein function. Axonal morphology is visualized by expressing CD8-GFP under the control of the same neuronal driver. Scale bar represents 10 μm.

Quantification of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles. Overexpression of dfmr1wt, dfmr1ΔCt, or dfmr1wt+NLS significantly reduced axonal branching area [****P < 0.0001; control 101.3 ± 29.76 SD, n = 30; UAS-dfmrwt 45.41 ± 21.69 SD, n = 29; UAS-dfmrΔCt 52.31 ± 14.67 SD, n = 32; UAS-dfmrwt+NLS 72.45 ± 21.83 SD, n = 29; UAS-dfmrΔCt+NLS 117.8 ± 36.96 SD, n = 28]. dfmr1ΔCt+NLS overexpression does not result in a significant change of axonal branching area compared to the background control [Pdf-Gal4, UAS-GFP] (P = 0.672). For quantification purposes, each branching area (n) was manually outlined, starting from the first point of defasculation of the axonal bundle. These measurements were normalized for variability in brain size across samples using LNv commissure length measurements for each brain. Two-tailed t-tests using Welch's correction were then used to compare controls with mutant phenotypes (GraphPad). Error bars represent mean values with SD. Dots visible for box plots of UAS-dfmrwt and UAS-dfmrΔCt represent samples that outlier the whiskers.

Overexpression of dfmr1ΔCt+NLS results in aberrant axonal termini morphology. Axonal guidance defects were frequently observed (arrowheads) and scored manually for bifurcations of the axon bundle (9/48), tangling of axons in the termini (13/48), and apparent looping of axons (7/48).
A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. (2015)
Figure 7

Subcellular localization of the Dfmrp variants in sLNv neurons

A–D Subcellular localization of transgenically expressed dfmr1wt, dfmr1ΔCt, dfmr1wt+NLS, and dfmr1ΔCt+NLS proteins in LNv neurons. DfmrpΔCt+NLS shows nuclear localization (A), colocalizing with the genetically encoded nuclear marker RedStinger, while Dfmrpwt, DfmrpΔCt, and Dfmrpwt+NLS are predominantly cytoplasmic (B–D). These results suggest that an intact C-terminus can mediate the nuclear export of the Dfmrp protein. Scale bar represents 5 μm.
Structure of the actin filament nucleator Arp2/3 complex bound to its activator SPIN90 (2018)
Figure 1

Figure 1: Structure and biochemical characterization of the C-terminus of SPIN90.

A. Domain organization of SPIN90 and SPIN90 constructs used in this study (SH3 - Src homology 3, PRD - proline rich domain, MS - middle segment, AR - armadillo repeat)

B. Ribbon diagram of SPIN90(351-722). Helices are colored according to their position within the repeat (1-yellow, 2-red, 3-cyan). Armadillo repeats are numbered 1-7. Disordered residues are indicated with dotted lines.

C. Surface representation of SPIN90(351-722) showing conservation scores derived from an alignment of WDS family proteins from 31 diverse species (see methods). Conservation score is indicated by color, with dark red > pink > light pink > grey > light cyan > cyan, and higher scores meaning greater conservation.

D. Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2/3 complex and 3 μM 15% pyrene labeled actin.

E. Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in (D).

F. Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2.5 μM SPIN90, 500 nM Arp2/3 complex, 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct.

G. Time courses of pyrene actin polymerization as described in panel (D), except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351-722 construct.
Structure of the actin filament nucleator Arp2/3 complex bound to its activator SPIN90 (2018)
Figure 4

Figure 4: Contacts of SPIN90 with the long C-terminal helix (αD) in ARPC4 are important for activation of Arp2/3 complex.

A. Ribbon diagram of co-complex structure with residues at the SPIN90-Arp2/3 interface depicted as Cα atom spheres colored according to side chain chemistry (grey: hydrophobic, red: basic, blue: acidic). Only the ARPC4 subunit of Arp2/3 complex is shown in the zoomed in panel for clarity.

B. Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90(269-722) to activate Arp2/3 complex. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex, and 20 μM wild type or mutant SPIN90(269-722).

C. Plot of maximum polymerization rate versus concentration of SPIN90(269-722) for reactions as shown in panel (B).
Structure of the actin filament nucleator Arp2/3 complex bound to its activator SPIN90 (2018)
Figure 5

Figure 5: Binding of the SPIN90 ARM domain to the ARPC4 long helix may stimulate movement of Arp2 into or toward the short pitch conformation.

A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666.

B. Cartoon of "rotation model" for movement of Arp2 into the short pitch conformation. White arrow shows the approximate position of a hinge point for the rotation.

C. Structural superposition showing the proposed hinge movement in ARPC4 and the position of bound SPIN90 relative to the hinge. Residues 156-168 from ARPC4 and residues 247-260 from ARPC2 in an inactive crystal structure (4JD2) were overlaid with the same residues from the molecular dynamics (MD) short pitch model of Arp2/3 complex (Dalhaimer & Pollard, 2010). ARPC4 from 4JD2 (blue) and from the MD simulation (cyan) are shown. The helical axis of the long alpha helix (αD) in ARPC4 is shown as a black line in the 4JD2 structure and as a green line in the MD simulation structure. The red line represents the helical axis of helix αD in ARPC4 in the SPIN90-bound co-complex overlaid as described above. For clarity, the cartoon representation of the ARPC4 subunit in the co-complex structure is not shown here, but is shown in Appendix Figure S10. The orientation of this panel is similar that depicted in panels (B and D).

D. Conceptual model of SPIN90-bound Arp2/3 complex in the short pitch conformation. The model was made by superposing subdomains 1 and 2 of Arp3 from the co-complex structure onto an actin subunit from a high-resolution structure of an actin filament (3J8I) (Galkin et al, 2015). Arp2 (red ribbon) was then moved into the short pitch position based on the filament structure. Arp2 in its original splayed position (as observed in the crystal structure) is shown in grey ribbon representation. Shortest Cα-Cα distances from SPIN90 to either subdomain 2 or subdomain 4 in Arp2 in the hypothetical short pitch conformation are indicated.

E. Comparison of the position of Arp2 relative to Arp3 in the short pitch arrangement (grey Arp2), the inactive splayed arrangement (red Arp2), and the SPIN90 bound x-ray crystal structure (cyan). The short pitch arrangement of Arp2-Arp3 was modeled by overlaying subdomains 1 and 2 of Arp3 on the same subdomains in an actin subunit in the actin filament structure 3G37(Murakami et al, 2010), and then superposing Arp2 from the 4JD2 structure onto the actin subunit in the short pitch position. The SPIN90-bound complex was then overlaid onto Arp3 subdomains 1 and 2 to determine the relative position of Arp2 in the SPIN90-bound complex. Blue line shows a potential trajectory of the center of mass of Arp2 when it moves from the splayed to the short pitch position.
Structure of the actin filament nucleator Arp2/3 complex bound to its activator SPIN90 (2018)
Figure 6

Figure 6: SPIN90 binds to the actin filament binding interface on Arp2/3 complex and blocks filament binding.

A. Ribbon diagram of the co-complex structure showing where an actin filament (grey semi-transparent surface model) binds to Arp2/3 complex. To model the bound filament, ARPC2 and ARPC4 from the EM reconstruction of Arp2/3 complex at a branch junction were overlaid onto ARPC2 and ARPC4 from the co-complex structure (Rouiller et al, 2008).

B. SDS-PAGE gel showing results of actin filament copelleting assay. Actin filaments at 3.5 μM were used to co-pellet 0.6 μM Bos taurus Arp2/3 complex in the presence or absence of the indicated concentrations of SPIN90(269-722) (S: Soluble fraction, P: Pellet)

C. Plot of co-pelleting data as shown in (B), run in quadruplicate with error bars showing the standard deviation.

D. Comparison of the contacts made by SPIN90 (left panel) and actin filaments (Rouiller et al, 2008) (right panel) with the αD helix in ARPC4 (ARPC4 is blue, with the αD helix highlighted in yellow). SPIN90 is colored according to the helix number within each repeat (helix 1 - cyan, helix 2 - yellow, helix 3 - red). The actin subunit from the filament that contacts ARPC4 is colored according to secondary structure (helix - red, strand - yellow). Select elements of secondary structure are labeled, as is the DNaseI binding loop of actin (DBL).

E. Superposition of subdomains 1 and 2 of Arp3 from the inactive GMF-bound structure (Luan & Nolen, 2013) (4JD2, orange) with Arp3 from the branch junction model (Rouiller et al, 2008) (grey) and Arp3 from the SPIN90-bound structure (cyan - shown only in right panel). Regions disordered in the structure are shown as dashed lines. Cyan arrow indicates the direction of motion in the transition from the twisted to flattened conformation.

F. Plot of the nucleotide cleft width versus the twisting dihedral angle in Arp3 from x-ray crystal structures, Arp3 from the branch junction EM reconstruction, or filamentous or monomeric actin structures. The pdb accession number is indicated for each data point. Orange: Arp3, x-ray crystal structures; cyan: monomeric actin x-ray crystal structures; black: actin filament fiber diffraction or cryoEM structures, green: Arp3 (rou-A) from branch junction reconstruction. Twist angle was defined as the dihedral made by connecting the center of mass off each subdomain 1-4. Nucleotide cleft distance was defined as the distance between Cα atoms in residues 67 and 222 (Arp3) or 59 and 207 (actin).
Ca2+-independent transmission at the central synapse formed between dorsal root ganglion and dorsal horn neurons (2022)
Figure 2

Figure 2. Imaging of CiVDS mediated synaptic transmission in co-cultured DRG and DH neurons.

A A representative photograph showing the co-cultured DRG and DH neurons. The DRG neurons were expressed with Spy-pHluorin for imaging the synaptic transmission. Scale bar, 10 μm.

B Images of a presynaptic bouton marked in (A) showing the Spy-pHluorin fluorescence at 20 s before (-20 s, pre-stimulus), 20 s, 40 s, and 120 s after electrical stimulation (post-stimulus) in 0Ca2+ (upper panel) or 2Ca2+ (lower panel) solution.

C Averaged fluorescence changes (ΔF/F0) of Spy-pHluorin in 0Ca2+ (left) or 2Ca2+ solution (right) in response to the same electrical stimulation (20 Hz, 20 s) (n = 45 puncta from 6 cells for 0Ca2+ and 57 puncta from 6 cells for 2Ca2+). The shadow in the traces represents the error bars (s.e.m) of each point.

D-F The same as in (A-C), but the experiments were performed in cultured hippocampal neurons (n = 70 puncta from 3 cells for 0Ca2+ and 72 puncta from 3 cells for 2Ca2+).
Ca2+-independent transmission at the central synapse formed between dorsal root ganglion and dorsal horn neurons (2022)
Figure 5

Figure 5. EPSCCiVDS are mediated by the SNARE complex and N-type Ca2+-channels.

A Left, diagram showing the protocol for EPSC recording from DH neurons co-cultured with Syb2-knockout (cKO) DRG neurons; right, cartoon of EPSC recording from DH neurons co-cultured with Syb2-cKO DRG neurons.

B Representative western blots (upper) and analysis (lower) of the expression levels of Syb2 in control (Ctrl) and Syb2-cKO DRG neurons (n = 3 per group). Control DRG neurons is from homozygous floxed Syb2-null mice infected with AAV5-CAG-EGFP virus.

C Evoked EPSCs and statistics from DH neurons co-cultured with control (Ctrl) or Syb2-cKO DRG cells (cKO) in 0Ca2+ solution (n = 13 cells for Ctrl and 11 for cKO).

D Evoked EPSCs and statistics from DH neurons co-cultured with DRG neurons before (black) and after (red) applying 1 μM ω-conotoxin-GVIA (GVIA) in 0Ca2+ bath (n = 9 cells).

E Upper, diagram showing the protocol for EPSC recordings from DH neurons co-cultured with Cav2.2 knockdown (KD) DRG neurons; lower, cartoon of EPSC recording from DH neurons co-cultured with Cav2.2-KD DRG neurons.

F Left panels, evoked EPSCs from DH neurons co-cultured with scrambled shRNA control (Ctrl) or Cav2.2-KD (sh-1/sh-2) DRG neurons in 0Ca2+ solution. Right panel, quantification of evoked EPSCs (n = 17 cells for Ctrl, 12 for sh-1, and 19 for sh-2). EPSCs were evoked by local electrical stimulation (Estim, at arrows).

Data information: Data are shown as the mean ± s.e.m.; paired Student's t test for B and D, unpaired Student's t test for C, and Kruskal-Wallis test followed by Dunn's multiple comparisons test for F; *p <0.05, **p <0.01, ***p <0.001.