EMBO/SourceData huggingface dataset:
Figure 6C. Representative PLA images of BRCA2-PALB2 PLA experiments showing more PLA events (white) in HeLa cells silenced for DPP9 (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.D. Quantification of PLAs experiments showing more BRCA2-PALB2 PLA events in cells silenced for DPP9 (siDPP9) in comparison to non-targeting controls (siNT). Each dot represents the number of PLA events in a single cell, from four biological replicates. The technical control samples (NgtCntrl) omitted the BRCA2 antibody. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).E. Representative PLA images of γH2Ax-BRCA2 PLA experiment showing a reduction in the number of PLA events (white) between γH2Ax and BRCA2 in HeLa DPP9 KD cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-γH2Ax: RRID:AB_2118009, anti-BRCA2: RRID:AB_2259370 F. Quantification of PLAs showing fewer MMC-induced γH2Ax-BRCA2 PLA events in HeLa DPP9 KD cells, in comparison to HeLa WT cells. Each dot represents the number of PLA events in a single cell, from three biological replicates. The technical control samples (NgtCntrl) omitted the γH2Ax antibody. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).
Figure 7A. Representative immunofluorescence images showing that re-expression of DPP9-SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9-SWT cells. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428. B. Graph showing the number of RAD51 foci following induction of DPP9-SWT expression, compared to uninduced HEK293 DPP9 KO + DPP9WT cells (-Dox). Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9-SS729A did not result in more RAD51 foci. Each dot represents the number of RAD51 foci in a single cell, from three biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).Representative images (C, and summarizing graph (D showing the number of RAD51 foci per nucleus in HeLa WT cells (C, D) Where stated, cells were treated with control siRNA (siNT) or silenced for BRCA2, and transiently transfected with the BRCA21‑3418 or BRCA23-3418 constructs. Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells (C, D). Each dot represents the number of RAD51 foci in a single cell, from three (D) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; ; anti-BRCA2: RRID:AB_2259370.Representative images , E) and summarizing graph F) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells (E, F). In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2∆MP3-3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21-3418 (E, F). Each dot represents the number of RAD51 foci in a single cell, from four (F) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; anti-BRCA2: RRID:AB_2259370.
Figure 1 B, C CDKL51-352 autophosphorylation is shown. WT CDKL5 uses ATPγS, KD is inactive, F89A uses benzyl-ATPγS with reduced efficiency, F89A/ C152A mutant has rescued activity. Quantification of Thio-P signal is normalized for CDKL5 levels (HA) and KD background is subtracted. Fisher's LSD: n = 4 replicates. bn = benzyl  E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5. 50 ng (40 nM) AS-CDKL5 phosphorylates 150 ng (50 nM) MAP1S very rapidly (E, F) Quantification of phosphorylated MAP1S is normalized to maximum intensity. n = 2 replicates E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5 In 30 minutes of incubation, 150 ng MAP1S is phosphorylated by small amounts of CDKL5 (G, H). Quantification of phosphorylated MAP1S is normalized to maximum intensity. n = 2 replicates I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutants ARHGEF2 S122A (I Substrate levels are shown by coommassie staining underneath I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant EB2 S222A (J Substrate levels are shown by coommassie staining underneath I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant MAP1S S786/812A (K). Substrate levels are shown by coommassie staining underneath. MAP1S phosphorylation is quantified in (L). Dunnett's multiple comparison: n = 3 replicates
Figure 2 A, B In vivo substrate phosphorylations are assessed by Western blotting of 5 WT (CDKL5 +/Y) and 5 CDKL5 KO (CDKL5 -/Y) mouse cortical lysates using phosphospecific antibodies. EB2 pS222 and MAP1S pS812 are dramatically reduced in KOs. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 5 animals per genotype C, D Developmental expression of CDKL5 and levels of its substrate phosphorylations in P4 - P50 cortex. CDKL5-dependent substrate phosphorylation is quantified by subtracting KO from WT levels for each age. n = 3 animals per age per genotype  E, F Expression of CDKL5 and levels of substrate phosphorylation in P8 Nex-Cre conditional KO cortex. CDKL5 protein expression is almost completely lost. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 3 animals per genotype  G CDKL5 WT and CDKL5 KO neurons in culture are co-stained with neuronal dendrite marker MAP2, EB2 and EB2 pS222. EB2 pS222 is lost in CDKL5 KO dendrites. Scale bar is 20 μm
Figure 3 A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations. 100 µm AP5 was used to block NMDAR activity during KCl treatment. Quantification of phosphorylated EB2 (B) and total CDKL5 (C) are normalized to 0 minutes time point. EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes. CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates D, E Protein lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations. Quantification of phosphorylated EB2 and total CDKL5 is normalized to 0 minutes time point. EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates
Figure 4 A Representative kymographs of EB3-tdTomato in cortical neuron dendrites at DIV14  B - D In CDKL5 KOs (CDKL5 -/Y), comet distance (B) and lifetime (C) is increased when compared to WT (CDKL5 +/Y). Comet velocity (D) is unchanged. Student's t-test: n = 16/615 and 15/381 neurons/comets in WT and KO, respectively  E Representative kymographs of CDKL5 WT and KO neurons transfected with control shRNA and MAP1S shRNA  F Comet lifetime is higher in control shRNA expressing KOs than WTs. MAP1S shRNA expression reduces comet lifetime in KO neurons. EB2 shRNA-mediated knockdown does not alter microtubule dynamics in WT or KO. Tukey's multiple comparison: n = 12-15 neurons, 388-741 comets per condition
Figure 5 A, B Representative stills (A) and kymographs (B) of TrkB-RFP overexpressed in dendrites of WT and CDKL5 KO DIV14 cortical neurons. Scale bar is 5 μm. C, D No significant change is observed in the ratio of dynamic vs stationary vesicles (C) or anterograde vs retrograde runs (D). S = stationary, D = dynamic, A = anterograde, R = retrograde. Vesicles with a net distance > 5 μm were considered dynamic. E, F Both anterograde and retrograde runs show a trend towards reduced velocity in CDKL5 KO neurons (E). Anterograde run lengths are significantly reduced in KO compared to WT neurons (F)
Figure 6 B COS-7 cells expressing HA-MAP1S LC, Flag-CDKL5 WT / kinase dead (KD) is shown with endogenous α-tubulin staining. MAP1S does not localize to microtubules when phosphorylated by CDKL5. Scale bar is 20 μm  C, D The percentage of cells where α-tubulin and MAP1S LC are colocalised (C) and the quantification of MAP1S LC and α-tubulin colocalisation by Pearson correlation coefficient (D). Tukey's multiple comparison: n = 22-24 cells per condition  E, F Taxol-stabilised microtubule co-sedimentation assay shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5. Quantification of soluble MAP1S in the supernatant (s/n) fraction is normalized to the MT-bound pellet fraction. 3:1 MT:MAP1S LC ratio used here. Tukey's multiple comparison: n = 3 technical repeats  G HA-MAP1S LC is overexpressed and stained with anti-HA antibody in cultured cortical neurons (red). Numerous loops at the dendrites' tips are observed (arrowheads). Scale bar is 5 μm
Figure 7 A, B Western blots for total EB2, pS223 and tubulin using protein lysates obtained from neurons derived from three patients with CDD and their related control. 1 = p.D135_F154del, male; 2 = p.R59X, male; 3 = p.R59X, female. Quantification of EB2 phosphorylation is normalized for total protein level. Student's t-test: n = 3 patients/controls. * p < 0.05, error bars are SEM  C Representative images of human iPSC-derived neurons co-stained with the neuronal dendrite marker MAP2, EB2 and EB2 pS223. EB2 phosphorylation is present in dendrites of control neurons but not in CDD neurons. Enlarged images of the boxed areas are shown on the right panel. Scale bar is 20 μm
Figure 1A. Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real-time RT-PCR. The results are shown as mean SEM of n=3 independent assays. The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp (TATA binding protein). The insert shows the expression of Lrig1 in embryonic E17.5 rat hippocampus examined by RT-PCR. Control sample without reverse transcriptase (-RT) is also shown.B. Localization of Lrig1 (red) in coronal sections from P15 ratbrain by immunofluorescence. Anti-Lrig1ECD antibodies label dentate gyrus cells, CA1-CA3 hippocampalneurons and pyramidal corticalneurons (*). Scale bar, 400 m.C-E. Confocal image of cortical (C) and hippocampal (D-E) pyramidal neurons stained with rabbit anti-Lrig1ECD antibody. Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1-CA3hippocampal and pyramidal cortical neurons (layer V). Scale bars, 20 m.F. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the neuronal marker ßIII tubulin (green) in dissociated hippocampal cells cultured for 7 DIV. Yellow indicates neuronal expression of Lrig1. Scale bar, 15 m.G. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the astrocytic marker S100 (green) in dissociated hippocampal cells cultured for 7 DIV. Scale bar, 20 m.H. Localization of Lrig1 (red) with anti-Lrig1ECD antibody and the somatodendritic marker MAP-2 (blue) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV. Scale bar, 20 m.
Figure 2A. Representative images of mouse hippocampal neurons transfected with either GFP-expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Scale bar, 15 m. Boxed area represents a higher-magnification image showing the profuse proximal dendritic arborization of Lrig1-shRNA transfected neurons.B. Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Data are shown as mean SEM of n=3 independent experiments. *p<0.05 and **p<0.01 by two-way ANOVA followed by Bonferroni multiple comparisons test.C-G. Quantification of primary (C), and secondary (D) dendrites as well as total dendritic branching (E), terminal dendritic points (F), and total dendritic length (G) of hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.H. Knockdown efficiency was analyzed by real-time RT-PCR in MN1 cells transfected with control or Lrig1 shRNA vectors. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs. Data are shown as individual values of a representative assay measured in triplicates. n=2 independent experiments were performed.I. Representative images of MAP-2 immunostained hippocampalneurons obtained from wild-type and Lrig1-deficient mice cultured for 7 days in vitro (7 DIV). Scale bar, 15 m.J. Sholl analysis of the dendritic arbor from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild type and Lrig1-deficient mice. Data are shown as mean SEM of n=3 independent experiments. *p<0.05 by two-way ANOVA followed by Bonferroni multiple comparisons test.K-M. Quantification of the number of primary dendrites (K), secondary dendrites (L) and total dendritic branching (M) from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild-type and Lrig1-deficient mice. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.
Figure 4A. Schematic diagram of the social interaction device indicating the social and the empty chambers.B, C. Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse (social enclosure, stranger 1). Lrig1-mutant mice exhibit social interaction defects as determined by the time spent interacting (sniffing) with the stranger enclosure (B) and the percentage of total interaction time with stranger in the three-chamber social interaction test (C). Dashed line in (C) represents chance-level performance (i.e. 50%) when mice equally explore the social enclosure and the empty container. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance between wt and knockout mice are *p<0.05 by Students t test.D. Schematic diagram of the social novelty test. In the test for social novelty, a second stranger (stranger 2) mouse was introduced in the empty container.E. In the preference for social novelty task, wt mice showed a preference for social novelty, while mutant mice showed no significant preference for novel target (stranger 2). Mutants also spent significantly less time interacting with the novel mice compared to controls. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance are as follow: *p<0.05 and **p<0.005 by ANOVA followed by Student-Neuman Keuls multiple comparison test.
Figure 5A. Schematic representation of Lrig1 mutants are shown on the left. Expression levels of these mutants were analysed in transfected cell extracts by immunoblotting with anti-Flag antibodies.B. Representative images of rathippocampal neurons transfected at DIV8 with empty vector, full length (FL) Flag-tagged Lrig1, or Lrig1 mutant lacking the LRR domain (LRR) in combination with an enhanced green fluorescent protein (GFP) expression vector. After transfection at 9 DIV, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h. Then, hippocampal cultures at 11 DIV were fixed and stained with anti-Flag antibodies to control Lrig1 expression. Scale bar, 15 m.C, D. Quantification of the effects of Flag-Lrig1 constructs on BDNF-induced primary (C) and secondary (D) dendrite formation of hippocampal neurons treated as indicated in A. The results are shown as mean SEM of 3 independent experiments. *p<0.05, **p<0.01 by one-way ANOVA followed by Tukey´s multiple comparison test. NS, not significant.E. Representative confocal images of dendritic shafts containing spines from hippocampal neurons transfected at 15 DIV with either control vector or Flag-Lrig1 construct together with GFP. After transfection, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h (15+3 DIV). Scale bar, 5 m.F. Quantification of the effect of Lrig1 overexpression on neurotrophin-induced spine density. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by one-way ANOVA followed by Student-Neuman Keuls multiple comparison test.
Figure 7A. Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous (+/-) and Lrig1 knockout (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control.B. Fold of TrkB activation relative to unstimulated control group (phospho-TrkB at Tyr705) in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).C. Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild-type (+/+) and Lrig1-deficient (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for the indicated times. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control. Fold of MAPK activation relative to tubulin is indicated.D. Fold of MAPK activation (P-MAPK) relative to untreated control group in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).E. Representative images of mousehippocampal neurons transfected at 4 DIV with either GFP-expressing control vector or Lrig1shRNA-GFP plasmid. After transfection, neurons were cultured in the absence or in the presence of BDNF (25 ng/ml) for 48 h (7 DIV). Arrows indicate branching points along the principal dendrite. Scale bar, 10 m.F-I. Quantification of primary dendrites (F), secondary dendrites (G), total branching (H) and branchig of the principal dendrite (I) of hippocampal neurons treated as indicated in E. The results are shown as individual values and the means of a representative assay. A total of 30 neurons were analyzed per experimental condition. Similar results were obtained in n=3 independent experiments.J. Lrig1+/+ and Lrig1-/- hippocampal lysates from postnatal day 11 (P11) mice were immunoblotted against phospho-TrkB (p-TrkB) at Tyr705 and total TrkB. Quantification (mean SD) of the p-TrkB/TrkB ratio between Lrig1+/+ (n=4) and Lrig1-/- (n=3) hippocampi is also shown.
Figure 1B, C, Analysis of GFP transfer after transplantation of Nrl::GFP donor photoreceptors into wildtype C57BL/6J (B) and Nrl-/- (C) recipient retinas after 14 (wt, n=9; Nrl-/-, n=7) and 21 (wt, n=6; Nrl-/-, n=4) and 90 (wt, n=5; Nrl-/-, n=4) days. Tissue is stained for GFP and nuclei (Hoechst). In this and all following figures, donor cells (D) are located above, and acceptor cells with GFP (A) are located below the outer limiting membrane (white dashed line), which delimits the apical border of the outer nuclear layer (ONL). In wildtype recipients, donor cells are typically located above the outer segment region (between the grey dashed lines). In Nrl-/- recipients, donor cells are located in the inner segment (IS) region. Plotted is the ratio of the numbers of GFP+ photoreceptors in the ONL (acceptor photoreceptors) and GFP+ donor photoreceptors in the subretinal space (SRS) over time.F, Co-transplanted P3-4 Crx::Cre and P3-5 ROSAmT/mG donor photoreceptors into C57BL/6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post-transplantation.
Figure 2A-C, Combined FISH and IHF to detect GFP (A, B) and GNAT1 (C) 21 days post-transplantation of Nrl::GFP photoreceptors into C57BL/6J (A) and Nrl-/- (B, C) recipients.F-K, FISH and IHF for GFP and GFP transcripts in C57BL/6J recipients transplanted with lentiviral-infected donor cells expressing cytoplasmic GFP (Lenti -GFP) (F, I) and nuclear-localized GFP (Lenti-nls-GFP) (G, J). H, MT index for cytosolic (Lenti-GFP, n=5) and nuclear localized (Lenti-nls-GFP, n=3). K, Quantification of transcripts in donor and acceptor photoreceptors. (Lenti-GFP, n=6 images from 3 transplanted animals; Lenti-nls-GFP, n=8 images from 4 transplanted animals).
Figure 3B-D'', Higher magnification of regions in A (indicated by yellow dashed boxes) showing colocalization of ATP5B and Mito-DsRed-labelled mitochondria in the outer segments (B), soma (C), and the axon (D) of acceptor photoreceptors.
Figure 4F, G, C57BL/6J cells with mitochondria labelled with MitoTrackerTM-Green (MTG) or MitoTrackerTM-Red (MTR) exchange mitochondria after 3 DIV (0 DIV, n=6; 3 DIV, n=6). White dashed line delimitates both cell somas and the protrusion connecting them (F). No exchange is observed when the two photoreceptor populations are separated by a transwell (n=3), nor in co-cultures of live MTG-labelled and lysed MTR-labelled cells (n=3) (G).
Figure 5A, Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl-/- recipient retina 21 days after transplantation with Nrl::GFP donor photoreceptors, showing protrusions (pink arrows) connecting donor and acceptor photoreceptors.D, E, Left: 3D reconstruction of two-photon (D) and light sheet microscopy images of intact cleared ROSAmT/mG eye showing that GFP+-donor and -acceptor photoreceptors are in the same area. D, Right: A high magnification image showing GFP+-donor and -acceptor photoreceptors connected by protrusions (pink arrows). E, Right: In detail, donor and acceptor photoreceptors also appear connected through cell protrusions (pink arrow).
Figure 6A, Confocal image shows a Lenti-GFP infected donor photoreceptor (yellow arrow) connected to an acceptor photoreceptor. The donor protrusion (pink arrow) co-localizes with α-tubulin but not the outer segment marker GNAT1.B, Nrl::GFP donor photoreceptors transplanted in a Prphr2-/- recipient retina exhibit polarized Prphr2 expression (yellow arrow) on the apical side of the donor photoreceptor. Basal protrusions (pink arrows) indicate donor protrusions that contact the host retina.C, D, Expression of β-tubulin (C), SV2 (D) and GFP in the protrusions connecting Lenti-GFP-infected C57BL/6J donor photoreceptors with acceptor photoreceptors. Pink arrows point to the protrusions connecting donor and acceptor cells.
Figure 7A, B, Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin+ (SirActin) and GFP+ (A) and tubulin+ (ViaFluor) and MitoTracker Green+ (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy.E, Time lapse imaging showing movement of MitoTracker Green+ puncta in photoreceptors connected by a protrusion. White arrows point MTG+ puncta inside of the protrusion during the time lapse imaging.