EMBO/SourceData huggingface dataset:
Figure 1(B) Representative emission spectra of mixtures containing ECFP-D4H, mVenus-Lact-C2, and liposomes containing indicated compositions of lipids. Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%) and that FRET signal was decreased when sphingomyelin (SM) (25%) was additionally incorporated into the liposomes. Asterisks indicate positions of emission maximum of ECFP (*477 nm) and FRET (**525 nm). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; SM, sphingomyelin (N-oleoyl-D-erythro-sphingosylphosphorylcholine). (C) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm (FRET/ECFP) (see Materials and Methods) from mixtures as shown in (D) (mean ± SEM, n = 4 independent experiments; Dunnett's multiple comparisons test, **P < 0.0001).(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing mCherry-GRAM1b. Cells were pre-incubated with either control imaging buffer (for control) or imaging buffer containing purified ECFP-D4H proteins (3 µM) (for masking accessible PM cholesterol) for 30 min at 37°C and imaged with the same buffer conditions. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 32 cells (Control), n = 35 cells (Pre-incubation w/ ECFP-D4H); data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].(E) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing EGFP-GRAM1b together with either mCherry for control or mCherry-Lact-C2 for PS masking. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 53 cells (mCherry), n = 32 cells (mCherry-Lact-C2 OE); data are pooled from four independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].
Figure 2(C-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (K161A, R191A, and K161A/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 3 independent experiments for all conditions in phosphatidylserine (PS) sensitivity, co-incidence detection, and anionic lipid selectivity assays; n = 12 independent experiments for WT, n = 3 independent experiments for K161A and R191A, and n = 6 independent experiments for K161A/R191A in the cholesterol sensitivity assay). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; PA, phosphatidic acid (1,2-dioleoyl-sn-glycero-3-phosphate); PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI, L-α-phosphatidylinositol.
Figure 4(B) Liposome tethering by purified GRAMD1b proteins. Left: Representative time course of liposome tethering, as assessed by turbidity of mixtures containing both LPM and LER together with either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Turbidity, as assessed by measuring absorbance values at 405 nm, reflects clustering of liposomes due to tethering of LPM and LER. Right: Values of turbidity corresponding to the end of the experiment as indicated by the arrow (mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for buffer alone, R189W, and RW/RA; Dunnett's multiple comparisons test, **P < 0.0001)(C) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer (FRET) between DHE and DNS-PE in LPM. A series of liposomes with different DHE mol% were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred (see Appendix Fig S2D and Materials and Methods). Right: Values of DHE transfer rate of GRAMD1b (WT), GRAMD1b (R189W), and GRAMD1b (RW/RA), as estimated by the FRET-based lipid transfer assay [mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for R189W and RW/RA
Figure 1A. PCR analysis of the human H49K variant of the ATPIF1 and rtTA constructs in wild-type (wt), ACTA1-rtTA (T), ATPIF1H49K-TRE (H) or double transgenic (T/H) mice.(B WB expression of the human (h) ATPIF1 protein in Skm (B extracts. hATPIF1 is only expressed in Skm from ATPIF1H49K|T/H mice. HSP60 are shown as loading controls. n= 3 mice/genotype.(C) Immunofluorescent staining of wt and ATPIF1H49K|T/H hindlimb muscle slices with hATPIF1 and βF1 antibodies. Images are representative of 3 mice/genotype, 5 fields/mouse. hATPIF1 H49K is only expressed in T/H mice.D) WB expression of the human + mouse (m+h) ATPIF1 protein in Skm D), brain, liver, and WAT (D) extracts. hATPIF1 is only expressed in Skm from ATPIF1H49K|T/H mice. α-tubulin are shown as loading controls. n= 3 mice/genotype.E. Polarographic profiles of isolated mitochondria from wt (lower trace) and ATPIF1H49K|T/H (upper trace) animals. Histograms show a reduction in state 3 respiration consistent with the inhibition of ATP synthase in ATPIF1H49K|T/H mice. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A.(F, G) Quantitative proteomic analysis (iTRAQ) of hindlimb Skm from wt and LowOXPHOS mice. The pie charts (F) and volcano plot (G) present the upregulated (log2[LowOXPHOS/wt]≥1, red) or downregulated (log2[LowOXPHOS/wt]≤-1, blue) proteins. A (-)log p value of >4 was considered statistically significant. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.H. Cytoscape representation of the most upregulated proteins in Skm from LowOXPHOS mice. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.K. Skm iTRAQ ratio of proteins from BCAA catabolism. Higher intensities of red or blue colors represent higher or lower LowOXPHOS/wt expression ratios, respectively. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.
Figure 5A. Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD (wt, n= 4; LowOXPHOS, n= 4; wt + HFD, n= 4; LowOXPHOS + HFD, n= 4).B. Polarographic profiles of isolated mitochondria from wt (lower trace) and LowOXPHOS (upper trace) animals using succinate (left graph) or palmitoyl-carnitine (right graph) as a substrate. Quantification of maximal respiration in the right histogram. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.(C-E) 2D-PAGE of Skm extracts (C, D) and acetylated proteins (E) from wt (n= 4) and LowOXPHOS (n= 3) mice. The isoelectric point (pI) of the CII subunits SDHB (C) and SDHA (D) was calculated by protein migration in pH 3-10 NL strips.F. Immunocapture (IP) of SDHA blotted with anti-acetyl-K antibody (upper panel) and of acetylated proteins blotted with anti-SDHA antibody (middle panel) in Skm isolated mitochondria from wt (n= 3) and LowOXPHOS (n= 3) mice.I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each ETC inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.J. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each CII inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.K. Representative blue native immunoblots (BN) and clear native in-gel activity (CN) of Skm mitochondrial membrane proteins from wt (n= 3) and LowOXPHOS (n= 3) 2-month-old mice. The migration of the respiratory complexes/supercomplexes CI-V is indicated. Subassembly of CI (with no activity, suggesting degradation) was observed in LowOXPHOS mice. VDAC is shown as a loading control.
Figure 6(A, B) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes. Palmitate is used as a substrate. A total of 41 hits were identified. Maximum respiration (Max R, % of untreated cells) of compounds categorized by their therapeutic use (B).(C, D) The schematic illustrates the screening process of the 41 hits on FFA β-oxidation using 9,103H(N) palmitic acid as a substrate in C2C12 myocytes. Ten enhancers were identified. D. Correlation between the maximum respiration (x) and palmitate β-oxidation (y). Colors identify classes of pharmaceuticals as in B. Edaravone was selected as a hit.E. Representative respiratory profile of C2C12 myocytes treated (green trace) or not treated (black trace) with 2 µM edaravone. The CPT1 inhibitor etomoxir was used as a negative control. OCR, oxygen consumption rate; OL, oligomycin; DNP, 2,4-dinitrophenol; Rot, rotenone; Ant A, antimycin A. Quantification in right histogram. Bars are the mean ± s.e.m. of 12 replicas/condition. Data information ;0.05 when compared to wt by ANOVA and Student's t-test.(F, G) FFA β-oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids (F) and in primary myotubes from wt and LowOXPHOS mice (G), treated (green bars) or not with 2 µM edaravone. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: *p <0.05 when compared to wt by ANOVA and Student's t-test.
Figure 1B Interaction of Atg18 with retromer subunits. Genomically tagged yomCherry-fusions of each retromer subunit were expressed in SEY6210 atg18∆, atg21∆, hsv2∆ cells, together with a plasmid expressing Atg18HA3yEGFP. Cell lysates were subjected to immunoprecipitation using RFP-Trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins.D Atg18-Vps35 interaction. Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed by SDS-PAGE and Western blotting using the antibodies indicated in brackets. E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled-down Vps35yomCherry. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** P < 0.001.F Atg18-Vps26 interaction. Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT, SEY6210 vps35∆ or SEY6210 vps29∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed as in D. G Proteins from F were quantified as in E. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** p < 0.001).
Figure 2B Salt-induced vacuole fission. The indicated cells were logarithmically grown in YPD and stained with FM4-64. Vacuole morphology was imaged as in A, before and after a mild salt shock with 0.5 M of NaCl for 15 min. The look-up table has been inverted to allow better representation of the clusters of extremely small vacuolar fragments in the SNX mutants. Scale bar: 5 µm. C The number of vacuoles per cell was quantified for the samples from B. n=3 experiments with at least 100 cells per condition were evaluated; Error bars represent the SEM.
Figure 3A Vps17 labilizes the Atg18-Vps26 interaction. Wildtype or vps17∆ cells expressing ATG18HA3-yEGFP from a centromeric plasmid were logarithmically grown in YPD. Genomically tagged Vps26yomCherry was pulled down from whole-cell extracts and analyzed for associated Atg18HA3-yEGFP by SDS-PAGE and Western blotting. Glucose-6-phosphate dehydrogenase (Zwf1) serves as a loading control. The intensity of the interacting Atg18HA3-yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry. Values of the wildtype interaction were used as the reference and set to 1. n = 4 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM, ** p < 0.01)B Epistasis of ATG18 and retromer genes concerning vacuolar morphology. The indicated cells were logarithmically grown in YPD medium, stained with FM4-64 and calcofluor white C Quantification of vacuole morphology. The number of vacuoles per cell was measured in the cells from B. The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles (n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. ** p < 0.01
Figure 4B Purified Atg18 and CSC-GFP were mixed in a 1:1 ratio, incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets.
Figure 5C Pull-down. Cells (SEY6210 atg18∆, atg21∆) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3-yEGFP variants were logarithmically grown in SC-URA media. Vps26yomCherry was pulled down from whole-cell extracts with RFP-trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins. Bands were quantified on a LICOR fluorescence imager. Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry. Vps1 served as a loading control. n = 3 independent replicates. Data were subjected to an unpaired t-test. Bars represent the mean and errors bars the SEM, *** P < 0.001).
Figure 6A, Effect of Atg18T56E on salt-induced vacuole fission. Cells expressing Atg18WTHA3-yEGFP or Atg18T56E-HA3-yEGFP from centromeric plasmids in a SEY6210 atg18∆, atg21∆ strain were logarithmically grown in SD-URA medium. They were stained and imaged before and after the induction of vacuole fission by a short salt shock Calcofluor white-stained cell walls are only represented in the merge (blue). Scale bar: 5 µm. B, Quantification of the number of vacuoles per cell from A, n = 3 biological experiments with at least 100 cells per condition were scored; bars represent the mean and error bars the SEM. C, Epistasis of atg18T56E over a vps17∆ mutation. The indicated variants of Atg18-HA3-yEGFP were expressed from plasmids in a SEY6210 atg18∆, atg21∆, vps17∆ strain. Cells were logarithmically grown in SD-URA and imaged Scale bar: 5 µm. D, Quantification of the experiments from C, n = 3 independent experiments with at least 100 cells per condition scored; bars represent the mean and error bars the SEM .
Figure 7A. Atg18-dependent recruitment of CSC-GFP to small liposomes. SUVs were incubated (10 min, 25°C) with purified CSC (1.5 µM) alone or in combination with the indicated recombinant Atg18 variants (1.5 µM). The vesicles were sedimented by centrifugation and supernatants (Sup.) and pellets were analyzed by SDS-PAGE and Coomassie staining. B. Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner. n = 3 independent experiments. Bars represent the mean and error bars the SEM, ** p < 0.01; * p < 0.05. C. Quantification as in B, but for the Atg18 variants.
Figure 9A Colocalization of WIPI1 with Vps26A. The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells, from which endogenous WIPI1 had been deleted (WIPI1-KO). The cells were analyzed by confocal microscopy. Scale bars: 10 μm. Insets show enlargements of the outlined areas. B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders' colocalization coefficient M2. The red line indicates the mean; n = 120 cells per condition, pooled from three independent experiments. P-values were calculated by t-test (analysis performed with 99% confidence ***p < 0.0001). C Expression levels of WIPI1mCherry variants. Lysates (50 μg of protein per sample) from the cells in A were analyzed by SDS-PAGE and Western blot against WIPI1 and tubulin.D Depletion of VPS35. HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 (VPS35 KD) or a control siRNA pool. Lysates (50 μg per sample) from the cells were analyzed by SDS-PAGE and Western blot against Vps35 and tubulin. E Blots from D were quantified on a LICOR Odyssey fluorescence imager. n=3. Red lines indicate the means, n = 3 independent experiments, using a Welch's t-test statistical analysis. Bars represent the mean and error bars the SD.F Cells from D were analyzed by confocal microscopy 18 h after transfection. Scale bar: 10 μm. G Quantification of tubule length in the cells from F. Data are means ± SD. of n=210 tubules per condition, from three independent experiments. p values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.
Figure 10A, Tf recycling. WIPI1-KO cells were transfected with WIPI1WT-eGFP, WIPI1S69E-eGFP or WIPI1S69A-eGFP for 18h. Then, they were serum-starved for 1 h, loaded with Alexa Fluor 568-conjugated Tf, chased at 37°C for 1 h without labeled Tf, and analyzed by confocal microscopy. Scale bar: 10 μm. White dashed lines delineate the circumference of the cells. B, Quantification of Tf-fluorescence in the cells from a that expressed WIPI1 variants. Total cell fluorescence was integrated and corrected for background fluorescence. Mean values ± SD are shown. n = 3 independent experiments with a total of 150 cells analyzed per condition. P values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.
Figure 1A. Confocal microscopy analysis showing the localization of endogenous Rab22a (green) and different endocytic markers (red): early endosomal marker EEA1, late endosome/lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state. The means ± SEM of the Pearson correlation coefficients (Pcc) from Rab22a and EEA1 (0.4317 ± 0.05101), Lamp1 (0.8412 ± 0.03562) and TfR (0.6409 ± 0.02999) were estimated from 15 images analyzed for each marker. Scale bars: 5 µm.B. Colocalization of endogenous Rab22a (red) and EEA1 (magenta) around endosomes containing fluorescent soluble OVA (OVA-FITC, green) after 30 min of internalization by BMDCs. More than 60% of the endosomes were positive for Rab22a and EEA1. The indicated boxes are shown at higher magnification in the insets. Scale bars: 5 µm.C. IF detection of endogenous Rab22a (green) and Lamp1 (red) after 1 h of phagocytosis of 3 µm latex beads (LB) in BMDCs. Around 50% of the phagosomes were double positive for Rab22a and Lamp1. Asterisks indicate the LB. Scale bars: 5 µm.D. BMDCs were incubated with 3 μm magnetic beads for 15 min at 37°C and chased for 0 or 45 min. The panel shows immunoblotting of purified phagosomes and the total cell lysates (TCL) analyzed for Lamp1 and Rab22a. A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL, respectively. The blot is representative of three independent experiments.
Figure 2A. Immunoblotting and densitometry quantification of Rab22a in JAWS-IIDCs infected with lentiviruses encoding a random sequence (Scramble) and a shRNA specific for silencing Rab22a (Rab22a KD). Data are representative of at least three independent experiments.B. RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A. Data show mean ± SEM of triplicate values and are representative of two independent experiments. ** P = 0.0047.C. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD JAWS-II DCs. Nuclei stained with DAPI and DIC images are shown on the left. Overlay is shown in the right panels. Scale bars: 5 µm. Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments.D. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (total) Scramble and Rab22a KD JAWS-II cells. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. P = 0.1082 (ns) and ***P = 0.0003.E. MHC-I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. ***P < 0.001 at 3 h and *P < 0.05 at 5 h between both DC types.F. MHC-I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. *P < 0.05 at 10 min and **P < 0.01 at 20 and 40 min.G. The transferrin (Tfn) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. P > 0.05 (ns) at 10, 20 and 40 min.