EMBO/SourceData huggingface dataset:
Figure 2Re-expression of FBXL4 into FBXL4-defective CRISPR lines reduces the levels of NIX and BNIP3 in FBLX4 deficient clones. FBXL4-deficient 2G10 and FBXL4-deficient 1D4 cell lines were stably transduced with a doxycycline-inducible FBXL4-HA construct. Cells were treated with doxycycline for the indicated times prior to immunoblotting with the specified antibodies.FBXL4 localises to the mitochondrial outer membrane. Cells transiently transfected with FBXL4-HA-C were treated with DFP for 24 h. Cells were stained with an anti-HA antibody (to recognise FBXL4) and either TOM20 (an outer mitochondrial membrane protein) or TIM50 (an inner mitochondrial membrane protein). The line scan intensity profiles for FBXL4 (green) and TOM20/TIM50 (magenta) represent fluorescence intensity (y-axis) plotted against distance (x-axis). Scale bars = 5 μm.FBXL4 is a proximity interactor of NIX and BNIP3. Cells expressing inducible BirA-BNIP3, BirA-NIX and BirA control were transduced with a lentiviral vector expressing FBXL4, as indicated. Cells were treated with doxycycline for 48 hours (to induce BirA-bait protein expression), biotin for 24 hours (for the biotinylation reaction), and, where indicated, MLN4924 for 24 hours (to stabilise NIX and BNIP3). Streptavidin-coupled beads were used to capture the biotinylated proteins. FBXL4 was specifically detected in the eluate from BirA-BNIP3 and BirA-NIX compared with BirA-alone.NIX and BNIP3 polyubiquitylation depends on FBXL4. U2OS or U2OS-FBXL4 KO cells were co-transfected with TR-TUBE and either myc-BNIP3 or myc-NIX, as indicated. Cell lysates obtained 48 hours post-transfection were immunoprecipitated with anti-FLAG beads, and the immunoprecipitates were analysed by immunoblotting using anti-myc antibody (to detect ubiquitylated NIX or BNIP3). The line on the right marks a ladder of bands corresponding to polyubiquitylated myc-BNIP3 or myc-NIX
Figure 3Loss of FBXL4 leads to an increase in mitophagy, which can be reduced by re-expression of FBXL4. U2OS mt-Keima FBXL4 KO clones (2G10 and 1D4) expressing doxycycline-inducible wild-type FBXL4-HA were treated with doxycycline for 72 hours. The emission signals obtained after excitation with the 458 nm laser (neutral pH) or 561 nm laser (acidic pH) are shown in green and magenta, respectively.Depleting NIX/BNIP3 via siRNA reduces the increased mitophagy caused by FBXL4 deficiency. U2OS mt-Keima cells and U2OS mt-Keima FBXL4 KO 2G10 cells were transfected with siRNAs targeting both NIX and BNIP3 (NIX/BNIP3 si) or non-targeting siRNA (NT si). UT=untransfected.Corresponding cells from D-E were harvested for immunoblotting to analyse the extent of NIX and BNIP3 reduction after siRNA transfection.Hyperstable NIX deletion mutants increase mitophagy compared with wild-type NIX. Hela Flp-in NIX KO mt-Keima cells stably expressing inducible NIX(WT), FLAG-NIX∆151-170, or FLAG-NIX∆171-184 were treated with doxycycline for 48 hours Mitophagy was visualised as in A and quantified as in B. Hyperstable BNIP3 deletion mutants increase mitophagy compared with wild-type NIX. Hela Flp-in NIX/BNIP3 DKO mt-Keima cells stably expressing BNIP3 deletion mutants were treated with doxycycline for 48 hours translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicate means +/- standard deviation. N=3. At least 200 cells were analysed per condition. P values were calculated based on the mean values using a one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001). Scale bars = 20 μm.
Figure 4FBXL4 patient-derived variants exhibit reduced efficiency compared to wild-type FBXL4 in mediating the downregulation and destabilization of NIX and BNIP3. U2OS FBXL4 KO (2G10) cells were rescued with constructs expressing wild-type FBXL4-HA, FBXL4(F-box mut), FBXL4(∆MTS), or specified patient variants. Cells were treated with cycloheximide for 3 hours prior to harvesting. Samples were lysed, and immunoblotting was performed. GFP serves as a marker of transduction efficiency/transgene expression. EV = empty vector.FBXL4-Arg482Trp and FBXL4-Gly568Ala patient variants are less efficient than FBXL4-wild-type at assembling into a complex with SKP1 and CUL1. FBXL4-KO cells expressing wild-type FBXL4-HA or FBXL4 variants were harvested and lysed. Whole-cell extracts were subjected to immunoprecipitation (IP) with anti-HA agarose beads and immunoblotting, as indicated.NIX/BNIP3 depletion mediated by siRNA reduces mitophagy in FBXL4-deficient patient fibroblast cells. FBXL4-deficient patient fibroblasts were transfected with siRNAs targeting both NIX and BNIP3 (NIX/BNIP3 si) or non-targeting siRNA (NT si) (left). Live-cell confocal microscopy was performed to visualise mitophagy and quantification was performed as in D (middle). The siRNA-mediated depletion of NIX and BNIP3 was evaluated by Western blotting (right). translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicates +/- standard deviation. in H, N=3. Over 100 cells were analysed per condition. P values were calculated based on the mean values using a one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001). Scale bar = 20 μm.­ Ubiquitylation of endogenous NIX is restored upon expression of FBXL4 in patient-derived cells. Patient derived fibroblasts were transfected with FLAG-tagged TR-TUBE, as indicated. FLAG-tagged TUBE protein was affinity purified from the corresponding lysates using FLAG-beads, and the precipitates were analysed by immunoblotting using antibodies to endogenous NIX.
Figure 1Jacob expression in human AD. The ratio of normalized levels of pJacob/Jacob is significantly reduced in the temporal cortex of AD patients as compared to the age-matched control group. All samples are normalized to Histone3 (H3). N=11-12 different subjects.(G, H) Jacob shRNA knockdown prevents Aβ-induced CREB shutoff. (G) Representative confocal images of hippocampal neurons transfected with Jacob-shRNA or scrambled (scr) shRNA control (both expressing GFP) and treated with oligomeric preparations of Aβ1-42 or Aβ3(pE)-42. (H) Neurons transfected with a Jacob knockdown construct did not display reduction of pCREB immunofluorescence intensity after treatment with Aβ1-42 or Aβ3(pE)-42. Bar plot of mean nuclear pCREB intensity normalized to untreated control. Scale bar: 10 µm. N=29-39 nuclei from 2 independent experiments.(J) Representative confocal images of slices immunolabeled against pCREB, co-labeled with NeuN and DAPI. Scale bar: 100 µm. (K) Bar plot of pCREB, N=17-21 slices.(L, M) The quantification of pCREB intensity in NeuN-positive cells revealed a statistically significant decrease in pCREB immunoreactivity in TBA2.1 but not in double transgenic animals (TBA2.1, -/-). (L) Representative confocal images of CA1 cryosections from 13 weeks old mice stained for NeuN, DAPI and pCREB. Scale bar: 100 µm. Data represented as cumulative frequency distribution. (M) Bar plot of average hippocampal pCREB nuclear immunoreactivity normalized to WT. N=34-38 hippocampal sections from 10-11 animals.(P) Double transgenic TBA2.1 Jacob/Nsmf knockout (TBA2.1, -/-) mice display significantly lower neuronal loss compared to TBA2.1 mice. The number of NeuN positive cells was normalized to WT group. N=36-47 CA1 images analyzed from 10-12 animals per genotype. Dotted line marks value of the normalized control group -100%. (Q) Jacob knockout rescues decrease in the BdnfIV gene transcription. Bar plot of mean BdnfIV transcript levels in hippocampal homogenates normalized to β-actin as reference transcript. N=5-10 hippocampi.
Figure 2(A) Pull-down assays confirm a direct interaction of MBP-Jacob-45-532 and His-SUMO-CREB.(C) The C-terminus (CREB-166-341-tagRFP) but not the N-terminus (CREB-1-165-tagRFP) of CREB closely associates with Jacob-GFP in FRET saturation experiments. (D) Both the N- (Jacob-1-228-GFP) and the C-terminus (Jacob-262-532-GFP) of Jacob are in close proximity to CREB-tagRFP, however, the Jacob-1-228-GFP association with CREB is significantly stronger. FRET efficiency is presented in arbitrary units from 5-6 independent experiments.(G) A proximity ligation assay revealed an interaction between Jacob and CREB in primary neurons. Dashed ROIs indicate neuronal nuclei.(M) Co-immunoprecipitation experiments to map the binding region of Jacob to the LIM1 domain of LMO4 revealed the association with Jacob-179-246-GFP, but not with Jacob-45-172-GFP (CREB-binding region).A Jacob-LMO4-binding mutant expressed in the nucleus does not induce CREB shutoff. Representative confocal images of hippocampal neurons transfected with ΔMyr-Jacob-GFP (Jacob targeted to the nucleus) or ΔMyr-Jacob-L175A-V176A-GFP. Scale bar: 10 µm. Lookup table indicates the pixel intensities from 0 to 255. (R, T) The mean of nuclear (T) CREB immunoreactivity in Jacob-expressing neurons was normalized to non-transfected controls. N =23-33 neuronal nuclei from two independent cell cultures.
Figure 3(B, C) FRET saturation experiments with (B) LMO4-GFP and CREB-1-165-tagRFP or (C) CREB-166-341-tagRFP and CREB-GFP and LIM1-1-80-tagRFP or LIM2-81-165-tagRFP revealed an association between the LIM1 domain of LMO4 and the C-terminus of CREB. N=8 independent experiments.(D) Recombinant His-SUMO-CREB directly binds to GST-LMO4 in pull-down experiments.(H-K) Knockdown of LMO4 reduces nuclear pCREB immunoreactivity. (H, J) Representative confocal images of hippocampal neurons transfected with LMO4 shRNA construct or scrambled control (both expressing GFP under CMV promoter as a transfection control). Scale bar: 10µm. Dot plots representing the mean of nuclear (I) pCREB or (K) CREB staining intensity normalized to scrambled control. N=30-37 nuclei analyzed from at least 3 independent cell cultures.The N-terminus of Jacob displaces LMO4 from CREB. (N) GST-LMO4 coupled to beads was preincubated with His-SUMO-CREB and subsequently incubated with an increasing amount of MBP-Jacob-45-228.
Figure 4(A) Pull-down experiments confirmed a direct interaction between recombinant MBP-Jacob-45-532 and His-PP1γ.(B) Mapping of the mCherry-PP1γ interaction region within the Jacob sequence revealed binding of a C-terminal fragment (Jacob-310-250-GFP) as well as the N-terminal part (Jacob-173-246-GFP) where the region between 213-246 aa is sufficient for immunoprecipitation. The pink boxes in the schematic indicate binding regions.Treatment of hippocampal primary neurons expressing phosphodeficient Jacob in the nucleus with OA rescues Jacob-induced CREB shutoff. Confocal images of pCREB immunostaining in DIV15 neurons overexpressing ΔMyr-Jacob-S180A-GFP with and without OA treatment. Scale bar: 20 μm. Lookup table indicates the pixel intensities from 0 to 255. N=14-17 nuclei analyzed from two independent cell cultures.(K) Treatment with staurosporine decreases Jacob phosphorylation level (S180), but increases its association with LMO4-tagRFP. Immunoblot of HEK293T cells extracts transfected with LMO4-tagRFP and ΔMyr-Jacob-GFP or GFP alone. (L) Treatment with staurosporine decreases the association of Jacob with CREB. Immunoblot of HEK293T cells extract transfected with CREB-tagRFP and ΔMyr-Jacob-GFP or GFP as a control. (
Figure 5Nitarsone disrupts binding of Jacob to LMO4 in a concentration-dependent manner. (H) MBP immunoreactivity normalized to input. N=6-12 independent experiments.(N, O) (N) 5 µM Nitarsone reduced Jacob-LMO4 complex formation in Aβ-treated (500 nM) primary neurons as revealed by proximity ligation assay. Scale bar: 5 µm. (O) Bar plot of normalized PLA signal intensity. N=20-24 neuronal nuclei from 3 cultures.
Figure 6(A, B) Co-application of Nitarsone prevents Aβ-induced CREB shutoff. DIV16 hippocampal cultures were treated with 5 μM Nitarsone, 500 nM Aβ1-42, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h and stained for pCREB, MAP2, and DAPI. (A) Representative confocal images. Scale bar: 10 µm. (B) Nuclear pCREB immunoreactivity normalized to control. N=63-67 nuclei from three independent cultures.(E, F) Treatment with Nitarsone rescues Aβ1-42-induced decrease of synaptic GluR1-immunoreactivity within Shank3. DIV16 dissociated, hippocampal cultures were treated with 5 μM Nitarsone, 500 nM Aβ1-42, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h and stained for Shank3, surface GluR1, and MAP2. (E) Representative confocal images of dendritic segments. Scale bar: 5 µm. (F) GluR1-immunoreactivity within Shank3 signal. N=39-61 of dendritic segments from four independent cell cultures.Nitarsone administration rescues mEPSC amplitude. (G) Analog traces of mEPSCs recorded in DIV16 hippocampal neurons treatd with 500 nM Aβ1-42, 5 μM Nitarsone, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h.
Figure 7(B, C) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of TBA2.1 mice. (B) Representative confocal images of cryosections from 11 weeks old mice stained for NeuN, DAPI, and pCREB. Scale bar: 10 µm (C) Bar plot of pCREB nuclear staining intensity. N=21-34 hippocampal sections from 6-9 animals.(D, E) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of 5xFAD mice. (D) Representative confocal images of CA1 cryosections from 18 weeks old mice stained for NeuN, DAPI, and pCREB. Scale bar: 10 µm. (E) Cumulative frequency distribution of pCREB nuclear staining intensity. N=22-27 hippocampal sections from 6-7 animals.(K, L) Nitarsone reduces number of varicosities in SLM of CA1 of TBA2.1 mice. (K) Representative, confocal images of dendrites filled with biocytin stained from 13 weeks old mice. Arrows indicate varicosities. Scale bar: 1 μm. (L) Number of dendritic swellings per 10 μm. N=19-22 dendrites from 2 animals per genotype.
Figure 8(A) Nitarsone rescues late CA1-LTP impairment in TBA2.1 mice. Insets show representative fEPSPs analog traces at indicated time points: 1 = baseline, 2 = late LTP. (B) Averaged fEPSP slopes recorded during the last 30 min. N=14-18 slices from 5-6 mice. (C) Nitarsone rescues late CA1-LTP impairment in 5xFAD mice. Insets show representative fEPSPs analog traces at indicated time points: 1 = baseline, 2 = late LTP. (D) Averaged fEPSP slopes recorded during the last 30 min. N=17-18 slices from 6 mice.(H-J) Nitarsone rescues short-term memory impairment in Y-maze object recognition task in (I) TBA2.1 N=9-14 and (J) 5xFAD mice. N=9-11
Figure 3(E) In C. elegans, all conserved ciliary cluster genes with available mutants as well as genes of particular interest for which mutants were generated in the course of this study were screened using the dye-fill assay to test for cilium structural integrity. 12 amphid neurons in the head and 4 phasmid neurons in the tail, both featuring non-motile sensory cilia, stain with the lipophilic dye DiI. Failure to take up dye is an indicator of ciliary structural defects. IFT mutants such as osm-5(p813) tend to display dye-fill null phenotypes, while other ciliary mutants present weaker phenotypes. Phenotypes were scored using a scale from 0 (no dye-fill phenotype) to 4 (null).(F) Scatter plot displaying the phenotypes of mutants in IFT and transition zone components. Each point on scatter plots represents one gene, with values shifted slightly to avoid overlap. (G) Scatter plot displaying the phenotypes for all known (grey) and previously uncharacterised genes (green) within the ciliary cluster, plotted as in (F).
Figure 4(B) Schematic and confocal microscopy images of scolopidia in chordotonal organ of the fly. Each scolopidium contains two cilia with their ends embedded in a cap cell. Scolopidium outline, cilia and ciliary dilation can be visualized by DIC. Immunofluorescence micrographs with Sas-4 and NompC antibodies used to mark basal bodies and ciliary tips, respectively. DIC images of flies depleted of candidate genes by RNAi reveal relatively few defects on par with IFT genes such as IFT140, with the exception of misplaced dilations for Cep135 and CFAP97D1 (arrows). Missing cilia (asterisks) as with SAS4 or IFT52 are not observed. NompC/Sas-4 staining reveals more subtle phenotypes, including mislocalized NompC signal (with CFAP97D1, Cep135, Elmod, Mapk15 and Tex9, arrowheads) and apparent ciliary structural defects (Cep104 and Cables1, arrows). At least 3 animals examined per condition.(E) Mature sperm can be dissected from the seminal vesicle and motility analyzed using high speed video capture in dark field microscopy. Sinusoidal motion can be seen in wild type. In RNAi depletions of certain known motility genes (here IQUB and GAS8), this motion is reduced or even absent. Arrowheads mark position of individual bends in sequential frames. At least 3 movies made from different animals per condition.(F) Spermatogenesis in wild-type and RNAi depletions of selected genes, highlighting the stage where morphogenesis first becomes noticeably abnormal. At least 6 testes from 3 different animals examined per condition.(G) Graph summarizing sperm defects observed for candidate genes chosen for further analysis, with selected known centriolar/ciliary genes shown for comparison. Yellow bar indicates stage where phenotypes first become apparent, blue bar indicates furthest progression observed. Any sperm accumulating in the seminal vesicle were scored for motility. The second graph plots the severity of sperm ciliary defects (stage at which defects were first observed) for all previously uncharacterized ciliary genes chosen for further analysis. Number of animals examined as in (E, F).
Figure 5(A, RNAi-mediated depletion/deletion of CABLES1, CCDC6, CEP104 in Drosophila chordotonal neurons (A) results in defects in ciliary ultrastructure similar to CEP135. In neurons, there is a loss of 9-fold symmetry towards the ciliary tips with displaced doublet microtubules (arrowheads) and axonemes frequently fail to reach the cap cell (asterisks). At least 3 animals examined per condition and tissue.B) RNAi-mediated depletion/deletion of CABLES1, CCDC6, CEP104 and TEX9 in Drosophila sperm (B) results in defects in ciliary ultrastructure similar to CEP135. In sperm, there is a loss of the inner pair of microtubules (asterisks), a phenotype hitherto only associated with CEP135. At least 3 animals examined per condition and tissue.(C) GFP fusions to TEX9, CCDC6 and CEP104 localize to the central lumen of sperm basal bodies marked by Ana1, while CABLES1 only faintly localizes to basal bodies.(D) TEX9, CCDC6 and CABLES1 are recruited to maturing basal bodies during Drosophila spermatogenesis. n >10 basal body pairs for each developmental stage.(E Localization interdependencies between CEP135, CEP104 and the novel components TEX9, CCDC6 and CABLES1 in spermatogenesis. CEP135 is required for the localization of TEX9, CCDC6 and CEP104 to mature basal bodies (E) n>6 basal body pairs per condition. Asterisks indicate statistically significant differences to the respective control (t-test, **P < 0.01,***P<0.001).F) Localization interdependencies between CEP135, CEP104 and the novel components TEX9, CCDC6 and CABLES1 in spermatogenesis. while CEP135 localization is independent of TEX9, CCDC6 and CABLES1 at all stages of spermatogenesis (F). Asterisks indicate statistically significant differences to the respective control (t-test, **P < 0.01,***P<0.001).
Figure 6(B). RNAi-mediated depletion/deletion of Drosophila CFAP97D1 and CFAP299 results in defects in ciliary ultrastructure in chordotonal neurons, including broken axonemes (asterisk) and perturbed 9-fold symmetry (arrowheads). At least 3 animals examined per condition.(C) A GFP fusion to CFAP97D1 localizes to the ciliary dilation but not basal body in chordotonal neurons. Insets acquired in Airyscan mode.(D) RNAi-mediated depletion/deletion of CFAP299 results in failure of membrane deposition around the developing sperm axoneme (arrowheads). At least 3 animals examined per condition.
Figure 7(A, B) Full gene deletions of mapk-15 and elmd-1 in C. elegans result in both shortened cilia and shortened dendrites, combining the signature phenotypes of IFT and transition zone mutants (here kap-1;osm-3 and ccep-290;nphp-4 double mutants, respectively). Representative images of phasmid (tail) neurons expressing OSM-6:GFP (A) and quantitation of cilium and dendrite lengths (B) in control and mutant animals as indicated. n >20 animals for all conditions. Asterisks indicate statistically significant difference to wild-type (cilium lengths, t-test, ***P < 0.001, dendrite lengths, Wilcoxon Mann-Whitney test, ***P < 0.001). mapk-15;elmd-1 double mutants do not display phenotypes stronger than either single mutant (t-test and Wilcoxon Mann-Whitney test as above, not significant).mapk-15 and elmd-1 mutants display defects in ciliary ultrastructure. (C) Cross-sectional views through the amphid channel reveal defects in cilium extension in both mutants, with fewer cilia protruding into the channel (asterisks). (D) Quantitation of cilium extension defects at the level of the proximal and distal amphid channel.mapk-15 and elmd-1 mutants display defects in ciliary ultrastructure. (E) Higher magnification views of individual cilia reveal defects (indicated by arrowheads) at the level of the proximal segment of the axoneme (fewer than 9 doublet microtubules), transition zone (disorganization of the central cylinder and overall architecture) and ciliary base (membrane blebs). Percentages reported in figure based on examination of 4 mapk-15 and 7 elmd-1 mutant animals.(F) Endogenous/Endogenous promoter GFP fusions to ELMD-1 and MAPK-15 localize to the ciliary base and periciliary membrane compartment in C. elegans phasmid neurons, the former marked by the IFT component CHE-11.Localization interdependencies between MAPK-15 and ELMD-1. (G) Loss of ELMD-1 results in a hyperaccumulation of MAPK-15 at the C. elegans ciliary base, while loss of MAPK-15 impairs localization of ELMD-1 Percentages reported in figure based on examination of >30 animals for each condition.
Figure 1A Immunofluorescence staining of KANK1 (red) and α-tubulin (blue) in Strep-GFP-CEP170B knock-in HeLa cells. Lower panels show enlargements of the peripheral (1) and central (2) cortical regions boxed in the upper panels. Data information: Scale bars, 5 μm.C Immunofluorescence staining of KANK1 in Strep-GFP-CEP170B knock-in HeLa cells treated with nocodazole. Lower panels show enlargements of boxed areas in the upper panels. D Immunofluorescence staining of CEP170B and α-tubulin in MCF7 cells. Lower panels show enlargements of boxed areas in the upper panels. Data information: Scale bars, 5 μm.E Images of fixed HeLa cells transiently expressing mCherry-CEP170B together with GFP-tagged KANK1, KANK2, liprin-α1, liprin-β1, ELKS, or LL5β. Insets show enlargements of the boxed areas. Data information: Scale bars, 5 μm.G Immunofluorescence staining of KANK1, KANK2, or liprin-α1 in control Strep-GFP-CEP170B knock-in HeLa cell line or indicated knockout (KO) cell lines that were generated based on the parental Strep-GFP-CEP170B knock-in HeLa cell line. Data information: Scale bars, 5 μ