EMBO/SourceData huggingface dataset:
figf5(a) Schematic presentation of NTAP-tagged (NT-) and HM-tagged (HM-) hiw transgenes encoding full-length, mutated or truncated Hiw proteins. Hiw functional domains are marked with colored boxes. The positions of amino-acid substitution in NT-HiwΔRING and the added amino acid residue necessary to maintain the reading frame in NT-Hiw-RCC1 and NT-Hiw-PHR are indicated. The right column summarizes the ability of the given hiw transgene to rescue the synaptic overgrowth phenotype in hiw mutants, to cause dominant negative overgrowth phenotype when expressed in wild-type background and to bind to Rae1. (b) Western blot analysis of fly brain lysate using antibody to TAP (PAP, Sigma) or Myc revealed the expression of wild-type and mutant hiw transgenes in predicted size.(c) Representative confocal images of muscle 4 synapses stained for DVGLUT (green) and FasII (red) in wild-type (+/hiwND8; +/+; GS-elav-Gal4/+), NT-Hiw overexpression (+/hiwND8; UAS-NT-hiw/+; GS-elav-Gal4/+), NT-Hiw-NT overexpression (+/hiwND8; UAS-NT-hiw-NT/+; GS-elav-Gal4/+), NT-Hiw-CT1000 overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT1000), NT-Hiw-PHR overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-PHR), HM-Hiw-HindIII overexpression (+/hiwND8; UAS-HW-hiw-HindIII/+; GS-elav-Gal4/+), NT-Hiw-RCC1 overexpression (NT-hiw-RCC1/hiwND8; +/+; GS-elav-Gal4/+), or NT-Hiw-CT overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT) 3rd larval NMJs. Overexpression is indicated by an up arrow. Scale bar represents 10 μm. (d) Quantification of the number of boutons at muscle 4 NMJs in wild-type larvae or larvae overexpressing wild-type or mutant hiw transgenes (n = 23, 24, 26, 27, 26, 21, 25 and 27, respectively; *P 0.001). Error bars denote s.e.m.
figf6(a) Indicated NTAP-tagged hiw transgenes (described in Fig. 5) and a TAP only transgene were expressed in neurons under the control of the BG380-Gal4 driver. Larval brain lysates from each sample were subject to IgG pulldown. Both the inputs and the IgG pulldown complexes were analyzed by western blot using antibody to Rae1. Rae1 was present in all of the pulldown complexes except for those from TAP only, NT-Hiw-CT1000 and NT-Hiw-NT. Full-length blots are presented in Supplementary Figure 10. (b) Quantification of the interaction between various Hiw transgenic proteins and Rae1. Rae1 intensities in TAP pulldown blots were normalized to both the intensities and molecular weights of the corresponding TAP pulldown proteins.(c) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type (BG380/Y;+/+; +/+), NT-Hiw-CT and GAP-GFP (BG380/Y; UAS-GAP-GFP/+; UAS-NT-hiw-CT/+), and NT-Hiw-CT and Rae1 (BG380/Y; +/+; UAS-NT-hiw-CT/UAS-GFP-Rae1) larvae. Scale bar represents 10 μm. (d) Quantification of number of boutons at muscle 4 NMJs in wild-type, NT-Hiw-CT overexpression (BG380/Y; +/+; UAS-NT-hiw-CT/+), NT-Hiw-CT and GAP-GFP, and NT-Hiw-CT and Rae1 (n = 24, 27, 32 and 26, respectively) larvae. Error bars denote s.e.m.; *P 0.001.
figf7(a) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene. Quantification of the number of boutons in wild-type, Rae1EX28, Rae1EX28; Hiw rescue (Res) (Rae1EX28; elav-Gal4/UAS-GFP-hiw), hiwΔN and hiwΔN; Rae1 rescue (hiwΔN; elav-Gal4/UAS-NTAP-Rae1) larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 7 and 11, respectively). (b) Representative confocal images of wild-type (BG380-Gal4/+; +/+; UAS-GFP-hiw/+) or Rae1 mutant (BG380-Gal4/+; Rae1EX28; UAS-GFP-hiw/+) ventral ganglions with UAS-GFP-hiw trangene expressed in neurons. The larvae were stained with antibodies to horseradish peroxidase (HRP, red) and GFP (green). Scale bar represents 10 μm.(c) Western blots of total proteins extracted from wild-type, hiwΔN, Rae1EX28 and Rae1EX28 rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) larval brains probed with indicated antibodies. Full-length blots are presented in Supplementary Figure 11.(d) Western blot analysis on total proteins extracted from hiwΔN, wild-type (C155-Gal4/+) and Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+) larval brains. Full-length blots are presented in Supplementary Figure 12. (e) Quantification of Hiw protein levels in the wild-type and the Rae1 expression larval brains (*P 0.001, based on seven independent experiments). Arrows indicate a 38-kDa endogenous Rae1 protein and the arrowheads indicate the 58-kDa transgenic NTAP-Rae1 protein. Error bars denote s.e.m.
figf8(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ wandering larvae. Scale bar represents 10 μm. (b) Quantification of the number of boutons in wild-type, atg1/+, atg2/+, atg18/+, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 12, 12, 16, 12 and 21, respectively).(c) Western blot analysis on total proteins extracted from wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains. The neural form of β-catenin (82 kDa, see Online Methods) was used as an internal control of neuronal proteins. Full-length blots are presented in Supplementary Figure 13. (d) Quantification of Hiw protein levels in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains (based on six independent experiments).(e) Representative confocal images of muscle 4synapses stained for both DVGLUT (green) and FasII (red) in wandering larvae of wild-type (C155-Gal4/+), Atg1Gap-GFP (C155-Gal4/+; UAS-Gap-GFP/+; UAS-Atg16B/+), and Atg1NTAP-Rae1 (C155-Gal4/+; UAS-NTAP-Rae1/+; UAS-Atg1/+). Scale bar represents 10 μm. (f) Quantification of the number of boutons at muscle 4synapses in wild-type, Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+), Atg1Gap-GFP and Atg1NTAP-Rae1 wandering larvae (segment A2-4, n = 28, 27, 33 and 34, respectively). Error bars denote s.e.m. *P 0.001, **P 0.05.
figf1a, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*P 0.02, **P 0.002, n = 3).b, TG levels in vector-infected (VEC) and siAtg5 cells in RM, OL or in MCDM (*P  0.001, n = 5). OL values are in mM.c, TG levels in wild-type (WT) or Atg5 knockout mice embryonic fibroblasts (Atg5-/-) (*P 0.01 or **P 0.0001, n = 5).df, Cells from a and b stained with BODIPY 493/503 (d). Nuclei are highlighted with 4,6-diamidino-2-phenylindole (DAPI; d). Error bars, s.e.m.df, Cells from a and b stained with oil red O (e) or visualized by electron microscopy (f). Right: quantifications of LD number and size (*P  0.01, **P  0.001 with untreated wild-type cells; #P  0.01, # #P  0.001 with cells in RM; n = 46).
figf2a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 34).c, d, Rates of TG decay in OL (c) and MCDM (d) (*P  0.05, **P  0.01, ***P  0.001 as compared to VEC cells, n = 37).e, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*P  0.00001 with DMSO-treated cells, #P  0.003 with 3MA-treated cells, n = 6). Error bars, s.e.m.f, Co-localization of BODIPY 493/503 (green) with LAMP1 (red) or LC3 (red) in hepatocytes in MCDM.g, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.
figf3a, Electron micrographs of cultured hepatocytes. dm, double-membrane cytosolic vesicles. Arrows indicate membranes in LDs. Insets show the double membrane in nucleus (N), lipid-containing vesicles (L) and mitochondria (mit), but not in LDs.b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P  0.01, **P  0.001 with cells in RM, n = 46). Left: representative examples. Error bars, s.e.m.
figf4a, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice. IκB, inhibitor of the nuclear factor of kappa light polypeptide gene enhancer; GPDH, glyceraldehyde-3-phosphate dehydrogenase.b, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P  0.001, **P  0.0001, n = 46). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid)c, Immunoblots of Hom, AV and lysosomes (Lys). L-1, LAMP1.d, Immunoblots of liver Hom and LDs from HFD-fed mice.e, Liver homogenate immunoblots. PDI, protein disulphide isomerase.f, Total hepatic TG and cholesterol content (*P 0.01, **P 0.00001, n = 817).g, Hepatic TG concentration (*P  0.05, n = 3).h, Percentage of cellular cholesterol in lysosomes (*P  0.02, n = 4).i, Immunoblots of homogenates and isolated LDs. Error bars, s.e.m.
f1The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).
f2Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO miceMEF (a) ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null mice MEF (b) ß-Actin was used as loading control.Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null neurons (d); ß-Actin was used as loading control.(e) Western blot against PINK1, GLUT1, GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples; α-Tubulin was used as loading control. Only representative western blots are shown per condition; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-Actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT. Thus, in all cases, the WT values were considered=1.00. *P0.05 versus the corresponding WT (Student's t-test; n=3-4 independent experiments).
f3(a) RT-qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α, or siControl (100 nM), after 72 h.(b) siHif1α effectively knockdowns expressed Hif1α cDNA WT, but not a Hif1α cDNA silent mutant form refractory to siHif1α (Hif1α cDNA REFR.) in mice MEF.(c) RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α. ß-Actin mRNA was used for normalization, and all data are expressed as the change relative to WT-siControl.(d) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α; ß-actin was used as loading control. As shown, siHif1α partially prevented the increase in GLUT1, HK2 and GAPDH protein abundances.The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α.siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information.
f4(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF.(c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control.d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl.Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF(g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal.(h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).
f5(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).(c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α).(e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3-4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.
f6a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons.(b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons.(c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).
Figure 1A-E Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) zygotene (Zyg.), (B) pachytene (Pac.), (C) diplotene (Dip.), (D) late diplotene (L. dip.), and (E) prometaphase I (ProM I).F-H Double-immunolabeling of Sororin (green) and SYCP1 (pseudocoloured in pink) on spread spermatocytes at (F) zygotene, (G) pachytene, and (H) diplotene.I-K Double-immunolabeling of Sororin (green) and HORMAD1 (pseudocoloured in blue) on spread spermatocytes at (I) zygotene, (J) pachytene, and (K) diplotene. The sex bivalent (XY) and their pseudoautosomal regions (arrows), as well as the round body (arrowheads) are indicated. Scale bar: 10 µm.
Figure 2A,C Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) early zygotene (E. zyg.) and (C) late zygotene (L. zyg.).B,D Double-immunolabeling of SMC3 (pseudocoloured in cyan) and SYCP3 (red) on spread spermatocytes at (B) early zygotene and (D) late zygotene.E Double-immunolabeling of Sororin (green) and REC8-myc (pseudocoloured in purple) on a spread mid zygotene spermatocyte. The sex univalents (X,Y) are indicated. Scale bar: 10 µm.
Figure 1C-E. Logistic statistical model based on the (C) single cell sequencing, (D) ImageStream (E) analysis or automated FISH of RPE-1 cells in untreated condition (blue circles) or treated with Auxin for 48 h (red squares). Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated (blue line) or Auxin-treated (red line) condition. Red asterisks (IAA) and Blue (Untreated) indicate significance over the respective mean using a binomial test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.F. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent a statistically significant difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. * = centromere 4 probe with image stream was reported to lead to non-specific signal (Worrall et al, 2018). Im. St. = image stream; Seq = single cell sequencing.
Figure 2B-C. (B) Micronuclei frequency formation by live cell imaging in untreated (NT) and Auxin-treated (IAA) condition. (C) Bar graph represents the type of chromosome mis-segregation (independently of the dCas9 signal) observed in the indicated conditions, with (IAA) or without (NT) auxin, in the cell lines expressing dCas9 mScarlet-I and sgRNA targeting chromosome 1 or 3. Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together (n = 45 - 149 cells).D. Representative live cell imaging of RPE-1 cells dCas9 mScarlet-I with sgRNA targeting chromosome 1 (red dots) starting from metaphase and showing example of correct (untreated) or mis-aligned chromosome (auxin) leading to the formation of a micronucleus. Yellow arrows mark mis-aligned- and micronucleus-containing chromosome 1. Cells were imaged every five minutes. The numbers indicate the time point at which the presented images were taken. Cells were fixed at the end of the movie to detect Cas9 with an antibody. Scale bar represents 10 µm.E. Bar graph representing the proportion of chromosome mis-segregation observed in both cell lines after auxin addition. Only movies in which the dCas9 mScarlet-I dots were clearly visible during the whole division were taken into consideration for the analysis (N = 6 - 10 cells).F. Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP-A depletion (28 h auxin). Chromosomes are stained using whole (X) or centromeric (6) FISH probes. Yellow arrows mark lagging chromosomes. Scale bar represents 5 µm. G-H. Bars represent the (G) proportion of chromosome mis-segregation and (H) the type of the different chromosome mis-segregations observed. Error bars represent the SEM of two replicates. n > 66 mis-segregation events. Paired t test lagging chromosomes vs mis-alignment chromosomes, *p = 0.0275.
Figure 3Bar plot showing the mean of (B) centromere length (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of the lengthBar plot showing the mean of (C) CENP-B boxes counts (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of counts of different HOR arrays CENP-B boxes counts are normalized to the average number of mapped reads in each replicate.D. Schematic using representative images of the mFISH labeling followed by CENP-B box FISH method used to identify and quantify centromere specific CENP-B boxes signal in (E). Scale bar represents 10 µm.E. Box and whisker plots of normalized CENP-B boxes intensity over the mean on metaphase spread from three independent experiments (n>50 cells) using the Tukey plot. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. **p < 0.01; ***p < 0.001; ****p < 0.0001..Scatter plot showing a non-significant or significant negative correlation between the mean of (F) centromere length (n = 4) and mis-segregation rate (from Fig. 1F) (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.Scatter plot showing a non-significant or significant negative correlation between the mean of (G) centromere CENP-B boxes FISH signal (n>50 cells) and mis-segregation rate, respectively (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.