EMBO/SourceData huggingface dataset:
Figure 4(A) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT (in green) and fluorescent markers of mitochondria (MitoDsRed, in red) and ER (Sec61β-BFP, in blue), and treated with DAPT to prevent C99 cleavage. Note the different distribution of C99WT or C99MUT forms. Scale bar = 10 µm. Insets show 5X amplifications of individual (C99 in green, and mitochondria in red) and merged images. Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize. Upon thresholding each channel, a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask, so the positive pixels found in both channels are shown in black.(B) Quantification of the degree of colocalization of C99WT or C99MUT forms with mitochondria or ER measured by ImageJ. Note the significantly decreased colocalization between C99MUT and mitochondria, when compared to that of C99WT. Unpaired t-test. (20-30 cells/condition from 3 independent experiments and 4-5 images/experiment; * p<0.05; n.s., not significant).(C) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT and ER and mitochondria markers as in (A) (upper panel). Individual zoomed single channels (middle panel) as well as areas of ER-mitochondria colocalization (bottom panel) are shown for each condition. Scale bar = 10 µm.(D) Quantification of ER-mitochondria colocalization per cell analyzed. Upon thresholding each channel, a mask for ER was generated and the mitochondria channel was superimposed on the ER mask, so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask. The number of cells analyzed is indicated in white text within each column. Levels of ER-mitochondria colocalization in APP-WT cells are shown for reference as a dashed line. EV, empty vector. One-way ANOVA (n=3 independent experiments and 5-8 images/experiment; * p< 0.05).
Figure 5(B) Representative immunoblot to reveal C99 levels in total homogenate (TH) and MAM fractions of APP-DKO cells expressing C99WT or C99MUT before (input) and after cholesterol pull-down. Acsl4 was used as a MAM marker. Streptavidin-HRP was used to detect total biotinylation (biotin conjugated to PhotoClick cholesterol). (C) Quantification of pulled-down C99 levels versus input from the experiment in B. Two-way repeated measures ANOVA (Fraction, mutation) (n=5; * p< 0.05, ** p< 0.01). (D) Immunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by western blot (shown in AppendixFig S1E).(E) Quantification of free cholesterol levels (FC) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol%). Graphs represent percentage over controls. Dashed line represents control levels. One-way ANOVA. (n=4; * p< 0.05)(F-G) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H-cholesterol and subsequent analysis of radiolabel incorporation. The dashed line indicates control levels. Graphs represent fold change over controls. Treatment with Sandoz 58-035, a specific ACAT1 inhibitor, caused a ~95% reduction in cholesterol esterification. EV, empty vector. One-sample t-test. (n=3-4; * p< 0.05).(H) Ratio of cholesteryl esters:free cholesterol (CE:FC) in the indicated cells.
Figure 1(A) Fed rat liver lysosomes were extracted with 1% of the indicated detergents and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were subjected to SDS-PAGE and immunoblotted for LAMP‐2A and flotillin‐1. Stars indicate the aliquots of the gradient of translucent appearance (highly enriched in lipids).(B) The distribution of LAMP‐2A through the sucrose gradient after extraction with 1% Triton X‐114 was calculated from the densitometric quantification of immunoblots as the one shown here. Values are expressed as percentage of the total lysosomal levels and are mean+s.e. of four experiments.
Figure 2(A) Lysosomes from livers of fed (Fed) or 24 h starved rats (Stv) were extracted with 1% Triton X‐114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent‐resistant (DR) fraction, and anything below this fraction was pooled, as the solubilized fraction (Sol) (B). Lanes 5-6 are one‐tenth of the amount of lysosomes loaded in the gradient.(C) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11-12; heavy: aliquots 13-14).(D) Fed ratliverlysosomes were incubated with MBCD or filipin, as described under Material and methods, and then processed as in (C). The mean value of the densitometric quantification of the LAMP‐ from two experiments is shown at the bottom in (C) and (D).
Figure 3(A) Lysosomalmembranes from fed ratlivers and their corresponding 1% Triton X‐114‐resistant regions, isolated as described under Material and methods, were subjected to one (left)‐ or two‐dimensional (right) electrophoresis and stained with Sypro Ruby.(B) Immunoblot for the indicated proteins of the detergent resistant (DR), intermediate (Int) and solubilized (Sol) fractions of lysosomal membranes obtained as in Figure 2.
Figure 4LAMP‐2A localizes in cholesterol‐ and GM‐1‐enriched intracellular membrane domains. Mouse fibroblasts grown on coverslips in the presence (+) or absence (−) of serum were fixed and labeled with filipin (red) (A) or incubated with Texas‐Red‐cholera toxin B subunit to label GM‐1‐enriched membrane regions (red) and then fixed.(B) Both groups of cells were then subjected to indirect immunofluorescence for LAMP‐2A (green). Where indicated, cells were treated with MBCD (25 mM) for 30 min before fixation. The merged images of both fluorophores are shown on the right. Insets show vesicles at higher magnification. Bar: 10 μm.The quantification of the colocalization of the two fluorophores is shown in (C). Values are expressed as the mean+s.e. of percentage of colocalization in 20-40 cells.
Figure 5(A, B) Regions of the lysosomalmembrane resistant to 1% Triton X‐114 recovered from the sucrose gradients were fixed and processed for electron microscopy (A) or visualized without fixation by electron microscopy (B). Arrows point to the membrane bilayer.(C) Intact ratliverlysosomes (top) and DR regions of the lysosomal membranes (bottom) were fixed and subjected to immunogold labeling for LAMP‐2A and LAMP‐2B, and a gold‐conjugated secondary antibody as described under Material and methods. Top right panels show 'clusters' of gold particles (black arrows) in individual lysosomes at higher magnification. Graph shows the labeling density (gold particles per μm) in the indicated fractions for both proteins. Values are mean+s.e. from the quantification of 15 different micrographs.(D) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP‐2A and flotillin‐1. Representative lysosomes are shown. Far right panel: zoomed‐in image of the distribution of LAMP‐2A in regions containing (black arrows) and not containing (white arrows) flotillin‐1. Bars: 0.2 μm.
Figure 6(A) Fed ratliverlysosomes were incubated in an isotonic buffer without additions, or in the presence of ratlivercytosol (25 μg), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (25 μg), GST‐hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X‐114 extraction, apyrase density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP‐2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3-6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP‐2A in lysosomes remained constant throughout the incubation.(B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP‐2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments.(C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP‐2A and hsc70) and of an unrelated lysosomalmembrane protein (LAMP‐1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP‐2A present in each fraction, determined by densitometric quantification of the immunoblots.
Figure 7(A) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples.(B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA).(C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples.(D) Lysosomal-mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal-mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.
Figure 8(A) Lysosomes from mouse fibroblasts transfected with wild‐type hLAMP‐2A and maintained in the presence (Serum+) or absence (serum−) of serum were collected by centrifugation and subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP‐2A. Lane 1: homogenate.(B) Amino-acid sequence of the transmembrane and cytosolic region of hLAMP-2A and the mutations performed. Replaced amino acids are shadowed.(C) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP‐2A and maintained in the presence of serum were processed as in (A).(D) The percentage of total lysosomal hLAMP‐2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here. Values, corrected for the amount of mLAMP‐2A in each fraction, are mean+s.e. of 3-4 experiments.(E) Changes in hLAMP‐2A distribution induced by serum removal. Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments. Changes in the three nonDR regions are shown together (no DR).(F) Proteolysis of a pool of 3[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild‐type (WT) or the indicated mutant forms of hLAMP‐2A. Values are the mean+s.e. of two experiments with triplicate samples.(G) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.
Figure 9(A) The Triton X‐114‐resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous (10-80%) sucrose density gradient, SDS-PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100-200 kDa); fraction 3 (200-400 kDa); fraction 4 (400-600 kDa); fraction 5 (>600 kDa).(B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot.(C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS-PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a).(D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments.
Figure 1(B) Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n=3 for each reporter.(C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells.(D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally.(E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm.
Figure 2(A) Box plot of average beta scores (similar to log-fold change) for tf-NBR1 flux in indicated deletion cell lines. Median values (central band), inner quartiles (boxed regions), 1.5 interquartile ranges (whiskers), and outliers (dots) are indicated. Representative hits across all three genetic backgrounds are indicated in blue.(B) Gene correlation plot of average beta scores for lipidation dependent autophagy (tf-NDP52, from (Shoemaker et al, 2019)) and lipidation independent autophagy (average of tf-NBR1 from ATG7KO, ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0.5 across all three lipidation-deficient cell lines. In black are lipidation components. Dashed lines, top 1% of beta scores.(C) Wild-type K562 cells and indicated deletion isolates expressing Cas9 and tf-NDP52 or tf-NBR1 were transduced with individual sgRNAs for indicated genes. The median red:green ratio of each population was used to calculate the fold-repression according to the following formula: (ratiosgGene-ratiosgATG9A)/(ratioNegativeCtl-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Deeper shades of red indicate stronger suppressor phenotypes. Genes were clustered on the basis of their patterns of genetic interactions.
Figure 3(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells).(B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm.(C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue.
Figure 4(A) Wild-type and indicated K562 knock-out cells expressing tf-NBR1 were transfected with TagBFP or TagBFP-RavZ, and BFP-positive cells were analyzed for red:green ratio by flow cytometry. The median red:green ratio of each sample was used to calculate flux relative to WT+TagBFP. Bar graphs represent mean +/- SD from three independent experiments. n > 7,500 cells per sample.(B) K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading. Bar graphs represent mean +/- SD from independent experiments (n=4 {NBR1, NDP52}; n=3 {SQSTM1}). p values for WT vs ATG9AKO was determined using a one sample test (theoretical mean = 1) with Bonferroni correction.(D) Wild-type and indicated K562 knock-out cells were treated The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4). (E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant
Figure 5(A) Extracts derived from wild-type and deletion K562 cells expressing tf-NBR1 were normalized for total protein by BCA. NBR1 was immunoprecipitated using GFP-Trap dynabeads. Input and eluate were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.(B) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue.(C) Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue.
Figure 6(C) HEK293T cells were transfected with either myc-TAX1BP1WT or myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.(D) HEK293T cells were transfected with myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.
Figure 7(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. (B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue.(C) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate. (E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units.
Figure 2(A) Soft or stiff 4T1 or MCF-7 cells were isolated by the microfluidic chip, and then seeded (500 cells) in 90 Pa soft 3D fibrin gel for 3 days. The percentage of colonies formed was calculated and the colony size was recorded. Scale bar, 100 μm. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. n = 3 independent experiments in (A).(F) NSG mice were intravenously injected with 100 soft or stiff B16 or MP-1 cells. Eight weeks later, the lung metastasis was analyzed by H&E staining. The metastasis index was defined as the percentage of total metastatic nodule area to the total lung area based on the calculation from 10 slides. Scale bar, 0.5 mm. n = 3 (for B16) or 5 (for MP-1) mice with metastatic tumor. Data information: Two-tailed Paired Student's t test The data represent mean ± SD.
Figure 3The 100 stiff ALDH+ or soft ALDH- 4T1 cells were injected into the mammary fat pads of NSG mice. Eight weeks later, the lung sections were H&E stained. metastasis index was calculated (D). Scale bar, 0.5 mm. n = 5 mice with metastatic tumor. Data information: two-tailed Paired Student's t test data represent mean ± SD.