Journal: Neuropsychopharmacology

Article Title: Identification of MicroRNA-124-3p as a Putative Epigenetic Signature of Major Depressive Disorder

doi: 10.1038/npp.2016.175

Figure Lengend Snippet: Functional validation of miR-124-3p targets. (a) Bar diagram represents the relative quantification of GRIA4, NR3C1, HSP90AB1, and AKT1S1 transcripts in SH-SY5Y neuroblastoma cell line transiently transfected with non-target-specific scramble control (n=3), mimic miR-124-3p overexpression oligo (n=3), anti-miR-124-3p overexpression oligo (n=3), and vehicle control (n=3). The expression status of respective genes was quantified in treatment groups relative to vehicle control using one-way analysis of variance, followed by post hoc Tukey's multiple comparison test. GAPDH was used as normalizer for target gene expression. Values are represented as ±SEM ‘a' and ‘b' denote significant differences when compared with vehicle. Overall group differences in the four groups (vehicle, scramble, mimic, and anti) are as follows: GRIA4: df=3.8; f=2.95, p=0.09; NR3C1: df=3.8; f=32.15, p<0.001; HSP90AB1: df=3.8; f=4.92, p=0.03; AKT1S1: df=3.8; f=6.61, p=0.015. ap<0.029; bp<0.001. (b) Schematic representation of Gria4 3′ untranslated region (UTR) cloning strategy with restriction enzyme digestion (SpeI and HindIII restriction enzyme digestion sites) in pMIR-Report vector downstream of firefly luciferase reporter gene. (c) Relative luciferase activity (normalized with Renilla luciferase activity) driven by Gria4 3′ UTR via miR-124-3p interaction was determined in HEK-293 lysate co-transfected with reporter clone and individually mimic miR-124-3p oligo (n=3) or antisense (anti) miR-124-3p oligo (n=3). Representative data were compared with nonspecific scramble control (n=3) and expressed as ±SEM. Overall group differences in the three groups (scramble, mimic, and anti) are as follows: df=2.6; f=33.22, p=0.001. ap=0.023; bp=0.011. (d–h) Involvement of endogenous miR-124-3p binding on single 7mer-m8 site of Gria4, two different sites of Nr3c1, one 8mer site of Akt1s1, one 8mer site of Gria3, and the proximal 7mer-A1 site of Grin2a on their 3′ UTR determined by RNA-induced silencing complex (RISC)-mediated immunoenrichment assay. Relative 3′ UTR enrichment normalized to 10% input in the immunoprecipitated ribonucleoprotein complex was analyzed with qPCR. Data are represented as ±SEM. Data were analyzed by independent-sample ‘t' test. ‘a' denotes significant difference between vehicle and corticosterone (CORT)-treated groups. Gria4 p=0.022, Nr3c1 7mer-m8 p=0.034, Nr3c1 7mer-A1 p=0.133, Akt1s1 p=0.58, Gria3 p=0.16 and Grin2a p=0.39.

Article Snippet: Amplified 3′ UTR was double-digested ( Spe I and Hind III) and directionally cloned in pMIR luciferase reporter vector (Addgene, Cambridge, MA, USA) ( Supplementary Table S1 ).

Techniques: Functional Assay, Transfection, Over Expression, Expressing, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) CW EPR spectra of TACC3 MTSL-C828 (black) and TACC3 MTSL-C662 (red) at 120 K. ( B-C ) Normalized four-pulse DEER trace at 50 K for (B) TACC3 MTSL-C828 and (C) TACC3 MTSL-C662. Inset, traces after subtraction of a mono-exponential decay. The weak oscillation is most likely from residual proton modulation.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques:

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) In vitro co-precipitation assays between immobilized NusA-TACC3 TACC domain and ch-TOG truncates. ( B ) Superposition of the top 20 ch-TOG 1817-1957 NMR structures using the structured core of helices, H1-H4 (residues 1826-1894) for alignment. Inset, structures aligned on ch-TOG residues 1905-1915. ( C ) Cartoon representation of the best NMR structure for ch-TOG 1817-1957 shown in the same orientation as in (B). Inset, stick representation of ch-TOG residues 1905-1915. Structures in (B-C) are colored by spectrum mode where the N-terminus is blue and the C-terminus is red.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: In Vitro

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Chemical shift perturbations observed on interaction of 2 H/ 15 N labeled ch-TOG 1817-1957 with a three-fold excess of TACC3 629-838 Δ699-765. Data extracted from the spectra shown in . ( B ) MST binding experiment of a synthetic ch-TOG H5 peptide with TACC3 629-838 Δ699-765. ( C ) Cartoon representation of an AlphaFold2 Multimer model of the complex between the TACC3 629-838 Δ699-765 dimer (wheat) and ch-TOG 1817-1957 (green). Arrows indicate the position of the deletion in TACC3. The model is shown below colored according to per residue confidence score (pLDDT) in rainbow colors from high (blue) to low (red) confidence. ( D ) Cartoon representations of the AlphaFold2 model showing the interface between the H5 region of ch-TOG (green) and TACC3 (wheat). Sidechains contributing to the interface from TACC3 protomer A (orange), TACC3 protomer B (yellow) and ch-TOG H5 (green), are shown in stick representation.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Labeling, Binding Assay, Residue

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: Plots show the protein alone (blue) and in the presence of a three-fold excess of TACC3 629-838 Δ699-765 (red).

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques:

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Predicted aligned error (PAE) plot for a model of the complex between TACC3 629-838 Δ699-765 and ch-TOG 1817-1957 generated by AlphaFold2 Multimer. ( B ) In vitro co-precipitation assays between immobilized His-NusA-ch-TOG 1817-1957 constructs and TACC3 629-838 (top). Binding of TACC3 was resolved by Western blot (bottom). ( C ) Sequence coverage map of TACC3 629-838 Δ699-765 in HDX-MS experiments. The yellow shaded regions in the thick bar at the top of the panel represent regions with sequence coverage while gray indicates regions that were not covered by detected peptides. Narrow yellow bars represent the individual peptides detected. ( D-F ) Woods plots showing the differences in deuterium uptake in TACC3 at three HDX timepoints (0.5, 5, 30 min of HDX), comparing TACC3 629-838 Δ699-765 alone with TACC3 629-838 Δ699-765 in the presence of Affimers E4 (D), E7 (E) and E8 (F). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides with no significant difference in exchange between conditions, determined using a 99 % confidence interval and a hybrid statistical test (dotted line), are shown in gray. A summary of key details of the HDX-MS experiment is shown in .

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Generated, In Vitro, Construct, Binding Assay, Western Blot, Sequencing

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) In vitro co-precipitation assay between Affimers, TACC3 and ch-TOG. C-terminal His-tagged Affimers were immobilized on nickel sepharose resin and incubated with TACC3 629-838 (TACC3 TD) or TACC3 629-838 Δ699-765 (TACC3 TDΔ). Binding of ch-TOG 1517-1957 in the presence of Affimer was assessed by the addition of ch-TOG to TACC3 TDΔ reactions. ( B ) ELISAs to assess binding between Affimers and TACC3 629-838 Δ699-765. Biotinylated TACC3 629-838 Δ699-765 was immobilized on Streptavidin coated plates and incubated with an Affimer dilution series (orange circles). Background binding of Affimers to the plate was measured by incubating the proteins in wells coated with PBS (gray squares). Data points are the mean ± standard error of the mean from two experiments. ( C ) Woods plots describing differences in deuterium uptake by residue, after 30 minutes of exchange, between TACC3 629-838 Δ699-765 in the absence of binding partner and in the presence of Affimers (as indicated). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides exhibiting no significant difference in exchange between conditions, determined using a 99% confidence interval and a hybrid statistical test (dotted line), are shown in gray. ( D ) Cartoon representation the TACC3 629-838 Δ699-765–ch-TOG complex model with TACC3 colored according to HDX behavior as in (C). The region of TACC3 protected from hydrogen/deuterium exchange in the presence of Affimers E4, E7 and E8 is colored blue, and ch-TOG H5 is colored pink. ( E ) Pull-down assay using cell extracts from asynchronously growing HeLa cells expressing GFP-TACC3 and the indicated mCherry-Affimers, or mCherry as a control. GFP-TACC3 was immunoprecipitated using GFP-trap and a representative Western blot from three experiments is shown. Blots were probed for TACC3, mCherry and ch-TOG, as indicated. ( F ) Quantification of mCherry and ch-TOG bands from pull-down assays. Each dot represents the mean intensity of the indicated protein band normalized to the mCherry condition, colored by experiment. Crossbar indicates the mean from three experiments.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: In Vitro, Incubation, Binding Assay, Residue, Generated, Pull Down Assay, Expressing, Control, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Representative confocal micrographs of metaphase HeLa cells (left) and CRISPR GFP-FKBP-TACC3 HeLa cells (right) expressing the indicated mCherry-Affimers (red). Cells were fixed with PTEMF or ice-cold methanol, as indicated. Where parental HeLa cells were used, an antibody to stain endogenous TACC3 (green) was used. ( B ) Induced relocalization of TACC3 Affimers to mitochondria. Representative confocal micrographs of HeLa cells at metaphase expressing the indicated FKBP-GFP-Affimers (green) with dark-MitoTrap, that were either treated or not with rapamycin (200 nM, 30 min) prior to fixation. Cells were stained for tubulin (not shown in merge) and TACC3 (red). DNA (blue) is shown in the merge. Relocalization of the Affimer to mitochondria can be seen in the rapamycin-treated cells compared to control, but no relocation of TACC3 is observed. ( C ) Quantification of Affimer (x-axis) and TACC3 (y-axis) spindle localization in untreated cells (salmon) and rapamycin treated cells (turquoise). Spindle localization was calculated as the ratio of spindle to cytoplasmic fluorescence shown on a log2 scale, n = 11-22 cells per condition. ( D ) Widefield micrographs of live HeLa cells in metaphase expressing GFP-TACC3 (green) and TACC3 Affimers (red) labeled with mCherry at either the C or N-terminus as indicated. Scale bars, 10 µm.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: CRISPR, Expressing, Staining, Control, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Representative confocal micrographs of untreated or MLN8237-treated (0.3 µM, 40 min) knock-in GFP-FKBP-TACC3 HeLa cells in metaphase. Cells were stained for ch-TOG (red), DNA (blue) and GFP-boost antibody was used to enhance the signal of GFP-FKBP-TACC3 (green). ( B ) Cells expressing mCherry or mCherry-Affimers, as labeled. Scale bars, 10 µm. ( C ) Quantification of spindle recruitment of TACC3 (green) and ch-TOG (red). Each dot represents a single cell, n = 40-45 cells per condition pooled from three independent experiments. Dashed line, no spindle enrichment. Large dot and bars, mean ± one standard deviation. Analysis of variance (ANOVA) followed by Tukey’s post hoc test is shown above each group, using the untransfected and untreated cells (black) and untransfected MLN8237-treated cells (purple) for comparison. ***, p < 0.001; NS, > 0.05.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Knock-In, Staining, Expressing, Labeling, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Representative confocal micrographs of untreated or MLN8237-treated (0.3 µM, 40 min) knock-in CLTA-FKBP-GFP HeLa cells at metaphase. Cells were fixed in PTEMF and stained for TACC3 (red), DNA (blue), and a GFP-boost antibody was used to enhance the signal of CLTA-FKBP-GFP (green). ( B ) Cells expressing the indicated mCherry-Affimers (grey, not shown in merge). Scale bar, 10 µm.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Knock-In, Staining, Expressing