EMBO/SourceData huggingface dataset:
figf3(a) Southern blot analysis of genomic DNA isolated from wild-type (WT, mAtg14L+/+) , mAtg14L+/- and knockout (KO, mAtg14L-/-) ES cells after digestion with PstI. Details of the probe are shown in Supplementary Information, Fig. S3.(c) The effect of Atg14L knockout on p62 turnover. WT, mAtg14L−/−, mAtg14L−/− + Atg14L, and mAtg5−/− ES cells were cultured in nutrient-rich (N) or starvation medium (S) for 3 h. Cell lysates were subjected to western blotting with the antibodies indicated.(d) Degradation of long-lived proteins is reduced in Atg14L knockout cells. WT, mAtg14L−/−, and mAtg5−/− ES cells were cultured in N or S medium or S medium with wortmannin (W, 100 nM) for 3 h and degradation of long-lived proteins was measured. The data are the mean ± s.d. of three independent experiments.(e) The effect of Atg14L knockout on LC3 turnover. WT, mAtg14L−/− and mAtg5−/− ES cells were cultured in N or S medium for 3 h in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1). Cell lysates were subjected to western blotting with the antibodies indicated. Full scans of the blots in b, c and e are available in Supplementary Information, Fig. S6.
figf4(a) The effect of Atg14L knockout (mAtg14−/−) on Atg16L dot formation. Wild-type (WT) and mAtg14−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with an anti-Atg16L antibody. Images of Atg16L staining (grey-scale) and Atg16L merged with Hoechst staining (colour) are shown.(c) Quantification of the number of Atg16L dots in a. The data are the mean ± s.d. of three independent experiments.(b) The effect of Atg14L knockout on LC3 dot formation. WT and mAtg14L−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with anti-LC3 antibody. Images of LC3 staining (grey-scale) and LC3 merged with Hoechst staining (colour) are shown. Scale bars, 20 μm. (d) Quantification of the number of LC3 dots in b. The data are the mean ± s.d. of three independent experiments. For each of the experiments in c and d at least 13 cells were counted.
figf5(a) Immunofluorescence using an anti-Atg16L antibody. A549 cells were transfected with adenovirus expressing GFP-Atg14L and cultured in nutrient-rich or starvation medium for 4 h. Cells were fixed and stained with an anti-Atg16L antibody.(b) Immunofluorescence with an anti-LC3 antibody. A549 cells were transfected with adenovirus expressing GFP-Atg14L and cultured in starvation medium for 4 h. Cells were fixed and stained with an anti-LC3 antibody. Scale bar, 10 μm.(c) Colocalization efficiency in a and b. The percentage of colocalization between Atg14L and Atg16L or LC3-positive dot is shown. For example, (Atg14L×Atg16L)/Atg16L means the ratio of colocalization of Atg14L and Atg16L puncta to all of the Atg16L-positive puncta. For colocalization between Atg14L and Atg16L the data are mean ± s.d. of 25 cells. For colocalization between Atg14L and LC3 the data are mean ± s.d. of 20 cells.d) Immunofluorescence with an anti-calnexin (ER) antibody. A549 cells transfected with adenovirus-based GFP-Atg14L. After 48 h, the cells were fixed and immunostained with calnexin (ER).Scale bar, 10 μm.(e) Immuno-EM analysis of GFP-Atg14L. A549 cells transfected with adenovirus-vector based GFP or GFP-Atg14L were subjected to immuno-EM analysis using an anti-GFP antibody. Scale bar, 500 nm. The arrows indicate the ER membrane and the arrowheads indicate the autophagosome membrane.
figf6(a) Double knockdown of Rubicon and Atg14L. A549 cells stably expressing GFP-LC3 were infected with adenovirus expressing either shRNA against Rubicon and Atg14L or control shRNA (luciferase). The image is available in Supplementary Information, Fig. S5a. The GFP-LC3 puncta were counted in nutrient-rich (N) or starvation (S) conditions. The data are mean ± s.d. of three independent experiments; at least 23 cells were counted for each experiment.(b) A549 cells were infected with adenovirus harbouring shRNA for Rubicon or control (luciferase). The cells were cultured in N or S medium in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) for 1-4 h. Cell lysates were subjected to western blotting with the antibodies indicated.(c) Quantification of Atg16L puncta in Rubicon knockdown cells. The images are available in Supplementary Information, Fig. S5c. The data are mean ± s.d., n = 60 (60 cells, sample of Medium: N, shRNA: Control), n = 68 (68 cells, sample of Medium: S, shRNA: Control), n = 78 (78 cells, sample of Medium: N, shRNA: Rubicon), n = 83 (83 cells sample of Medium: S, shRNA: Rubicon).d) Bulk degradation activity of Rubicon knockdown cells. The bulk degradation activity in control and Rubicon knockdown cells in nutrient-rich medium was measured. Wortmannin (W, 100 nM) was added as a negative control. The data are mean ± s.d. of three independent experiments.(e) A549 cells infected with adenovirus harbouring shRNA for Rubicon and control (luciferase) were cultured in N or S medium for 4 h and cell lysates were subjected to immunoblotting with the antibodies indicated. Full scans of the blots in b and e are available in Supplementary Information, Fig. S6.
figf7(a) A549 cells transfected with a GFP-Rubicon expression plasmid and were subjected to immunofluorescence with various organelle marker antibodies. Scale bar, 10 μm.(b) A549 cells stably expressing GFP-LC3 were subjected to Rubicon knockdown (KD) and then transfected with mStrawberry, mStrawberry-Rubicon or mStrawberry-Rubicon mutant lacking the Beclin 1 binding region (Δ393849). Cells with strong expression of mStrawberry-Rubicon showed an overexpression-dependent dominant-negative phenotype; therefore, we selected cells with modest expression. The number of GFP-LC3 puncta was counted and mean ± s.d. are shown. mStrawberry, n = 12 (12 cells); mStrawberry-Mm-Rubicon, n = 14 (14 cells); mStrawberry-Mm-RubiconΔ393-849, n = 13 (13 cells).
figf8(a) The maturation of autophagosomes is enhanced in Rubicon knockdown cells. A549 cells stably expressing GFP-LC3, with Rubicon or control shRNA, were incubated in nutrient-rich medium with or without the protease inhibitors (PI) E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) treatment for 4 h. The cells were stained with an anti-Lamp1 antibody (upper panels) and colocalization efficiency between GFP-LC3 and Lamp1 was counted (lower panel). The data are mean ± s.d. of 30 cells.(b). Electron microscopy analysis of Rubicon knockdown cells. Control (luciferase) or Rubicon shRNA A549 cells grown in nutrient-rich medium was subjected to electron microscopy analysis (left panels). The arrows indicate autophagosomes and the arrowheads indicate autolysosomes. Scale bar, 1 μm. Autophagosomes and autolysosomes were counted in 21 cells, and the mean ± s.d. are shown (right panel).(c) EGFR degradation in Rubicon knockdown cells. Control or Rubicon shRNA A549 cells were treated with EGF (200 ng ml−1) for the indicated periods and the lysates were subjected to western blotting with the antibodies indicated (left panel). The band intensity was measured in three independent experiments and the mean ± s.d. are shown (right panel).(d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP-Rubicon. After 24 h, EGF was added to cells expressing GFP-Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies.(d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP-Rubicon. After 24 h, EGF was added to cells expressing GFP-Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies.
Figure 1D) SDS-PAGE analysis of purified tubulin from brains of all mouse models used in the current study, stained with Coomassie Brilliant-Blue. Alpha- and β-tubulin are separated using a specific SDS-PAGE protocolE) Immunoblot analysis of all purified tubulin variants. The molecular weight in (D) and (E) is indicated in kDa.
Figure 2B) Representative IRM and TIRF microscopy images showing microtubule positions and Tau-meGFP intensities for different PTM variants (grey: wild type; blue: Ttll1-/-; yellow: Ttll7-/-; green: Ttll1-/-Ttll7-/-; pink: Atat1‑/‑). In the last column, false-coloured TIRF images highlight differences of Tau-meGFP intensities on different types of microtubules (Movie EV1). C) Quantification of integrated intensity of Tau-meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples. Single assays (Fig EV2A) of one experiment were combined after normalisation to the wild-type microtubule values (A.U. = arbitrary units). Each data point represents the normalised fluorescence intensity value of one microtubule. D) Summary quantification the three independent experiments shown in (C). Each point is a normalised mean of the triplicates, error bars represent standard deviation.E) Saturation curve of increasing concentration Tau-mCherry binding to GMPCPP wild-type and Ttll1-/-Ttll7-/- microtubules. Each data point is shown as mean ± standard deviation of the raw integrated intensity values (A.U. ×104) of three experiments with independently purified tubulin samples (Fig EV2D). Dissociation constants Kd and R2 coefficient from non-linear fits of each microtubule type are indicated below the plot.
Figure 3C) Decay curves of normalised microtubule length plotted against time upon incubation with katanin (A.U. = arbitrary units) for single microtubules highlighted by arrow heads with yellow borders in (B). D) Half-life times (seconds) of all microtubules from the assays of which representative images are displayed in (B) and representative decay curves are shown in (C). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown. Mann-Whitney test, p-values displayed.E) Analysis of normalised microtubule half-lifetimes from 14 independent experiments (Fig EV3). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.
Figure 4B) Kymographs showing single Kif5B-EGFP motor molecules moving on one single microtubule of known PTM state (Movie EV4). Horizontal scale bars 2 µm; vertical scale bars 5 C) Scatter plots showing the average velocities (µm/s) exhibited by single Kif5b-EGFP molecules on the different microtubule types (each data point represents one Kif5b-EGFP molecule). Data pooled from 29 independent experiments (Appendix Fig S2). Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.D,E) Survival probability (Kaplan-Meier plots, taking varying microtubule lengths into account; see Methods) of the interaction time (D) and run length (E) of Kif5b-EGFP molecules on different microtubule types. Data in (C,D,E) were pooled from individual experiments (Appendix Fig S2) without normalisation. The 50% probability values are show in the plots. Statistical analysis displayed in the tables below: hazard ratios with 95% confidence intervals pairing all data from different microtubule types (*pairs differing with significance level p<0.05).
Figure 1B: Hippocampal neurons transduced with GFP-TDP-25 constructs (DIV5+8) or GFP-TDP-43 (DIV5+4 due to higher toxicity) were sequentially extracted with RIPA buffer followed by 2% SDS buffer and analyzed via immuoblotting. Left panel shows representative immunoblot for GFP and loading control Calnexin. Right panel shows quantification of the ratio of the respective GFP densiometric signal in SDS extract to RIPA extract. Barplots showing mean ± SD from n=3 independent experiments. TDP-43 (0.039 ± 0.029, mean ± CI) vs TDP-25 wt (1.22 ± 0.296) vs TDP-25 mut (0.928 ± 0.564): F(2,6)=51.4, ***p=0.000168, η²=0.94. TDP-43 vs TDP-25 wt: ***p=1.67*10-4, TDP-43 vs TDP-25 mut: p=7.983*10-4, TDP-25 wt vs TDP-25 mut: p=0.1182,
Figure 2(A-D) Primary rat hippocampal neurons were transduced with GFP-TDP-25 variants, GFP-NPM1 or Htt97Q-GFP and analyzed by fluorescence recovery after photobleaching (FRAP) at DIV5+8, except Htt97Q-GFP which was analyzed at DIV5+6 to avoid excessive toxicity. Example images with indicated bleach regions (dashed circles) are shown (A, C). Scale bar = 1.5 µm. Resulting normalized FRAP curves (relative fluorescence intensity over time) with values representing means ± SD are shown in (B, D). (B) Comparison of the averaged recovery fraction of the last six timepoints obtained from three independent experiments. GFP-NPM1 (0.982 ± 0.019, mean ± CI, n=37 cells) vs GFP-TDP-25 wt (0.097 ± 0.013, n=35 cells) vs GFP-TDP-25 mut (0.085 ± 0.006, n=38 cells): H(1)=74.134, df=2, ***p<2.2*10-16, η²[H]=0.674, Kruskal-Wallis Test. GFP-NPM1 vs GFP-TDP-25 wt: ***p<2*10-16, GFP-NPM1 vs GFP-TDP-25 mut: p<2*10-16, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.11, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction. In (D), the recovery fraction averaged at >20 min timepoints was compared from four independent experiments. Htt97Q-GFP (0.094 ± 0.017, n=25 cells) vs GFP-TDP-25 wt (0.312 ± 0.041, n=23 cells) vs GFP-TDP-25 mut (0.271 ± 0.029, n=24 cells): H(1)=46.376, df=2, ***p=8.501*10-11, η²[H]=0.643, Kruskal-Wallis Test. Htt97Q-GFP vs GFP-TDP-25 wt ***p=1.9*10-11, Htt97Q-GFP vs GFP-TDP-25 mut ***p=4.3*10-12, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.12, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.
Figure 3E: Immunofluorescence of hippocampal neurons transduced with GFP, GFP-TDP-25 wild-type or mutant and treated with MG132 (10 µM, 14h, DIV5+8). Representative images of GFP-TDP-25 inclusions and diffuse cytoplasmic staining. Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei (omitted here for clarity) in MG132 treated cells compared to DMSO control. Counterstain to label the neuronal cytoskeleton (MAP2). Scale bar = 10 µm. Box plot as in Fig. 3C. GFP (1.23 ± 0.393, n=5 independent experiments) vs GFP-TDP-25 wt (2.11 ± 0.804, n=5) vs GFP-TDP-25 mut (2.36 ± 1.16, n=5): H(1)=7.34, df=2, p=0.02548, η²[H]=0.445, Kruskal-Wallis test. GFP vs GFP-TDP-25 wt: *p=0.048, GFP vs GFP-TDP-25 mut: *p=0.048, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.690, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.
Figure 1A, Reconstructed MSOT images (800 nm) without and with linear unmixing data in the neck and the upper and lower abdominal areas, showing the ROIs for the liver, kidney, iBAT, rpWAT, aorta, and SV. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value neck/upper abdominal/lower abdominal: Hb: 2.5/3.5/0.9; HbO2:2.9/4.9/2.5; Lipid: 24000/19000/13000) Scale bar: 4 mm.
Figure 2A, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value chow/HFD: Hb: 1.3/0.6; HbO2: 1.8/0.9; Lipid: 27000/27000). Scale bar: 4 mm.D, Tissue oxygenation (sO2) and total blood volume (TBV) results from liver, kidney, iBAT, and rpWAT, Each dot represents data from one animal, in total 8 animals (n = 8). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units.
Figure 3A, Oil-red-O staining of control (healthy) and steatotic liver. Scale bar: 100 µm.B, Reconstructed MSOT image (800 nm) with linear unmixing and difference value of lipid in lower abdominal section. Unmixing result: blue for Hb, red for HbO2, yellow for lipid, jet for 700 nm - 930 nm difference. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: Hb: 1.1/0.5; HbO2: 1.9/0.8; Lipid: 21000/24000; 700-900nm: 3000/3000) Scale bar: 4 mm.
Figure 4A, HE staining of livers with grade 0 - 3 steatosis. Scale bar: 100 µm.E, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value chow 1 month/ chow 2 month/ chow 3 month/HFD 1 month/HFD 2 month/HFD 3 month: Hb: 2.5/1.9/2.9/2.7/1.8/1.3; HbO2: 2.8/2.6/2.8/3.1/2.1/1.9; Lipid: 27000/19000/29000/ 32000/26000/27000). Scale bar: 4 mm.F, Longitudinal track of lipid in mouse livers. Each line represents data from one animal. Each data point is from a single measurement of each animal at each time point. All animals have grade 3 steatosis at the endpoint confirmed by histology.
Figure 5A, Reconstructed MSOT image (800 nm) with linear unmixing data of ICG from mice with and without steatosis. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: 0.07/0.02) Scale bar: 4 mm.E, Representative fluorescence microscopy and HE staining images of control and steatotic liver. Scale bar: 100 µm.
Figure 1(A) Ookinetes sandwiched between two hydrogel surfaces.(B) Percentage of ookinetes moving for more than one parasite length within the time of observation (5-10 min) if sandwiched between soft, medium (med) or stiff hydrogel or glass. Bars represent the mean and error bars the standard deviation of at least three independent experiments. (C) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B.(D) Percentage of motile ookinetes after 20-26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in (B). Shown is the average ± S.D. from at least two independent experiments. (E) Speed of motile ookinetes after 20-26 hours of incubation between hydrogels. Speed on glass was not analyzed (n.a.) due to the low fraction of motile ookinetes at this timepoint.(F) Migration patterns of ookinetes. Image: Overlay of tracked migration paths (black) and DIC image showing ookinetes at the end of the recorded image sequence. Graph: Quantitative analysis of the indicated different migration patterns on soft, medium (med) and stiff hydrogels.(G) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints.
Figure 2(A) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel.(B) Overlay of sporozoite tracks (black) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence. Scale bar, 20 µm.(C) Speed of WT sporozoites moving in the skin in vivo (Douglas et al, 2018b) and in soft hydrogels with small or large pores.(D, E) Speed of indicated mutant and control (Ctrl) parasites in hydrogels with small (D) and large (E) pores.(F, G) Speed of drug-treated and control parasites in hydrogels with small (F) and large (G) pores. Data information: Note the faster speed in gels with larger pores. Data from at least two independent experiments. Numbers above bars indicate the number of sporozoites analyzed. Significance determined by Mann-Whitney test. Box-and-whisker plots (C-G) depict the 25% quantile, median, 75% quantile and nearest observations within 1.5 times the interquartile range (whiskers). Outliers beyond this range are shown as black dots.
Figure 3(A) Percentage of motile sporozoites on endothelial cells (LSECs) versus glass. Shown is average ± S.D. from three independent experiments. Significance determined using an unpaired t-test.(B) Left: Merged fluorescence images showing sporozoites on cultured HFF cells. Actin filaments of HFF cells were stained with phalloidin (shown in red), nuclei were stained with Hoechst (blue) and sporozoites expressed GFP (green). Scale bar, 50 µm. Right: Maximum projection of sporozoites moving for 3 min on cultured HFF cells. Scale bar, 50 µm.(C) Percentage of motile sporozoites on confluent layers of endothelial cells (HUVEC), fibroblasts (HFF) and on glass. Shown is average ± S.D. from two independent experiments. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.(D) Sporozoite on planar uncoated hydrogels.(E) Overlay of sporozoite tracks (black) and DIC image showing sporozoites on a hydrogel. Scale bar, 50 µm.(F) Percentage of motile sporozoites on soft, medium (med) or stiff hydrogel or glass 30 min after activation with BSA. Shown is the average ± S.D. from at least three independent experiments. Significance determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.(G) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA. All sporozoites moving for more than one parasite length within the time of observation were analyzed, even if they stopped moving or detached during imaging. Significance determined by Kruskal-Wallis test with Dunn's Multiple Comparison test.(H) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses. Note that not all sporozoites analyzed in (G) complied with that requirement. To be able to analyze a high number of sporozoites, movies were taken 10 to 45 min after activation with BSA. Speed of sporozoites on soft hydrogels was not analyzed (n.a.) due to the low fraction of motile sporozoites on these hydrogels. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.
Figure 1C, Immunostaining of endogenous Trnp1 and DAPI in P19 cells and E14 cortical cells at 1 day in vitro (div).D, Co-IP with anti-GFP and WB with anti-Trnp1 antibodies in lysates from P19 cells transfected with plasmids expressing Trnp1-GFP fusion protein and/or Trnp1-IRES-RFP for 24h. Mw (molecular weights): 55 kDa (black line); 35 kDa (blue line); 25 kDa (red line). Input: 0,1%; co-IP: all immunoprecipitated proteins.E, Representative phase contrast images of in vitro phase separation of the recombinant proteins YFP (upper row) or Trnp1 (lower row) at the indicated concentration in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT with the crowding agent dextran or RNA when depicted.