EMBO/SourceData huggingface dataset:
Figure 5A Average distance to center as measure of anxiety in the open field (ANOVA: genotype F1,24=16.24 p=0.0005, age F2,24=5.217 p=0.0131). Mutants show increased anxiety levels at 9- and 14-months.B Estimated frequency of rearing activity as measure of exploratory drive in the open field (ANOVA: genotype F1, 24=39.40 p<0.0001, age x genotype F2,26=8.204 p=0.0017). Mutants showed little exploratory activity at 9- and 14-months.C Light-dark transition test, % time spent in the bright compartment (ANOVA: genotype F1,16=7.393 p=0.0152). Reduced scores at 9-months old animals indicate increased anxiety.D Exploratory corner visits in dark and light phase during adaptation to the IntelliCage (ANOVA: phase F1,18=617.2 p<0.0001). Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9-months old animals.E Learning in the hippocampus-dependent IntelliCage serial reversal task (ANOVA: day F2,56=26.71 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.F Learning in the hippocampus-dependent IntelliCage chaining task (ANOVA: day F2,54=450.1 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.G Water-maze place navigation, swim path during acquisition and reversal (ANOVA: day F4,104=21.14 p<0.0001, genotype F1,26=13.11 p=0.0013, age x genotype F2,26=8.204 p=0.0017). All groups learned: controls showed reduced performance with increasing age, mutants showed poor performance already at 9- and 14-months.H Number of wall approaches as indicator of poor coping with the test and non-spatial strategy choice (ANOVA: genotype F1,26=18.31 p=0.0002, age F2,26=4.486 p=0.0212, age x genotype F2,26=7.068 p=0.0035). Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9- and 14-months.Water-maze probe trial, spatial retention score for target zones 1-3 comprising 5, 10 and 10% of the pool surface versus remaining 75% of surface (ANOVA: zone F3,78=36.04 p<0.0001, genotype x zone F3,78=7.807 p=0.0001). Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9- and 14-months.
Figure 6Noise exposure and ABR measurements. Exposure of mice to broadband noise (2-10 kHz) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds. Thresholds in Mrps5V338Y/V338Y mutant mice (black bars) were significantly higher after exposure than thresholds in littermates (open bars), (p<0.0001, two-way ANOVA for combined 108 and 110 dB data). Frequencies of 12, 24 and 32 kHz were selected for measurement to cover the mid-frequency to high-frequency regions of the cochlea. n=6 for baseline littermates; n=6 for baseline mutants; n=3 for all noise exposures.
Figure 1B. ITC binding assays of the UVR8 and HY5 VP-peptides versus the COP1 WD40 domain. The top panel represents the heats detected during each injection. The bottom panel represents the integrated heats of each injection, fitted to a one-site binding model (solid line). The following concentrations were typically used (titrant into cell): UVR8 - COP1 (2500 μM in 175 μM); HY5 - COP1 (1500 μM in 175 μM). The insets show the dissociation constants (Kd) and stoichiometries of binding (N) (± standard deviation).E. ITC assays of the HY5 and UVR8 VP-peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used (titrant into cell): UVR8 - COP1Lys422Ala (1000 μM in 100 μM); UVR8 - COP1Tyr441Ala (2500 μM in 90 μM); UVR8 - COP1Trp467Ala (2500 μM in 90 μM); HY5 - COP1Lys422Ala (1500 μM in 175 μM); HY5 - COP1Tyr441Ala (1600 μM in 138 μM); HY5 - COP1Trp467Ala (1600 μM in 112 μM). The insets show the dissociation constants (Kd). The stoichiometries of binding (N) for UVR8 - COP1Lys422Ala = 0.87 ± 0.2; HY5 - COP1Lys422Ala = 0.83 ± 0.2; HY5 - COP1Tyr441Ala = 0.92 ± 0.3 (All measurements ± standard deviation; n.d.: no detectable binding).Images of representative individuals (F) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Data Information , lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.quantification of hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.Quantitative real-time PCR analysis of (H) HY5 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.Quantitative real-time PCR analysis of (I) RUP2 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.
Figure 2A. ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1 - UVR8 +UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation).B. Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.D. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.E. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.
Figure 3B. Representative image showing the phenotype of wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 seedlings grown for 4 days under white light (- UV-B) or white light supplemented with UV-B (+ UV-B). The scale bar represents 5 mm.C. Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median.D. Anthocyanin accumulation in seedlings Average and SEM are shown (n = 3).E. Quantitative real-time PCR analysis of RUP2, CHS, HY5 and ELIP2 expression in wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 4-day old seedlings grown under white light in response to 2h of UV-B (+UV-B vs. -UV-B). Error bars represent SEM of 3 biological replicates. Note: the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera.F. Yeast 3-hybrid analysis of the COP1-HY5 interaction in the presence of the UVR8HY5C44 chimera. (Left pane) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Right panel) Representative filter lift assays of the yeast analyzed in left panel. AD, activation domain; BD, DNA binding domain; Met, methionine.G. Binding kinetics of the UVR8HY5C44 chimera pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8HY5C44 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8HY5C44 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.H. Size-exclusion chromatography assay of purified, recombinant chimeric proteins expressed in Sf9 insect cells in the presence and absence of UV-B.
Figure 4A. Table summarizing affinities (Kd, dissociation constant) of VP-containing peptides versus the COP1 WD40 domain (WT) and COP1Lys422Ala as determined by ITC. All values are μM (± standard deviation).D. ITC assays between the COL3 VP-peptide (left) and CRY1 VP-peptide (right) versus the COP1 WD40 and COP1Lys422Ala, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): COL3 - COP1 (2240 μM in 195 μM); COL3 - COP1Lys422Ala (1500 μM in 175 μM); CRY1 - COP1 (750 μM in 75 μM); CRY1 - COP1Lys422Ala (1500 μM in 175 μM). The inset shows the dissociation constant (Kd). The stoichiometries of binding (N) for COL3 - COP1 = 0.75 ± 0.2; COL3 - COP1Lys422Ala = 0.90 ± 0.2; CRY1 - COP1 = 0.99 ± 0.3; CRY1 - COP1Lys422Ala = 0.98 ± 0.1 (All measurements ± standard deviation).
Figure 5A. Representative image of wild-type (Ws), cop1-5/Pro35S:YFP-COP1, cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala and cop1-5/Pro35S:YFP-COP1Trp467Ala transgenic lines grown for 39 days in long day conditions.B. Representative image of individual wild-type (Ws), co-11, cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala plants grown for 39 days in long days conditions.C. Quantification of flowering time. Means and SD are shown (n = 14). D. Number of rosette and cauline leaves at flowering. Means and SD are shown (n = 14). E. Immunoblot analysis of CRY2 and actin (loading control) protein levels in wild-type (Col), cry2-1, wild-type (Ws), cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala seedlings grown for 4 days in darkness.F. ITC assays between the CO VP-peptide (left) and CRY2 VP-peptide (right) versus the COP1 WD40 and the COP1Lys422Ala WD40 domains, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): CO - COP1 (2240 μM in 195 μM); CO - COP1Lys422Ala (1200 μM in 120 μM); CRY2 - COP1 (750 μM in 70 μM); CRY2 - COP1Lys422Ala (3000 μM in 175 μM). The stoichiometries of binding (N) for CO - COP1 = 1.03 ± 0.00; CO - COP1Lys422Ala = 0.89 ± 0.03; CRY2 - COP1 = 1.04 ± 0.20; CRY2 - COP1Lys422Ala = 1 (fixed) (All measurements ± standard deviation).G, H. Binding kinetics of the (G) full-length activated CRY2 and (H) CRY2NC80 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of protein injected are shown in red, with their respective heterogenous ligand binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - CRY2 (highest concentration 14 μM), CRY2NC80 (highest concentration 450 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.
Figure 1A) Western blot analysis of Mpl in healthy donors' (HD) platelets shows two distinct bands representing the mature glycoprotein (top band) and the ER‐core glycosylated form (bottom band). The ratio is ~ 1.B) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F, alone or cotransfected with cDNA coding for Mpl.C) Summary of the ratios of mature:immature Mpl for at least three independent (2 for the JAK2V617F transfection) blotting experiments under the protocol shown in (B). Based on two−tailed Student t−test statistical analysis, there is a significant difference (p = 0.0001) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells. Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V/V617F by comparison to controls (p = 0.0159 and p = 0.0009, respectively). The mature:immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V/V617F (p < 0.01).D) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl, alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F.E) Summary of data in (A), (B) and (D), showing the relative band intensity of mature and immature Mpl and normalized for levels of β‐actin in each lane.F) Immunoprecipitation of the Mpl‐Jak2 complex in different cell lines analyzed by immunoblotting with anti‐Mpl and anti‐JAK2 antibodies.
Figure 2Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno‐fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B).C) Confocal fluorescence image of K562 cells (JAK2WT+/+) transiently transfected with cDNA coding for MplmOrange2. Red arrow in this image points to plasma membrane localization.D and E) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno‐fluorescent labeling of endogenous JAK2 in K562 (D) or HEL (E) cells. Profiles of fluorescence (across individual cells and indicated by a white arrow) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane (black arrow heads) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL.F and G) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine (F) or JAK2V617FmCitrine (G) in K562 cells. Profiles of fluorescence (white arrows) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine, especially at the plasma membrane (black arrow heads), but a very poor colocalization with JAK2V617FmCitrine (grey arrow heads). Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.
Figure 3A) K562 cells labeled live for 30 min with an anti‐CD235a‐Atto655 (glycophorin A) antibody before treatment with the Duolink reagents.B) K562Mpl‐mKO2 cells non‐treated with the Duolink reagents.C and D) HEL cells primed live for 30 min with an anti‐CD235a‐Atto655 antibody before treatment with the Duolink reagents.E and F) K562Mpl‐mKO2 cells submitted to the Duolink reaction. White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane, while green arrow heads are pointing at positive Duolink reactions within the cytoplasm. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.
Figure 4HEL (A) and K562 (B) cells treated with non‐targeting (NT) or anti‐JAK2 siRNA analyzed by western blot.C) Confocal live cell (upper panel), or fixed cells (lower left panel), imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti‐JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells. After treatment with anti‐JAK2 siRNA, Mpl strongly colocalizes with the ER marker, calnexin. This colocalization in treated vs non‐treated cells is quantified and shown in Figure C lower right panel. Nuclei are labeled with Hoechst and scale bars = 5 µm.
Figure 5Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.
Figure 6Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 (A) and HEL (B) cells. Electron micrographs of K562 (C) and HEL (D) cells transiently expressing MplmKO2‐miniSOG. Dark precipitate indicates the presence of the miniSOG‐tagged Mpl protein after photo‐precipitation of DAB. White boxes indicate original location of higher magnification images with corresponding letters.E-G) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C, showing MplmKO2‐miniSOG dark precipitate into autophagic carrier vesicles (black arrows, Figure E). Osmiophilic reaction products also heavily stain mature AP in these preparations (black arrows, Figure G).H) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D, cells transiently expressing MplmKO2-miniSOG , showing different stage of formation of APs (black arrows numbered from stages 1-4). Scale bars = 5 µm (A-D) or 100 nm (E-H).
Figure 7A) Western blot analysis and quantification of streptavidin‐immunoprecipitated surface‐biotinylated proteins, probed with an anti‐Mpl antibodyB and C) Confocal images of K562 cells transiently expressing MplmOrange2. Cells in (B) are untreated. Cells in (C) were stimulated 60' with Tpo, washed and chased for 15 min before imaging.D) Quantification of plasma membrane fluorescence in resting and Tpo‐stimulated cells. Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background. A two‐tailed Student t‐test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal. Scale bars = 5 µm.E) Western blot analysis of protein phosphorylation in HEL, K562Mpl‐mKO2 and K562Mpl‐mOrange2 cells after Tpo stimulation (50 ng/mL Tpo, 10 min).
Figure 8A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl‐mKO2 cells after treatments for 60 min with Tpo, ionomycin (I), PMA, Mon or A23187.C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl‐mKO2 cells treated with Mon or A23187 for 15 h.D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng/mL for 30 min. The quantification of the blot is shown.E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3−MA (3−Methyladenine) or Rp.F) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R‐V5 6 h post‐transfection.G) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55-V5 or HA-STX5.
Figure 1B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.