EMBO/SourceData huggingface dataset:
Figure 2 Figure 2. DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK-293T cells were treated with DMSO or okadaic acid (OA), lysed and analyzed by Western blot. B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST-proteins. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry. Low abundance peptides not included. Refer to Table EV1 for a complete peptide list.E HEK-293T cells expressing Flag-DENND2B were treated with DMSO, 250 nM OA or with increasing concentrations of PKD inhibitor CID755673. Cell lysates were analyzed by Western blot.F HEK-293T cells expressing Flag-DENND2B or control cells were treated with 250 nM OA or DMSO, incubated with protein G beads coupled to anti-Flag and analyzed by Western blot.G Lysates from HEK-293T cells expressing Flag-DENND2B and PKD-dominant negative (DN) were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.H HEK-293T cells co-expressing Flag-DENND2B with pcDNA3 or PKD-constitutively active (CA), were incubated with protein G beads or protein G beads coupled to anti-Flag and bound proteins were detected by Western blot.I Quantification of H where PKD-CA treatment is normalized to pcDNA3 control. Mean +/- SD. Welch's t-test *p=0.016, n=6 for both treatment groups from 3 independent experiments.
Figure 3 Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.C Quantification of B. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test **p=0.006 n=8 for both treatment groups from 5 independent experimentsD HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.E Quantification of D. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test *p=0.040. n=5 for both treatment groups from 3 independent experimentsF GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.
Figure 4 Figure 4. DENND2B promotes EGFR recycling A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.  Figure 4. DENND2B promotes EGFR recycling A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.C Transduced MCF10A cells were quantified by real-time PCR. Mean +/- SD, ANOVA with Dunnett post-hoc test ***p=0.000 for both comparisons, n=3 from 3 independent experiments.D Transduced HEK-293T cells expressing Flag-DENND2B were analyzed by Western blot.E Transduced MCF10A cells, were biotinylated and cell lysates were incubated with streptavidin beads. Bound proteins were detected by Western blot.F Quantification of E. where surface EGFR is normalized to total EGFR. Mean +/- SD, Anova with Dunnett post-hoc test *p=0.013 and ***p=0.000. n=6 for all treatment groups from 4 independent experimentsG Quantification of E where total EGFR is normalized to Hsc70. Mean +/- SD n=3 for all treatment groups from 3 independent experimentsI MCF10A cells expressing GFP-DENND2B were labelled for surface EGFR on ice, incubated at 16°C to allow internalization, acid washed to remove remaining labelled-EGFR and processed for immunofluorescence to visualize the internal pool of EGFR (post-endocytosis, top panel) or incubated at 37°C to allow recycling, acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR (post-recycling, bottom panel).J Quantification of I top panel, internal EGFR post-endocytosis. Mean fluorescence per cell +/- SD, n=13 for GFP-DENND2B and n=21 for control cells pooled from 2 independent experiments.K Quantification of I bottom panel, internal EGFR post-recycling. Mean fluorescence per cell +/- SD, n=10 for GFP-DENND2B and n=26 for control cells pooled from 2 independent experiments.
Figure 5 Figure 5. EGF treatment dissociates DENND2B from ITSN A HEK-293T cells were serum starved, treated with 100 ng/mL EGF for the indicated times and cell lysates were analyzed by Western blot. B Quantification of A where P-PKD is normalized to Hsc70. Mean +/- SD. ANOVA with Dunnett post-test *p=0.015 and ** p=0.003 n=3 from 3 independent experiments.C HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.D Quantification of B where P-PKD substrate is normalized to Flag-DENND2B. Mean +/- SD Welch's t-test *p=0.036, n=4 from 3 independent experiments.E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.F Quantification of E where bound Flag-DENND2B is normalized to SM. Mean +/- SD, Welch's t-test *p=0.012, n=6 from 3 independent experiments.
Figure 1(A, B) In situ cryo-electron tomogram displaying the proximal region of a mature mother centriole (A) and procentriole (B). Mature centriole, MC; procentriole, PC; mitochondria, mito; vacuole, vac; white dashed line, lamella edge. Scale bars, 100 nm.(C) Side view z-projection of cartwheels containing the central hub and several CIDs from a mature centriole. Central hub, CH; cartwheel inner densities, CID. Scale bar, 20 nm.(D) Cross section of the cartwheel-containing regions from a mature centriole. Microtubule triplet, MTT; spokes, SP. Scale bar, 200 nm.(E) Side view z-projection of cartwheels containing the central hub and several CIDs from a procentriole. Scale bar, 20 nm.(F) Cross section of the cartwheel-containing regions from a procentriole. Scale bar, 200 nm.(G) Nine-fold symmetrized cross sections of the cartwheel-containing region from a mature centriole (left side) and a procentriole (right side). Dashed white circle, central hub. Scale bars, 100 nm.(H) Longitudinal periodicity measurements of the central hub and CIDs. Central hub, blue; CID, red. Mean values are displayed above the data range. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. Mature centriole, central hub, n = 30, mean = 4.1 +/- 0.67 (S.D); mature centriole, cartwheel inner density, n = 18, mean = 8.7 +/- 1.6 (S.D); procentriole, central hub, n = 10, mean = 4.0 +/- 0.66 (S.D); procentriole, cartwheel inner density, n = 5, mean = 8.6 +/- 1.2 (S.D).(I, J) Nine-fold symmetrized central hub z-projections, starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5.4 nm steps in a mature centriole (I) and a procentriole (J). Red arrow, CID; blue arrow; central hub. Scale bar: 20 nm.
Figure 4(A) Cryo-electron tomogram of P. tetraurelia centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (D, E). Red lines and arrows indicate the position and the direction of the cross-sections made in (B). Scale bar, 100 nm.(B) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in P. tetraurelia. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in C. Scale bar, 60 nm.(C) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of P. tetraurelia along the proximal-distal axis. Each panel corresponds to the above image from panel B. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.(D) Side view showing the transition from pinhead to inner scaffold in P. tetraurelia.(G) Cryo-electron tomogram of C. reinhardtii centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (J, K). Red lines and arrows indicate the position and the direction of the cross-sections made in (H). Scale bar, 100 nm.(H) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in C. reinhardtii. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in I. Scale bar, 60 nm.(I) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of C. reinhardtii along the proximal-distal axis. Each panel corresponds to the above image from panel H. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.(J, K) Side views showing the transition from pinhead to inner scaffold in C. reinhardtii from two consecutive sections from a tomogram.(M, N) Positioning of the different structures along the proximal length from representative P. tetraurelia (M) and C. reinhardtii (N) centrioles. Distance between the ends of the pinhead and cartwheel regions is denoted by zone 1 (for quantification, see Figure EV4E). Distance between end of the pinhead region and start of the inner scaffold region is denoted by zone 2(O) Cartwheel and A-C linker length in C. reinhardtii (n= 5 centrioles), P. tetraurelia (n= 16 centrioles), N. gruberi (n= 11 centrioles), and H. sapiens (n= 5 centrioles). Means and standard deviations of the mean are displayed above the range. A-C linker, turquoise; cartwheel, blue; microtubule triplets, grey.
Figure 1The differentially expressed RBPs during embryoid body (EB) differentiation, neural differentiation and cardiac differentiation, which are classified as different clusters. The right line plots show the dynamic expression pattern of each cluster, gray lines represent the expression of all genes in individual cluster, while the red line represents the average expression level. The number on the graph indicates the number of RBPs in the cluster. The expression value was firstly z-score scaled before plotting. EBD4: embryoid body at day 4; EBD8: embryoid body at day8; NSC: Neural Stem Cells; NPC: Neural Progenitor Cell; MES: mesodermal cells; CP: Cardiac Precursors; CM: Cardiomyocytes.Heatmap showing the expression pattern of the 29 RBPs during EB differentiation.
Figure 2AP staining of 11 RBP-KO ES cell lines maintained under ES cells culture conditions. Scale bar, 200 μm.EB formation from WT and RBP-KO ES cell lines. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; most of RBP-KO ES cell-derived EBs were more compact. Scale bar, 600 μm.Time-course analysis of Oct4 expression and three lineage differentiation markers (Hand1, Brachyury and FGF5) during EB formation. It was quantified by qPCR, the relative expression of genes was calculated by the double delta Ct (ΔΔCt) method, normalized to the expression value at day 0. The relative expression data was represented as the heatmap.AP staining of WT and 11 RBP-KO ES cell lines after LIF withdrawal. Scale bar, 200μm.RBP ranking based on ES cell abilities to self-renewal (determined by colony formation (±LIF) and Oct4 expression) and exit-from-pluripotency (determined by AP staining, EB formation and marker genes expression) in the context of RBP KO. The expression value of Oct4, FGF5, Hand1 and Brachyury was represented as the relative level of these genes compared to their expressions in WT ES cells. The quantitation of other phenotypes, such as colony formation, AP staining and EB formation was manually defined.
Figure 3Immunofluorescence staining of Oct4 and hnRNPLL in WT and hnRNPLL-KO ES cells from two clones (H8 and G6). hnRNPLL was not expressed in either of the hnRNPLL-KO clones. DAPI stained the nuclei. Scale bar, 100 μm.EB formation from WT and the two hnRNPLL-KO ES cell clones. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; While both hnRNPLL-KO ES cell clones-derived EBs were more compact. Scale bar, 200 μm.AP staining of WT and both hnRNPLL-KO ES cell clones after LIF withdrawal. Scale bar, 200 μm.Cumulative distribution showing the relative expression of WT-differentially expressed genes in hnRNPLL-KO ES cells during EB differentiation. Red lines and green lines represent the up- and down-regulated genes during EB differentiation of WT ES cells, while the black line stands for all expressed genes in WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance. The relative expression ratio was log2-transformed before plotting.Heatmap of key pluripotency and differentiation associated gene expression during EB formation from WT and hnRNPLL-KO clones (H8 and G6).Hematoxylin and eosin labeling and Oct4 immunohistochemical staining of teratomas derived from WT and hnRNPLL-KO ES cells (left). Representative Oct4-positive regions are shown. The percentage of Oct4 positive region (%) is plotted (right). The data represent the means ± SD, (n=3, from biological repeats). *P＜0.05; ***P＜0.001, T-test was used to determine the significance. Scale bar, 100 μm.AP staining of WT and hnRNPLL-overexpression ES cell lines maintained under ES cells culture conditions and colony formation assay. Scale bar, 200 μm. The data represent the means ± SD (n=3 biological repeats).Relative expression of Oct4 and Sox2 after hnRNPLL overexpression. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.
Figure 4Birth rate of the different hnRNPLL genotypes, the birth rate of hnRNPLL-/- mice was significantly lower than that of WT hnRNPLL+/+ mice. The data represent the means ± SD. T-test was used to determine the significance.Representative images of hnRNPLL+/+ and hnRNPLL-/- littermates at 3 weeks.Body weight growth curve of mice at different postnatal days. The data represent the means ± SD (n=5-10). ***P＜0.001. T-test was used to determine the significance.Survival curve analysis for hnRNPLL+/+, hnRNPLL+/-and hnRNPLL-/-mice (ten mice in hnRNPLL+/+/+/-group and seven mice in hnRNPLL-/-group).Genotypes and representative images of hnRNPLL+/+ embryo and retarded hnRNPLL-/- embryos at E11.5 which generated from hnRNPLL+/- intercrosses. Scale bar, 1mm.
Figure 5Silver nitrate-stained NuPAGE gel of hnRNPLL interacting proteins. Red star indicate hnRNPLL specific interacting proteins compared to IgG.Mass spectrum identification of the hnRNPLL interactome in ES cells. RNA splicing associated proteins are well enriched by hnRNPLL IP. The enrichment score was log2-transformed before plotting.Western blot validation the interactions between hnRNPLL and the indicated splicing associated proteins.Cumulative distribution of PSI with exon skipping events between WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance.Volcano plot of differential exon skipping events between WT and hnRNPLL-KO ES cells. Indicated AS events were labeled in purple (up-regulated) and green (down-regulated) with gene names.Western blot analysis of hnRNPLL protein during EB formation of WT ES cells at indicated days.Heatmap of the 38 and 25 divergent exon skipping events in Figure 5I. * indicates the different exon skipping events of the same gene.RT-PCR validation of partial divergent exon skipping events shown in Figure 5J. Red boxes represent ESC preferred exons, blue boxes indicate EB preferred exons.
Figure 6RIP-PCR validation of the transcripts that interact with hnRNPLL. The left panel shows the results of western blots verification of hnRNPLL Immunoprecipitation efficiency. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.Purification of hnRNPLL-RNA complex and detection of the RNA targets in the complex by PCR. The left panel shows the membrane position labeled as the yellow square which was cut to extract the hnRNPLL-RNA complex. The bands around 70KD indicate hnRNPLL proteins, the band around 50KD indicate the heavy chain of antibodies used in Immunoprecipitation. The right plot indicates the enrichment of these RNA targets in the hnRNPLL-RNA complex. The data represent the means ± SD (n=3, from biological repeats). *P＜0.05; ***P＜0.001. T-test was used to determine the significance.Detection of Bptf and Tbx3 exon skipping events during EB formation in WT and hnRNPLL-KO ES cells (left) and in hnRNPLL-overexpression ES cells under ES cells culture conditions (right) by RT-PCR.OE, hnRNPLL overexpression.RT-PCR validation of Bptf exon24a and Tbx3 exon2a deletion in WT ES cells.AP staining of Bptf E24a KO or Tbx3 E2a KO ES cells maintained under normal ES cells culture conditions (upper). Scale bar, 200 μm. Colony formation assay of Bptf E24a KO or Tbx3 E2a KO ES cells under ES cells culture condition (lower left). The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. Colony diameter of Tbx3 E2a KO ES cells (lower right). The data represent the means ± SD (n＞80). ***P＜0.001. T-test was used to determine the significance.Relative expression of Oct4 and Rex1, determined by qRT-PCR. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.
Figure 7RT-PCR validation of Bptf and Tbx3 alternative exon deletion, separately in hnRNPLL-KO H8 cells (upper) and simultaneously in hnRNPLL-KO H8 cells (lower).AP staining of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal. hnRNPLL-H8, hnRNPLL-KO ES cells; H8-Bptf KO, alternative Bptf exon was deleted in hnRNPLL-KO H8 cells; H8-Tbx3 KO, alternative Tbx3 exon was deleted in hnRNPLL-KO H8 cells; H8-Bptf/Tbx3 KO, alternative Bptf or Tbx3 exons were simultaneously deleted in hnRNPLL-KO H8 cells. Scale bar, 200 μm.Colony formation assay of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal. The data represent the means± SD (n=3, from biological repeats).Hematoxylin and eosin labeling of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. The blue triangle represent ectoderm, the dark one represent mesoderm and the green one represent endoderm. Scale bar, 100 μm.Oct4 immunohistochemical staining of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. Representative Oct4-positive regions are shown (left). Scale bar, 100 µm. The percentage Oct4-positive region (%) is shown (right). The data represent the means± SD (n=3 biological repeats). **P＜0.01; ns, not significant. T-test was used to determine the significance.
Figure 1(A) Overlay of 15N-1H HSQC spectra of 15N-labeled Ca2+/CaM by itself (red peaks) and after addition of saturating, unlabeled PSD-95 (1-21) (blue peaks) or saturating, unlabeled PSD-95_pT19 (green peaks). All NMR experiments were recorded at 600 MHz 1H frequency.(B) Chemical shift perturbation of CaM amide resonances in the absence vs. presence of PSD-95_pT19. Chemical shift difference (CSD) plotted on the vertical axis was defined as CSD = {(HNA - HNB)2 + (15NA - 15NB)2}1/2 , where "A" and "B" designate free and bound states of CaM. HN and 15N represent amide 1H and 15N chemical shifts, respectively. Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation (see Methods).(C) Representative data from IPAP [15N-1H]-HSQC spectra of 15N-labeled Ca2+/CaM bound to PSD-95_pT19 in the absence (blue) and presence of 17 mg/ml filamentous bacteriophage Pf1 (red). Residual dipolar couplings (RDCs) for backbone HN resonances are calculated (in Hertz) as the difference in splitting for the 15N-{1H} doublet components (red peaks) relative to the isotropic 1JNH coupling (blue peaks). RDC values are calculated to be +40, +30, +20, +20, and -20 Hz for I100, I27, N137, D64, and I130, respectively.(D) Correlation between observed RDCs (panel C) versus RDCs calculated from the final NMR derived structure of Ca2+/CaM bound to PSD-95_pT19. The correlation coefficient (R) equals 0.99 and Q is 0.11.
Figure 2(A) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres.(B) NMR structure of Ca2+/CaM bound to PSD-95 phosphorylated at T19 (PDB accession no. 5J7J). Residues 14-19 of phosphorylated PSD-95 adopt a hook-like structure (highlighted in red) not seen in unphosphorylated PSD-95. This hook-like structure is stabilized by PSD-95 residues E17 and pT19 forming salt bridges with R126 and K115, respectively. Side-chain atoms are depicted by a stick representation for PSD-95 residues Q15, E17 and T19 (red) and CaM residues K115, T117, and R126 (blue).
Figure 3(A) FP titrations showing the binding of wild-type (WT) CaM to PSD-95 (1-21) peptides, either WT (blue), Q15A (salmon), E17R (black), T19E (green), or T19K (yellow). The solid lines represent optimal theoretical curves calculated using a one-site binding model described in Methods. The calculated dissociation constants in each case are given in Table 1.(B) FP titrations showing the binding of R126E CaM to PSD-95 peptides, either WT (blue) or E17R (black).(C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).
Figure 4 Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads. (A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down.(B) Quantification of PSD-95 palmitoylation. Seven samples from three independent sets of cultures were analyzed per condition (**p<0.01; t-test).