EMBO/SourceData huggingface dataset:
Figure 2 (A) Localization of TIP60 was analysed employing U2OS cells stably expressing FLAG- TIP60WT, FLAG-TIP60S90A or FLAG-TIP60S86A. Nucleoplasm (nuc.) and chromatin (chr.) fractions were generated and were subjected to Western blot analysis  (B) The experiment as described in (A) was repeated twice with U2OS cells and twice with Ba/F3 cells and the blots were quantified. Each dot represents the ration of FLAG-TiIP60 to H4 in the chromatin fraction of altogether four individual experiments as shown in (A)  (C) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT or TIP60S90A, or FLAG-tagged chromodomain mutants TIP60F50A, TIP60Y47A, TIP60Y44F or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting  (D) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S90E, TIP60S90D or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting (E) U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A or empty vector (vec) were treated with 2 µM SNS-032 (SNS) for 3 hours as indicated. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting
Figure 3 (A) FLAG- immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S86A or empty vector (vec). Immunoprecipitated material and input lysate were subjected to Western blot analysis (B) The experiment as shown in (A) was repeated three times. The graphs and error bars represent mean and standard deviation of these three individual experiments (one-way ANOVA, Fisher"s LSD test, WT vs. S86A p=0,5992, WT vs. S90A p=0,0429, S90A vs. S86A p=0,0176 (*p < 0,05; n.s. not significant, p > 0,05))  (C) HEK293 cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG-TIP60S90A or empty vector. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis  (D) U2OS cells stably expressing FLAG-TIP60WT or empty vector were treated with 2µM SNS-032 (SNS) for 2 hours as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. This material and input lysate were subjected to Western blot analysis  (E) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were subjected to ChIP using anti-FLAG antibody. Immunoprecipitated DNA and input were subjected to quantitative real-time PCR using primers annealing in the myc locus about 50bp upstream (myc+50) or about 500 bp downstream (myc-500) of the transcription start site. Promoter occupancy of FLAG-TIP60WT or FLAG-TIP60S90A was determined as percent input in relation to FLAG -TIP60WT. The graphs and error bars represent mean and standard deviation of three individual experiments (one-way ANOVA, Bonferroni"s multiple comparison test, myc+50 WT vs. S90A p=0,0289, myc-500 WT vs. S90A p=0,0155 (*p < 0,05))
Figure 4 (A) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were treated with 2µM SNS-032 (SNS), 2µM JQ1 (JQ1) or the solvent DMSO (d.) for 2h as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis (B) U2OS cells stably expressing FLAG-TIP60WT were treated with 2µM JQ1 (JQ1) for 2h. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting  (C) HEK293 cells were transiently transfected with pcDNA4 encoding Brd4 or GFP. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting
Figure 5 (A) U2OS cells were transfected with a siRNA pool targeting human TIP60 (Dharmacon) or luciferase (Dharmacon). The nuclear lysates were analysed by Western blotting 24h after transfection  (B) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A, FLAG-TIP60S86A or empty vector (vec) were analysed by Western blotting 60 hours after viral transduction
Figure 4(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HCT116Bax/Bak -/- cells stained with AF647-anti-GFP-nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Gallery of typical GFP-Bax WT non-random structures during apoptosis on HCT116Bax/Bak -/- cells. Scale bars, 100 nm.(C) Comparison of confocal (left) and STED (right) images of GFP-Bax overexpressing HeLa cells 2h after STS treatment. STED reveals a notable increase in resolution. Scale bars, 500 nm.(D) Representative GFP-Bax non-random structures found with STED (lines, arcs, rings, double lines). Scale bars, 100 nm.
Figure 7(A) AFM image of a control SLB prepared from LUVs with a mitochondrial-like lipid composition. The bilayer looks flat, without aggregates or defects. Scale bar, 1 µm.(B) AFM image of a SLB prepared from LUVs with a mitochondrial-like lipid composition pre-incubated with heat-activated Bax. The green arrows indicate the presence of membrane pores, which are heterogeneous in size and shape. The edges of the pores present protrusions corresponding to Bax clusters. Scale bar, 1 µm.(C) 3D AFM topography of a Bax arc (left) and ring (right). Both images reveal a circular dark hole that spans the lipid membrane (green). Bax molecules around the pore rim (magenta and white) protrude 3,97 ± 1,02 nm above the membrane plane, as confirmed by the height profiles shown below each image (corresponding to the white line in the 2D image insets). The topography of the arc structure reveals a pore only partially surrounded by Bax molecules, while lipids alone form the rest of the pore rim. Images are shown in a 42° tilted representation.(D) Quantitative analysis of the distinct structures found for activated Bax on SLBs. Data show the total number of structures in all the measurements.
Figure 1(C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm.(F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.
Figure 2The number of sim-expressing nuclei as counted by automated spot detection over time (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression.(D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).
Figure 4(D-E) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant (Hairless H2) and under overexpression of Twist (Twist OE) The observed outcomes are most consistent with the predictions from the state-dependent inactivation model.
Figure 1(A) In vitro pull-downs between strep(II)-tagged DDB1-DCAF12 and untagged wild type (WT) CCT5 or CCT5 (1-529) (Δ(Ct)) seen on a Coomassie-stained SDS-PAGE gel.(B) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1-DCAF12 or terbium-coupled streptavidin (Tb-SA) (n=3). Signal originating in the absence of TbDDB1-DCAF12 is unspecific and was subtracted in subsequent experiments.(C) TR-FRET counter-titration of unlabeled wild type (WT) CCT5 or an unlabeled CCT520 peptide into pre-assembled TbDDB1-DCAF12488 (n=3).(D) TR-FRET counter-titrations of label-free CCT5 C-terminal peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position and corresponding CCT5 amino acid number. (E) Counter-titration of unlabeled CCT510 mutant peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position. D
Figure 3(D) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type (WT) or mutant TbDDB1-DCAF12 complexes (n=3). Unspecific signal arising from the Tb-SA label was subtracted.(E) In vitro ubiquitination of CCT5 by wild type (WT) or mutant CRL4DCAF12 complexes in the presence of ubiquitin, ATP, E1 and E2 enzymes.
Figure 5(A) Counter-titration of unlabeled CCT5 or unlabeled TRiC into pre-assembled TbDDB1-DCAF12488 (n=3).(B) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin, ATP, E1 and E2 enzymes. CCT5, but not TRiC, is modified with poly-ubiquitin (Ubn) chains by CRL4DCAF12. Free CCT5 protein carries an N-terminal strep(II) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC.
Figure 1Representative false colour images of leaf surface temperature captured by a thermal camera from WT, stp1-1, stp4-1, stp1stp4, stp13, stp1stp13 and stp4stp13 plants.Normalized leaf surface temperature over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.Whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.Whole-plant recordings of changes in stomatal conductance (gs) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.
Figure 2Content of soluble sugars in guard cell-enriched epidermal peels of WT and stp1stp4 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 µmol m-2 s-1 following the EoN. Data for two independent experiments are shown; means ± SEM; n ≥ 7 per genotype and time point.Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants. Scale bar, 10 µm.Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point.
Figure 3Representative false colour images of photosystem II (PSII) operating efficiency (ΦPSII) captured by chlorophyll fluorescence imaging from WT, stp1-1, stp4-1 and stp1stp4 plants. ΦPSII was measured at a photosynthetically active radiation (PAR) of 440 µmol m-2 s-1.   ΦPSII quantified over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.   Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point.Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 7) WT, stp1-1, stp4-1 and stp1stp4 plants.Projected rosette area over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.
Figure 4Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype.Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2.Projected rosette area over the phenotyping period. Data shown are from two independent experiments; means ± SEM; n ≥ 24 per genotype and time point.Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm.Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.
Figure 1(A) RNAscope fluorescence in-situ hybridization of Ch25h transcripts (red) in the spinal cord of non-immunized mice (NI) (left panels) and mice 17 days after EAE immunization (peak disease) (2 right panels) shown in endothelial cells (Isolectin B4 (IsoB4) green top panels) and activated macrophages/microglia (Ionized calcium biding adaptor molecule 1 (IBA1) green lower panels). Nuclei are shown in blue. (B) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages/microglia (MG) and endothelial cells (EC), n = 6 biological replicates/group.(E) Percentage of eGFP+ CNS ECs in the same condition as in (D). Symbols indicate individual mice and bars indicate mean ± SD. Ch25hfl/fl NI: n = 4 biological replicates, Ch25hfl/fl EAE: n = 5 biological replicates, Ch25hECKO NI: n = 4 biological replicates, Ch25hECKO EAE: n = 4 biological replicates. Representative results of 1 experiment.
Figure 2(A) EAE disease course in Ch25hECKO and Cre negative littermates (Ch25hfl/fl). Top panel: EAE clinical score. Bars indicate mean ± SEM. Bottom panel: Survival analysis of EAE, depicting disease incidence. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (B) As in (A) except that Ch25h fl/fl- Pdgfb-iCreERT2 mice that express Cre recombinase in blood endothelial cells (Ch25hBECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (C) As in (A) except that Ch25hfl/fl- Prox1-CreERT2 mice that express Cre recombinase in lymphatic endothelial cells (Ch25hLECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.(D, E) Histopathological quantifications (D) and representatives staining (E) of spinal cord sections of immunized Ch25hfl/fl mice (n = 7 biological replicates) and Ch25hECKO mice (n = 10 biological replicates) at day 21 post-immunization for cellular infiltration (HE). Five sections per mouse were quantified. Scale bars 200 µm (top panels), 100 µm (bottom panels).
Figure 3(A) RT-qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells (pMBMECs) isolated from Ch25hfl/fl and Ch25hECKO mice. Cells were left unstimulated (NS) or stimulated with IL-1β. n = 5 biological replicates/group. The experiment was performed 3 times.(F) RT-qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl/fl mice and stimulated with IL-1β. n = 4 biological replicates/group. The experiment was realized once.(G) RT-qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl/fl mice during EAE (day 10 after immunization). Ch25hECKO n = 3 biological replicates, Ch25hfl/fl n = 4 biological replicates. The experiment was performed once.
Figure 4(C) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC concentration in the same conditions as in (B). Bars represent mean ± SD. n = 6 biological replicates/group except for Ch25fl/fl IL-1β were n = 5 biological replicates. The experiment was performed once. (D) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC levels in spinal cord tissue extracted from Ch25hfl/fl (n = 8), Ch25hECKO (n = 3) and Ch25hBBBKO (n = 6) at the peak of EAE.(G) Prostaglandin concentration (nM) in the supernatant of pMBMECs comparing conditions mentioned in (A). n = 5 biological replicates/group and bars represent mean ± SD.
Figure 5(C) Impact of CNS PMN-MDSC and BM-PMN on CD4+ T cell proliferation assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the CNS (right panel) and BM (left panel) of WT mice at the peak of EAE. NS = non stimulated. n = 4 biological replicates/group. Bars indicate mean ± SD. CNS PMN-MDSC/CD4+ T cells: representative results of 2 independent experiments; BM-PMN: 1 experiment.(H) Percentage (upper left panel) and absolute number (lower left panel) of CNS live cells CD45+CD11b+Ly6CintLy6G+ PMN-MDSC; Percentage of live CD4+CD44+Ki67+ cells (upper right panel) and ratio (lower right panel) of absolute number of PMN-MDSC and proliferating CD4+ T cells in the CNS at the peak of the disease of Ch25hfl/fl (n = 9) and Ch25hECKO mice (n = 8). Symbols depict individual mice and bars indicate mean ± SD. Combined results of 2 independent experiments.(I) EAE disease course in Ch25hBBBKO mice (n = 11) where the Cre-recombinase is expressed in endothelial cells of the CNS and Cre negative littermates (Ch25hfl/fl: n = 7 and Ch25hfl/wt : n = 2). Bars indicate mean ± SEM. Representative results of 2 independent experiments.
Figure 6(B) Survival analysis in the same conditions as in (A). Ch25hfl/fl isotype: n = 7, Ch25hECKO isotype: n = 8, Ch25hfl/fl Combo: n = 8, Ch25hECKO Combo: n = 9.(C) Representative contour plots of PMN-MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in (A). (D) As in (C) except that statistical analysis and percentage of PMN-MDSC in live cells are shown. Symbols depict individual mice and bars mean ± SD. Ch25hfl/fl Isotype n = 4, Ch25hECKO isotype n = 4, Ch25hfl/fl Combo, n = 3, Ch25hECKO Combo n = 4. ((E) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry. Ch25hECKO and Ch25hfl/fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE (day 14 after immunization). (F) As in (E) except that statistical analysis is shown. Symbols depict individual mice, bars mean ± SD. Ch25hfl/lfl isotype n = 7, Ch25hECKO isotype n = 5, Ch25hfl/lfl Combo n = 6, Ch25hECKO Combo n = 5. The experiment was performed once. (
Figure 1Reverse Transcriptase PCR showing that bd1971, and control gene dnaK, are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus. RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E. coli cells. Primers were designed to anneal specifically to the gene of interest. L = 100bp DNA ladder, AP = Attack Phase cells, 15-45 = 15-45 minutes respectively since infection, 1h-4h = hours respectively since infection. Ec = E. coli strain S17-1 RNA (negative control: no Bdellovibrio RNA); NT = control with no template RNA; gen = B. bacteriovorus HD100 genomic DNA (positive control). The cartoon above represents each stage in the predatory cycle.Epifluorescent images of E. coli invaded by B. bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection. Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images (see methods) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation. Fluorescence images and merges with phase contrast images are shown. Scale bars are 2 µm. Images are representative of three independent experiments.
Figure 2Relative levels of c-di-GMP in matched cell biomass host-independent (HI) cultures of bd1971::kan compared with fliC1 merodiploid KnR HI control strain (Wild-Type for bd1971). The bd1971 mutant strain contains significantly more c-di-GMP than the control. Data are from 3 independent experiments.Relative levels of c-di-GMP in matched cell biomass wild-type predatory cultures of HD100 strain, silent Bd1971 deletion strain (Δ1971), Bd1971D306307A strain (bd1971DD) and Bd1971R67D strain (bd1971R). All mutant strains had higher levels of c-di-GMP compared to the wild type strain. Error bars show SEM. Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants.
Figure 5Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation.HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.
Figure 8ITC titration curves and relative heats of binding for (A) cAMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.