EMBO/SourceData huggingface dataset:
Figure 1Photograph 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.
Figure 2A-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.
Figure 3Transfected 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 patientFMRP.Transfected HEK293 cells expressing a modified version of GFP-tagged patientFMRP (GFP-FMR1G-ins. [NLSmutated]). The novel amino acid sequence in the C-terminus of patientFMRP [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 patientFMRP, strongly suggesting that the novel amino acid sequence present in the patientFMRP C-terminus is a functional nuclear localization signal (NLS).
Figure 4Transfected 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.
Figure 5The 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).
Figure 6Overexpression 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).
Figure 7A-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.
Figure 3LC-MS-MS analysis of in-gel-digested HEK293 cells transfected with hNICD-GFP, and subjected to immunoprecipitation using GFP antibody, identified multiple phosphorylation sites in NICD, highlighted in green.Phosphorylation of serine 2513, but not serine 2516, appears to be essential for the NICD-FBXW7 interaction. hNICD-GFP phospho-mutant peptides encoding non-phosphorylatable residues at S2513 and/or 2516 (serine to alanine) were expressed in HEK293 cells. The exogenously expressed protein was subsequently immunoprecipitated with anti-GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody. Wild-type hNICD-GFP and GFP only vectors were included as positive and negative controls, respectively. Western blot using GFP antibody served as immunoprecipitation efficiency control. β-Actin has been used as loading control for the input lanes.
Figure 4HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK2 (+) followed by western blot for NICD, CDK2, CDK1, CDK4 and CDK8. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01.   HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK1 (+) followed by western blot for NICD, CDK1, CDK2, CDK4 and CDK8. β-Actin has been used as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.   Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here. At both 1 and 10µM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1µM) inhibits more than 94% of CDK2 activity. At both 1 and 10 µM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor DatabaseHEK293 cells were treated with three highly selective CDK2 inhibitors (10 µM of Roscovitine, 0.1µM of Purvalanol B and 10µM of GSK650394A) for 3 hours. Endogenous levels of NICD were detected by western blot. β-Actin served as loading control.   Quantification of the density of western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed, with *p≤0.05, **p≤0.01, and ns= not significant.   HEK293 cells were treated with RO-3306, a specific CDK1 inhibitor, for 3 hours at 10 µM. Levels of NICD, FBXW7, phospho-Histone H3 and Histone H3 were detected by western blot. β-Actin was used as loading control.   Quantification of the density of western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.
Figure 5Cell cycle profile for HEK293 cells released from synchronization after double thymidine block. Cells were released and harvested at the indicated time points (AS= asynchronous). Analysis of cell cycle arrest and release was performed using propidium iodide (PI) staining and flow cytometry. A representative experiment of three performed is shown.Expression of the indicated proteins in HEK293 cells was examined by western blotting, and β-Actin was used as loading control. This summary is a representation of three independent experiments.Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01 (ns=not significant).Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05 and **p≤0.01.
Figure 6iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU (marked in red within the NICD panel). EdU incorporation occurs during S phase (marked by yellow in the NICD panel). Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase. M phase cells are distinguishable by their chromatin condensation, observable in the DAPI panel (dividing cells are indicated with white in the NICD panel). G1 cells do not have any of the features described above (blue in NICD panel). Scale bars are 20 µm.G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells (p≤0.001). All groups were compared against each other with a one-way ANOVA analysis, followed by Turkey test. M phase cells have the lowest NICD. Each point in the box-plot represents the normalized intensity of a single cell. Data from three different experiments (n=3) was randomised collated and plotted.
Figure 7Purvalanol B treatment reduced the NICD-FBXW7 interaction. 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0.1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin has been used as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.   E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of Purvalanol B (1 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin was used as loading control.E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of RO-3306 (10 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin has been used as loading control.Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the "-" explant. n=18. Scale bar is 100 µm.Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 10 µM of RO-3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. RO-3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the "-" explant. n=10. Scale bar is 100 µm.The mean log2 fold change in successive somite length at position +1 (last formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.0437 for RO-3006 and p= 0.0524 for PurB, compared to DMSO. n=14.   The mean log2 fold change in successive somite length at position +2 (penultimate formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.6409 for RO-3006 and p= 0.4065 for PurB, compared to DMSO. n=14.
Figure 1(A) Abnormal distribution of Cux1-positive neurons in CAMDI-KO mice. Expression of Cux1 in the somatosensory cortex was compared between P2 wild-type (WT) and CAMDI-KO (KO) mice. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT = 788 cells, KO = 2,139 cells). *, p<0.05, **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.(C) Delay in neuronal migration by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Scale bar, 100 μm.(D) Quantification of the number of EGFP-positive neurons. Note the abnormal distribution of neurons in lower cortical layers of CAMDI-KO mice. n = 3 for WT mice (546 cells), n = 4 for KO mice (680 cells). *, p<0.05, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.(E) Aberrant axonal projection and terminal arbors by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Representative pictures show an aberrant axonal projection of corticostriatal neurons in CAMDI-KO mice. Sections were stained with Hoechst 33258 and anti-EGFP antibody. Scale bar, 1 mm.(F) Quantification of the striatal intensity. n = 3 mice/genotype. ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.
Figure 2(A, B) Representative traces from adult control (WT) and CAMDI-KO (KO) in open field test. Locomotor activity (A) and distance traveled (B) for 15 min. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.53). Data are presented as mean ± SEM.(C) Repetitive jumping behavior in open field test. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.09). Data are presented as mean ± SEM.(D) Three-chamber social interaction test. The time spent sniffing with stranger mouse. n = 19 for WT mice, n = 15 for KO mice. *, p<0.05, ***, p<0.001,Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 63)=5.79, main effect of chamber F(1, 63)=90.47, interaction F(1, 63)=5.29). Data are presented as mean ± SEM.(E) Resident-intruder test (social sniffing). n = 7 for WT mice, n = 8 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.15). Data are presented as mean ± SEM.(F) Resident-intruder test (digging). n = 7 for WT mice, n = 8 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 10.97). Data are presented as mean ± SEM.(G) Marble burying test. n = 9 for WT mice, n = 25 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 32) = 5.39). Data are presented as mean ± SEM.(H) Forced swimming test. n = 10 for WT mice, n = 13 for KO mice. **, p<0.01, ***, p<0.001 Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 42)=84.30, main effect of session F(1, 42)=33.88, interaction F(1, 42)=4.92). Data are presented as mean ± SEM.(I, J) Light-dark test. Time in light chamber (I) and Latency to light chamber (J). n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test, ((I) F(1, 13) = 8.75, (J) F(1, 13)=9.73). Data are presented as mean ± SEM.(K) Hot plate test.. n = 8 for WT mice, n = 12 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 18) = 17.51). Data are presented as mean ± SEM.
Figure 3(A) Co-expressed FLAG-CAMDI and HDAC6-HA interacted with each other in SH-SY5Y cells. n=3 independent experiments.(B) Endogenous interaction of CAMDI with HDAC6 in the mousebrain. Co-IP assay was performed with the indicated antibodies using E16 mousebrain. n=3 independent experiments.(C, D) N-terminal region of CAMDI recognizes the second HDAC domain of HDAC6. SH-SY5Y cells were co-transfected with HDAC6-HA and each FLAG-CAMDI fragment (C) (fragment 1, 1-250; 2, 251-500; 3, 501-750; 4, 751-1000; 5, 1001-1451 amino acids) or co-transfected with FLAG-CAMDI and each HDAC6-HA fragment (D) (fragment 1 (DD1), 1-440; 2 (DD2), 441-840; 3, 841-1215 amino acids). Co-IP assay was performed with the indicated antibodies. n=3 independent experiments. **, p<0.01, ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.
Figure 4(A) CAMDI and CAMDI Fragment 1 overexpression enhanced α-tubulin acetylation. HeLa cells overexpressing FLAG-CAMDI and FLAG-CAMDI frag.1 were immunostained with anti-FLAG or anti-Ac-tubulin antibodies. Ratio line-scans along the line (from x- to y-axis) from images. Scale bar, 10 μm.(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (B) Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and Ac-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (C) CAMDI N-terminal domain alone increased Ac-tubulin level. n=3 independent experiments. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.(D) Deletion of CAMDI N-terminal domain (1-250 amino acids) failed to inhibit HDAC6. n = 3 independent experiments. N.S., not significant. Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.
Figure 5(A) Effect of HDAC6 over-expression or CAMDI co-expression on the level of acetylated α-tubulin in the centrosome. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-acetylated α-tubulin antibodies (red). Scale bar, 1 μm.(B) Quantification of intensity of acetylated α-tubulin at centrosome. n = 25, 30, 30 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.(C) Effects of HDAC6 overexpression or CAMDI co-expression on the centrosomal localization of γ-tubulin. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). DNA was stained with Hoechst 33258. Scale bar, 1 μm.(D) Quantification of intensity of γ-tubulin at centrosome. n = 39, 16, 17 cells. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.(E) CAMDI stabilizes centrosomal γ-tubulin through HDAC6 inhibition. Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.(F) Effects of CAMDI knockdown on the centrosomal localization of γ-tubulin in vivo.Coronal sections through the somatosensory cortex of E17.5 were analyzed following in utero electroporation of EGFP and CAMDI-sh plasmid at E14.5. The sections were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). Representative images of EGFP at Cortical Plateneuron expressing CAMDI-sh stained for endogenous γ-tubulin and with Hoechst (blue). Z-stack number 6 indicates centrosome of EGFP-positive CAMDI-sh-expressing neuron. Z-stack number 19 indicates centrosome of a nearby non-electroporated neuron. Quantification of γ-tubulin intensity. EGFP (-) = 12 cells, EGFP (+) (CAMDI-sh) = 12 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM. Scale bar, 5 μm.(G) Reduced γ-tubulin in the centrosome fraction of CAMDI-KO brain. The centrosomal fraction was isolated from brain lysates of WT and CAMDI-KO mice and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.