EMBO/soda-nel huggingface dataset:
Depressive patient-derived GABA interneurons reveal abnormal neural activity associated with HTR2C (2022)
Figure 1

Figure 1. Differentiation of GINs and abnormal neural morphology and GINs subtypes expression in sMDD GINs.

Scheme illustrating the generation of GINs from CTRL and sMDD patient-derived iPSCs (5 cell lines from 5 CTRLs and 6 cell lines from 5 patients).

Representative images of GINs cultures showing GABA and TUJ1 expression. Scale bar = 100 μm.

The proportion of GABA+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at day 35. (CTRL, n=120 fields were counted from 5 cell lines; sMDD, n=134 fields were counted from 6 cell lines). Mean ratio ± SEM.

Representative images and schematic morphometric Sholl analysis from CTRL and sMDD GINs at day 35. Scale bar = 50 μm.

Sholl intersection number of CTRL and sMDD GINs at day 35 (Each red box is from 5 CTRL cell lines, n=25 independently differentiations from 5 cell lines, each point represents mean value of neurons from five independent experiments, n=226 neurons from CTRL groups. Each blue box is from 6 sMDD cell lines, n=30 differentiations independently from 6 cell lines, each point represents mean value of neurons from five independent experiments, n=260 neurons from sMDD groups). Two-way ANOVA for different distance from soma, ****p＜0.0001 for 10, 30 and 50 μm. The center line shows the median the box shows SEM and the whiskers represent the maximum and minimum.

Representative images of GINs from CTRL and sMDD at day 65. Scale bar = 50 μm.

Quantification of primary branch numbers of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65, n≥11, Nested t-test, **p=0.0016 for CTRL versus sMDD. The center line shows the median. Mean ratio ± SEM.

Quantification of longest neurite length of GINs shown in 5 CTRL cell lines and 6 sMDD cell lines at day 65, n≥11, Nested t-test, **p=0.0075 for CTRL versus sMDD. Mean ratio ± SEM.

Representative images of GINs subtype calretinin (CR) expression. Scale bar = 50μm.

The proportion of CR+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at days 35, 40 and 50, respectively. (Red bar from 5 CTRL cell lines, blue bar from 6 sMDD cell lines). n=5 in each cell line. Two-way ANOVA for timepoints, ns: no significant at day 35, ****p＜0.0001 at days 40 and 50. Mean ratio ± SEM.

Representative images of GINs subtype somatostatin (SST) expression co-staining with mature neuron marker MAP2 in CTRL and sMDD GINs at day 65. Scale bar = 50μm.

The proportion of SST+ neurons of MAP2+ cells from CTRL and sMDD iPSCs derived GINs at day 65. n=43 in CTRL groups and n=37 in sMDD groups. T-test, ****p＜0.0001. Mean ratio ± SEM.
Depressive patient-derived GABA interneurons reveal abnormal neural activity associated with HTR2C (2022)
Figure 3

Figure 3. Defects in calcium signaling both in sMDD GINs and ventral forebrain organoids.

Schematic diagram of calcium imaging for neurons with Fluo-4 indicator dyes.

Representative images of calcium imaging in CTRL and sMDD GINs at each timepoint. Scale bar = 20 μm.

The representative trajectory of average intensity changes over time from CTRL (red line) and sMDD (blue line) neurons in response to 67 mM KCL. CTRL, n=6 neurons; sMDD, n=6 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Mean ratio ± SEM.

Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=155 neurons derived from 5 CTRL cell lines, n=198 neurons derived from 6 sMDD cell lines). Nested t-test, **p=0.0022 for CTRL versus sMDD. Mean ratio ± SEM.

Schematic illustrating the generation of human ventral forebrain organoids from hiPS cells.

Immunostaining for the GABA interneuron marker GAD67, neural progenitor marker SOX2 and ventral prosencephalic progenitor marker NKX2.1 at day 30 in CTRL and sMDD ventral forebrain organoids. Scale bar = 50 μm.

Schematic diagram of calcium imaging for ventral forebrain organoids with Fluo-4 indicator dyes.

Representative images of calcium imaging in CTRL and sMDD ventral forebrain organoids at different states. Scale bar = 20 μm.

Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=156 neurons derived from 4 CTRL cell lines, n=88 neurons derived from 4 sMDD cell lines). Nested t-test, ***p=0.0005 for CTRL versus sMDD. Mean ratio ± SEM.
Depressive patient-derived GABA interneurons reveal abnormal neural activity associated with HTR2C (2022)
Figure 5

Figure 5. Overexpression of HTR2C and Trzd partially reverses defects of sMDD GINs in single-cell transcriptome level.

Schematic diagram of injecting overexpression virus into ventral forebrain organoids followed by neurons adherent.

Calcium imaging was performed on GFP+ neurons in the overexpression group. The representative image shows the rest state of GFP+ cells and the state of cells after KCl stimulation. The pseudo-color showed Rhod-4 calcium fluorescent dye, and the selected cells are GFP+ neurons. Scale bar=20 μm.

The representative trajectory of average intensity changes over time in Vehicle virus and OE virus groups. CTRL, n=10 neurons; sMDD, n=10 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Mean ratio ± SEM.

Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per virus group. n=166 for Vehicle virus, n=81 for OE virus, respectively from 3 batches. T-test, **p=0.0012. Mean ratio ± SEM.

Representative images of calcium imaging in CTRL (from RC01001-C) and sMDD (from SA004) GINs at resting and stimulus state with different treatments. Scale bar = 20 μm.

Quantification of peak [Ca2+] (Fmax-F0)/F0 shown with different treatments including BDNF, Trzd, PNU, Ro at concentrations of 1 μM and 10 μM. T-test, **p=0.0042 for sMDD+BDNF versus sMDD; one-way ANOVA, ****p＜0.0001 for sMDD+10 μM Trzd versus sMDD. n≥7 neurons from each group. Mean ratio ± SEM.

Schematic diagram of single cell sequencing from CTRL, sMDD and sMDD+10 μM Trzd GINs followed by assaying.

Gene set enrichment analysis (GSEA) results indicating altered GO terms after Trzd treatment (adjusted p-value < 0.05). Upregulated terms are colored with red while downregulated terms are colored with blue.

Dot plots showing the expression level and percentage of genes selected from functional gene sets across CTRL, sMDD and sMDD with Trzd GINs clusters.

Top: Violin plots showing the expression level of CAMK2N1 and CAMK2N2 in three groups. Bottom: Scatter plot showing the correlations of foldchanges according to the expression of genes encoding CaMK family proteins between CTRL GINs and sMDD GINs and between sMDD with Trzd GINs and sMDD GINs. A linear regression line is added to the plot based on these two variables. sMDD+Trzd vs. sMDD, n=14 genes; CTRL vs. sMDD, n=14 genes.
CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization (2018)
Figure 1

Figure 1: M18BP1-1161-580 is sufficient for centromere localization in metaphase egg extract

A) Representative immunofluorescence images showing localization of Myc-tagged M18BP1-1 truncations schematized in (B) to metaphase sperm centromeres in M18BP1-depleted extract. Truncations analyzed are indicated on the left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X.

B) Schematic of M18BP1-1 truncations used to define the metaphase targeting domain in (A) and (C).

C) Quantification of the average Myc intensity at centromeres from (A). Values are normalized to centromeric signal of full-length M18BP1-1. Graph shows the mean ± SEM of at least three experiments. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.
CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization (2018)
Figure 2

Figure 2: Mutation of the conserved SANTA domain disrupts M18BP1-1 binding to CENP-C and metaphase centromere localization

A) Alignment of SANTA domain among select eukaryotes. Red circles indicate conserved hydrophobic residues predicted to play a role in protein-protein interactions that were mutated in this study (Zhang, et al. 2006). Darker shades of blue represent increased conservation of amino acids in alignment of ~300 M18BP1 homologues. Secondary structure prediction was reported by Zhang, et al. (2006).

B) Representative Western blot showing disruption of the interaction between M18BP1-1 and CENP-C by mutation of the SANTA domain by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Flag-M18BP1-1WT or Flag-M18BP1-1SANTA. The M18BP1-1 species added to each reaction and the cell cycle state of the extract is indicated above. The immunoblotted species is indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.

C) Representative images showing that mutation of the SANTA domain in M18BP1-1 causes decreased localization to sperm centromeres. Metaphase extracts were depleted of endogenous M18BP1 and either mock-depleted or depleted of CENP-C. The extracts were then complemented with WT or SANTA mutant M18BP1-1. M18BP1-1 species and depletion conditions are indicated at left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X.

D) Quantification of the average Flag-M18BP1-1 intensity at centromeres from (C). Values are normalized to centromeric signal of M18BP1-1WT in mock-depleted extract. Graph shows the mean ± SEM of three experiments. Significance determined by Welch's unpaired two-tailed t-test, ** p<0.005.
CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization (2018)
Figure 3

Figure 3: M18BP1-1161-580 recapitulates metaphase-specific binding to CENP-C

A) A representative autoradiograph from a GST-pulldown of CENP-C to map the CENP-C binding domain of M18BP1-1. Radiolabeled Myc-M18BP1-1 truncations (amino acids indicated at the top) were mixed with recombinant GST-CENP-C1191-1400. Material bound to glutathione agarose was resolved by SDS-PAGE and visualized by autoradiography (see also Figure EV 2).

B) Schematic showing the cognate binding domains on M18BP1-1 (top) and CENP-C (bottom), indicated by the black boxes, relative to other functional domains.

C) Quantification of (A). Bound material as a fraction of the input was calculated from autoradiographs. The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1-1161-580, the CENP-C binding domain.

D) Representative Western blot showing metaphase-specific binding of M18BP1-1161-580 to CENP-C by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Myc-M18BP1-1 or Myc-M18BP1-1161-580. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated above. Immunoblotted species indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.

E) Representative immunofluorescence images showing Myc-M18BP1-1161-580 localization at sperm centromeres in extract depleted of endogenous M18BP1. In addition, extract was mock-depleted or immunodepleted of CENP-C (indicated at left). Cell cycle state indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets magnified 3X.

F) Quantification of (E). Graph shows mean Myc-M18BP1-1161-580 intensity at centromeres ± SEM of two independent experiments normalized to the mock-depleted, metaphase condition.
CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization (2018)
Figure 4

Figure 4: Identification of metaphase phosphorylation sites in M18BP1-1 by mass spectrometry

A, B) Metaphase binding of M18BP1-1 to CENP-C requires phosphorylation. Either full-length Myc-M18BP1-1 (A) or Myc-M18BP1-1161-580 (B) was immunoprecipitated from metaphase extract. Half of the immunoprecipitate was treated with λ-protein phosphatase (+ LPPase) to assess whether binding to CENP-C required phosphorylation. Immunoprecipitation with an equivalent amount of mouse IgG served as a negative control. Immunoblotted species is indicated at left.

C) Cdk inhibition with flavopiridol prevents M18BP1-1161-580 localization at metaphase centromeres. Representative images showing Myc-M18BP1-1161-580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors. Inhibitor treatment is indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X. See Figure EV 3 for demonstration of inhibitor efficacy.

D) Schematic of phosphorylation sites in M18BP1-1161-580 mutated for this study. M18BP1-1 residue numbers indicated above. Positions of phosphorylation sites are indicated by orange lines and labels below. Bold indicates consensus Cdk phosphorylation sites.

E) Quantification of immunofluorescence experiments examining localization of M18BP1-1161-580 phosphorylation site mutants to sperm centromeres in metaphase extract depleted of endogenous M18BP1. Graph shows mean centromere intensity ± SEM of three independent experiments normalized to the WT condition. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005. See also Figure EV 4B.
CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization (2018)
Figure 5

Figure 5: Phosphorylation of T166 in M18BP1 regulates metaphase localization and binding to CENP-C

A) Representative immunofluorescence images showing M18BP1-1 mutant localization. M18BP1-depleted metaphase extract was supplemented with in vitro translated full-length Flag-M18BP1-1 mutants and sperm chromatin. In addition, extract was either mock-depleted or CENP-C depleted to assess CENP-C dependent metaphase localization. M18BP1-1 species and CENP-C depletion status are indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.

B) Representative Western blot showing co-immunoprecipitation of CENP-C with phosphorylation site mutants of M18BP1-1. M18BP1-depleted metaphase or interphase extract was supplemented with in vitro translated Flag-M18BP1-1 mutants. M18BP1-1 mutation and cell cycle state indicated above, immunoblotted species indicated at right. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.

C) Quantification of (A). Flag-M18BP1-1 mutant centromere intensity normalized to WT localization in the mock-depleted condition. Error bars represent SEM from three independent experiments. Significance determined by Welch's unpaired two-tailed t-test, ** p<0.005.

D) Model showing phosphorylation-dependent binding of Xenopus M18BP1 to CENP-C during mitosis (top) and the transition to CENP-A-dependent binding in interphase (bottom).
CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization (2018)
Figure 6

Figure 6: Metaphase targeting mutants of M18BP1-1 do not support robust CENP-A assembly

A) Representative immunofluorescence images showing incorporation of exogenous Myc-CENP-A at centromeres. Sperm nuclei were incubated in M18BP1-depleted interphase Xenopus egg extracts complemented with the indicated M18BP1-1 protein. Extracts were supplemented with RNA encoding Myc-CENP-A to track new CENP-A assembly and in vitro translated HJURP. Scale bar, 10 μm. Insets are magnified 3X.

B) Quantification of Myc-CENP-A loading at Xenopus sperm centromeres in (A). Values are normalized to the centromere signals in mock-depleted extract. Dashed lines indicate the Myc-CENP-A assembly signal observed upon M18BP1 depletion (bottom) and Flag-M18BP1-1WT add-back (top) as points of reference for mutant rescue. Graph shows the mean ± SEM of four independent experiments. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.

C) Interphase Mis18 complex formation is unaffected by M18BP1-1 mutations that prevent binding to CENP-C. Extract depleted of endogenous M18BP1 was supplemented with Myc-Mis18α, Myc-Mis18β, and Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated at the top. Co-immunoprecipitation of Myc-Mis18α/β was assessed by α-Myc immunoblot following Flag precipitation.

D) HJURP association with M18BP1-1 is unaffected by mutations that prevent CENP-C binding. Extract depleted of endogenous M18BP1 was supplemented with Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract are indicated at the top. Coimmunoprecipitation of Flag-M18BP1-1 was assessed by α-Flag immunoblot following HJURP precipitation.
CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization (2018)
Figure 7

Figure 7: Metaphase targeting mutants of M18BP1-1 inhibit nuclear localization during interphase

A) Representative images showing G1 localization of human M18BP1WT or human M18BP1SANTA in sfGFP-AID-M18BP1 DLD1 cells treated with 1 mM IAA for 24 h to remove endogenous M18BP1. Centromeric localization is indicated by localization with ACA (α-centromere autoantibody serum), early G1 cell cycle state is indicated by midbody staining in the tubulin channel. M18BP1 species indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X.

B) Representative immunofluorescence images showing localization of full-length Flag-M18BP1-1 (mutant species indicated at left) without (left panels) and with (right panels) fusion to an SV40 NLS to sperm centromeres in interphase extract depleted of endogenous M18BP1. Immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X. Quantification of immunofluorescence intensity at centromeres as total integrated fluorescence signal normalized to wild type levels. The values shown on each panel are represented as a percentage of wild-type Flag-M18BP1-1 ± SEM from three independent experiments.

C) Quantification of Myc-CENP-A assembly in M18BP1-depleted extract complemented with the indicated M18BP1-1 species. Graph shows mean immunofluorescence intensity at centromeres normalized to reactions complemented with WT M18BP1-1. Error bars show SEM of at least three independent experiments. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.

D) Representative Western blot of CENP-A assembly reactions in (B). Efficient M18BP1 depletion is indicated by comparing lanes 1 and 10. Add-back of wild-type or mutant M18BP1-1 is near endogenous levels.

E) Model showing that metaphase localization of M18BP1 (green) promotes M18BP1 retention on chromosomes during nuclear envelope formation to promote centromeric localization during interphase.

Expanded View Figure Legends
Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling (2020)
Figure 2

Figure 2 - Homozygosity and compound heterozygosity for null mutations in GRK2 in ATD.

(A) Sanger sequence trace showing homozygosity for the c.469C>T mutation, predicting a stop codon in exon 6 of GRK2 (arrow).

(B) Exome analysis showing the biallelic changes c. 1348_1349del and c.555 +1G>A. Red arrow points to the place of mutation. Orange and green boxes indicate the reference nucleotide and its substitution, respectively.

(C) Domain composition of GRK2 and the corresponding location of the mutations (RGS, regulator of G protein signaling domain; PH, pleckstrin homology domain).

(D) Expression of GRK2 as measured by RT-PCR in three replicates each of control and R05-365A fibroblasts, demonstrating absence of GRK2 transcript in ATD cells. GAPDH served as a positive control.

(E) Two control and ATD fibroblast samples were immunoblotted for GRK2. Note the absence of GRK2 protein in the ATD cells. Actin served as a loading control. The data shown are representative of three independent experiments.
Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling (2020)
Figure 3

Figure 3 - GRK2 is essential for skeletal growth plate development.

(A) GRK2 immunohistochemistry in a control 17-week gestational age distal femur growth plate demonstrating widespread expression, particularly in the bone (B), proliferating zone of the growth plate (P), and the perichondrium (arrowheads). Non-reactive antibody was used as a control (Ab control). R, reserve zone. H, hypertrophic zone. Scale bars, 100 μm.

(B) Picrosirius red staining of the femoral growth plates of a control and the ATD R05-365A fetus. The control growth plate shows normal architecture with clearly distinguishable reserve (R), proliferating (P) and hypertrophic (H) zones. A profound disruption of growth plate architecture is obvious in the GRK2-/- growth plate, which shows irregularly-shaped reserve zone chondrocytes, a lack of discernable proliferating chondrocytes and a significantly under-developed hypertrophic zone. Scale bars, 100 μm.

(C) Shortened long bones in the chicken wings injected with CMPD101. The right limb bud was injected with CMPD101, the left one was left as a control. Note the shortened humerus (H), ulna (U) and radius (R) in the CMPD101-injected wing. Mean ± SEM. Mann-Whitney U test; number of animals is indicated. Scale bar, 150 μm.

(D) Alcian blue staining of the femoral growth plates of a control and the ATD R05-365A fetus. Note the significantly lesser blue staining of the R05-365A growth plate stained by alcian blue pH 1.0, suggesting impaired sulfation of the extracellular matrix. Scale bars, 200 μm.

(E) Alcian blue staining of the chicken limb bud micromasses treated with GRK2 inhibitor CMPD101. Note the gradual inhibition of the cartilage nodules with increasing concentration of CMPD101, quantified below. Mean ± SEM. Mann-Whitney U test; number of micromasses is indicated. A representative experiment of four (pH 2.5) and three (pH 1.0) is shown. Scale bar, 100 μm.

(F) Alcian blue staining of the micromasses generated from Grk2+/+ and Grk2-/- NIH3T3 cells. Note the mildly weaker staining by alcian blue pH 2.5 and the nearly absent sulfation stained by alcian blue pH 1.0 in the Grk2-/- micromasses. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of micromasses is indicated. Scale bar, 1 mm.