Article Figures & Data
Figures
- Figure S1. ESCRT-I depletion causes enlargement of lysosomes in RKO cells.
(A) Western blots showing the depletion efficiencies of ESCRT-I subunits, Tsg101, or Vps28 (using two single siRNAs for each subunit, #1 or #2), as compared to control conditions (nontargeting siRNAs, Ctrl#1 or #2), in RKO cells. The GAPDH protein is shown as a gel loading control. (B) Maximum intensity projection confocal images of fixed RKO cells showing the effects of ESCRT-I depletion on the intracellular distribution of LAMP1 (red) as compared with control conditions. Cell nuclei marked with DAPI stain (blue). Scale bar, 50 μm. Dot plot on the right showing the mean area of LAMP1-positive vesicles in control or ESCRT-I–depleted cells, calculated based on confocal microscopy images including those shown on the left. Values derived from independent experiments (dots) and their means (n = 3 ± SEM) are presented. Statistical significance tested by comparison to averaged values measured for siCtrl#1 and #2. *P < 0.05, **P < 0.01. (C) Western blots showing the efficiency of ESCRT-I depletion due to the CRISPR/Cas9–mediated knockout of TSG101 in RKO cells transduced with two single-guide RNAs (gRNAs, gTsg101#1 and #2) as compared with nontargeting gRNAs (gCtrl#1 and #2). Tubulin is shown as a gel-loading control. (D) Maximum intensity projection confocal images of live RKO cells showing the effects of TSG101 knockout on the intracellular distribution of lysosomes stained with LysoTracker dye (red). Cell nuclei marked with Hoechst stain (blue). Scale bar, 50 μm. The dot plot on the right shows mean area of detected lysosomal structures in control or TSG101 knockout cells, calculated based on live cell microscopy images (including those shown on the left). Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to averaged values measured for gCtrl#1 and #2. *P < 0.05, **P < 0.01.
- Figure 1. ESCRT-I dysfunction causes enlargement of lysosomes in RKO cells.
(A) Maximum intensity projection confocal images of live RKO cells, showing the intracellular distribution of lysosomes stained with LysoTracker dye (red) under control conditions (nontargeting siRNAs, Ctrl#1 or #2) and upon depletion of ESCRT-I subunits, Tsg101, or Vps28 (using two single siRNAs for each subunit, #1 or #2). Cell nuclei marked with Hoechst stain (blue). Scale bar, 50 μm. (A, B) Dot plots showing average fluorescence intensity of LysoTracker expressed in arbitrary units (a.u.; top panel) and the mean area (bottom panel) of detected lysosomal structures in control or ESCRT-depleted cells, calculated based on live-cell microscopy images (shown in A). Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to averaged values measured for siCtrl#1 and #2. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Maximum intensity projection confocal images of fixed control and ESCRT-I–depleted cells showing intracellular distribution of cathepsin D, stained with pepstatin A conjugated to BODIPY FL (green), as compared to late endosomes/lysosomes, detected using anti-LAMP-1 antibody (red). Cell nuclei marked with DAPI stain (blue). Scale bar, 20 μm. The dot plot on the right shows average BODIPY FL fluorescence intensity of LAMP-1–positive structures calculated based on confocal images. Values derived from independent experiments and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to averaged value measured for siCtrl-treated cells (AveCtrl). *P < 0.05, **P < 0.01. (D) Representative EM images of control and ESCRT-I–depleted cells showing the morphology and size of lysosomes (indicated by yellow arrowheads). Scale bar, 500 nm.
- Figure S2. ESCRT-I dysfunction causes enlargement of lysosomes in DLD-1 cells.
(A) Western blots showing the depletion efficiencies of ESCRT-I subunits, Tsg101, or Vps28 (using single siRNAs for each subunit, #1 or #2) as compared with control conditions (nontargeting siRNAs Ctrl#1 or #2). The GAPDH protein is shown as a loading control. (B) Maximum intensity projection confocal images of live cells, showing the intracellular distribution of lysosomes marked with LysoTracker dye (red) in control or ESCRT-I–depleted cells. Cell nuclei marked with Hoechst stain (blue). Scale bar, 50 μm. (B, C, D) Dot plots showing average fluorescence intensities of LysoTracker expressed in arbitrary units (a.u., C) and mean area (D) of detected lysosomal structures in control or ESCRT-depleted cells, calculated based on live-cell microscopy images (shown in B). Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to averaged values measured for siCtrl#1 and #2. *P < 0.05, **P < 0.01, ***P < 0.001.
- Figure S3. ESCRT-I dysfunction leads to accumulation of enlarged endosomes and autophagosomes with nondegraded content.
(A, B) Representative EM images of control (siCtrl#1 or #2, nontargeting siRNAs) and ESCRT-I–depleted (siTsg101#1 or siVps28#2) cells showing the morphology and size of multivesicular bodies—MVBs, small endosomes—e, enlarged endosomes—E and autophagosomes (in A), and multilamellar bodies—MLBs (in B). Scale bar, 500 nm.
- Figure 2. ESCRT-I mediates the degradation of lysosomal membrane proteins in RKO cells.
(A) Maximum intensity projection confocal images of fixed RKO cells showing intracellular distribution of ubiquitin (green), LysoTracker dye (red), and LAMP1 (gray) in ESCRT-I–depleted (siTsg101#1 or #2, siVps28#1 or #2) or control (Ctrl#1, #2, nontargeting siRNAs) cells. Cell nuclei marked with DAPI stain (gray). Enlarged lysosomes enriched in ubiquitinated proteins at lysosomal outer membranes, marked with LAMP1, are indicated by arrowheads. Scale bar, 10 μm. (B) Representative Western blots (upper panel) showing the levels of ectopically expressed GFP-MCOLN1 (detected by anti-GFP antibodies) and ESCRT-I components in control or ESCRT-I–depleted RKO cells. The graph (lower panel) shows GFP-MCOLN1 levels expressed as fold change with respect to averaged values measured for siCtrl#1 and #2 by densitometry analysis of Western blotting bands. Vinculin was used as a gel-loading control. Values derived from independent experiments and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1. **P < 0.01, ***P < 0.001. (C) Representative single confocal plane images of fixed cells showing the effect of ESCRT-I depletion and/or 18 h bafilomycin A1 (BafA1) treatment on the intracellular distribution of ectopically expressed GFP-MCOLN1 (green), with respect to LAMP1 (red) and LysoTracker dye (gray). In control cells, GFP-MCOLN1 accumulation on LAMP1-positive vesicles indicated by arrows. GFP-MCOLN1 accumulation on enlarged LAMP1-positive lysosomal structures in ESCRT-I–depleted cells indicated by arrowheads. Cell nuclei marked with DAPI stain (gray). Scale bar, 10 μm. (C, D) Dot plot showing the fluorescence intensity of GFP-MCOLN1 colocalizing with LAMP1-positive vesicles expressed in arbitrary units (a.u.) in confocal microscopy images of control or ESCRT-I–depleted cells and/or upon18 h bafilomycin A1 (BafA1) treatment (shown in C). Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to averaged values measured for siCtrl#1 and #2 (AveCtrl). ns—nonsignificant, #P < 0.1, *P < 0.05, **P < 0.01. (E) Representative Western blots showing the abundance of GFP-MCOLN1, Tsg101, and Vps28 proteins in control and Tsg101-depleted cells treated with cycloheximide (CHX, 100 μg/ml) for the indicated time periods. Graphs on the right show fold change of GFP-MCOLN1 levels measured by densitometry analysis of Western blotting bands, including those shown on the left. Values derived from independent experiments and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1- or siTsg101#2-treated samples.
- Figure 3. Depletion of ESCRT-I induces the expression of genes annotated to autophagy and cholesterol biosynthesis.
(A) Gene ontology (GO) analysis of top biological processes identified by annotation of genes with up-regulated expression (≥1.5-fold; adjusted P-value < 0.05), detected by RNA-Seq in RKO cells depleted of Tsg101 or Vps28 (siTsg101#1 or #2, siVps28#1 or #2), as compared to control cells (treated with nontargeting siRNAs, Ctrl#1 or #2). RNA-Seq data analysis was performed based on three independent experiments. (B) Heatmap visualizing expression of genes annotated to “autophagy” (GO:0006914) process, whose mRNA levels were detected by RNA-Seq as up-regulated after Tsg101 or Vps28 depletion in RKO cells. Established MiT-TFE target genes are indicated by asterisks. (C) Heatmap visualizing expression of genes annotated to “cholesterol metabolic processes” (GO:0008203) process, whose mRNA levels were detected by RNA-Seq as up-regulated after Tsg101 or Vps28 depletion in RKO cells. Established cholesterol biosynthesis genes are indicated by asterisks.
- Figure 4. Depletion of ESCRT-I induces prolonged activation of TFEB/TFE3 signaling.
(A) Western blots showing levels of TFEB and TFE3 proteins in whole-cell lysates (W) and nuclear fractions (N) of RKO cells depleted of Tsg101 or Vps28 (siTsg101#2, siVps28#2), as compared to control cells (treated with nontargeting siRNA, Ctrl#1). To examine the fraction purity, the levels of vinculin (cytosolic marker) were probed. The H3 histone protein was used as a loading control for nuclear fractions. (B) qPCR results showing the expression of MiT-TFE target genes upon ESCRT-I and/or MiT-TFE depletion (using single siRNAs for Tsg101, Vps28, TFEB, or TFE3) presented as fold changes with respect to control cells. Values derived from independent experiments and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to siTsg101 or siVps28 conditions. *P < 0.05, **P < 0.01, ****P < 0.0001. (C) Maximum intensity projection confocal images of fixed RKO cells at 48 or 72 h post transfection (hpt) showing intracellular distribution of TFEB or TFE3 (green) in ESCRT-I–depleted or control RKO cells. Cell nuclei marked with DAPI (blue). Dot plots on the right show percentage of cells with nuclear TFEB or TFE3 localization. Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1 and/or siTsg101#2 conditions. ns, nonsignificant (P ≥ 0.05), **P < 0.01, ***P < 0.001, ****P < 0.0001.
- Figure S4. Depletion of ESCRT-I leads to nuclear accumulation of TFEB and TFE3 in DLD-1 and HEK293 cells.
(A, B) Western blots showing levels of TFEB and TFE3 proteins in whole-cell lysates (W) and nuclear fractions (N) of DLD-1 (in A) or HEK293 (in B) cells treated with control nontargeting siRNAs (siCtrl#1 or 2) or siRNA targeting ESCRT-I (siTsg101#1 or #2, Vps28#1 or #2). To examine the fraction purity, the levels of vinculin (cytosolic marker) were probed. The lamin protein was used as a gel loading control for nuclear fractions.
- Figure 5.
ESCRT-I deficiency leads to endolysosomal accumulation of cholesterol that does not contribute to activation of TFEB/TFE3 signaling. (A) Maximum intensity projection confocal images of fixed RKO cells at 48 h post transfection (48 hpt) showing the effect of culture in delipidated medium (for 40 h) on the intracellular distribution of free cholesterol marked with filipin dye (red) with respect to LAMP1 protein (green) in control (treated with nontargeting siRNA, Ctrl#1) or ESCRT-I–depleted (siTsg101#2 or siVps28#1) cells. Cell nuclei marked with DRAQ7 dye (blue). Scale bar, 50 μm. The dot plot on the bottom shows the average filipin fluorescence intensity per LAMP1-positive structure (expressed in arbitrary units, a.u.), as compared to control cells. Values derived from independent experiments (dots) and their means (n = 3 ± SEM) are presented. Statistical significance tested by comparison to averaged values measured for control cells (AveCtrl) and/or siTsg101#2 conditions. **P < 0.01, ***P < 0.001. (B) Maximum intensity projection confocal images of fixed RKO cells at 48 hpt showing the effect of culture in delipidated medium on the intracellular distribution of TFEB or TFE3 (green) in control or ESCRT-I–depleted cells. Cell nuclei marked with DAPI (blue). Dot plots on the right show the percentage of cells with nuclear TFEB or TFE3 localization. Values derived from independent experiments (dots) and their means (n = 3 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1 conditions. *P < 0.05, **P < 0.01, ***P < 0.001.
- Figure S5. Cholesterol supplementation prevents the induced expression of cholesterol biogenesis genes upon ESCRT-I deficiency.
qPCR results showing the expression of the indicated cholesterol biosynthesis genes in ESCRT-I–depleted (siTsg101#2 or siVps28#2) or control (siCtrl#1,#2 nontargeting siRNAs) RKO cells at 72 h post transfection with or without 48 h supplementation with water-soluble cholesterol (20 μM). Data are presented as fold changes with respect to siCtrl#1. Values derived from independent experiments (dots) and their means (n = 3 ± SEM) are presented. Statistical significance tested by comparison to averaged values measured for control cells (AveCtrl). ns, nonsignificant (P ≥ 0.05), **P < 0.01, ***P < 0.001.
- Figure S6. Calcineurin-dependent signaling is not required for TFEB/TFE3 nuclear localization in cells lacking Tsg101.
Maximum intensity projection confocal images of fixed RKO cells at 48 h post transfection showing intracellular distribution of TFEB or TFE3 (green) in ESCRT-I–depleted (siTsg101#2) or control (Ctrl#1, nontargeting siRNA) cells upon 2 h treatment with vehicle (Vh, DMSO) or 25 μM cyclosporin A (CsA). Cell nuclei marked with DAPI stain (blue). Scale bar, 50 μm. Dot plots on the right showing the percentage of cells with nuclear TFEB or TFE3 localization. Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1 conditions. ns, nonsignificant, **P < 0.01.
- Figure 6. Ca2+-dependent signaling is required for TFEB/TFE3 nuclear localization but not for TFEB dephosphorylation in cells lacking Tsg101.
(A) Maximum intensity projection confocal images of fixed RKO cells at 48 h post transfection (48 hpt) showing intracellular distribution of TFEB or TFE3 (green) in ESCRT-I–depleted (siTsg101#2) or control (Ctrl#1, nontargeting siRNA) cells upon 2 h treatment with vehicle (Vh, DMSO), BAPTA-AM (10 μM), or ML-SI1 (25 μM). Cell nuclei marked with DAPI stain (blue). Scale bar, 50 μm. Dot plots on the right show the percentage of cells with nuclear TFEB or TFE3 localization. Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1 conditions. ns, nonsignificant, *P < 0.05, **P < 0.01. (B) Representative Western blots showing levels of phosphorylated (at Ser122 or Ser211) or total TFEB and of Tsg101 and vinculin (loading control) in lysates obtained 48 hpt from control (siCtrl#1) or Tsg101-depleted RKO cells treated for 3 h with the indicated reagents: vehicle (Vh, DMSO), 10 or 20 μM BAPTA, 25 μM ML-SI1or 25 μM CsA.
- Figure 7. TFEB/TFE3 signaling in ESCRT-I–deficient cells is activated due to the inhibition of the Rag GTPase–dependent mTORC1 pathway.
(A) Western blots showing levels of phosphorylation of TFEB at Ser122 and the indicated canonical mTORC1 targets in cells lacking ESCRT-I (transfected with siTsg101#2 or siVps28#1) as compared with control cells (siCtrl#1 or #2, nontargeting siRNAs) at 48 h post transfection (48 hpt) upon culture in regular medium (EMEM) or nutrient-deficient medium (EBSS) for 2 h. Vinculin was used as a loading control. (A, B) Graphs showing fold levels of indicated phosphorylations measured by densitometry analysis of Western blotting bands, including those shown in (A). Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1. ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001. (C) Single-plane confocal images of fixed RKO cells at 48 hpt showing intracellular distribution of TFEB (green) with respect to LAMP1 protein (red) or DAPI-stained nuclei (blue) in ESCRT-I–depleted (siTsg101#2) or control (siCtrl#1) cells upon 2 h treatment with vehicle (Vh, DMSO) or 1 μM INK128. Scale bar, 100 μm. (D, E) Dot plots showing the percentage of cells with nuclear TFEB localization (D) or TFEB colocalization with LAMP1-positive structures (E) in ESCRT-I–depleted (siTsg101#2) or control (siCtrl#1) cells. Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. ns—nonsignificant, *P < 0.05, **P < 0.01, ****P < 0.0001. (F) Single-plane confocal images of fixed RKO cells at 48 hpt showing intracellular distribution of TFEB or TFE3 (green) with respect to DAPI-stained nuclei (blue) in control or ESCRT-I–depleted cells expressing or not wild-type (WT) or constitutively active (S75L) HA-GST-RagC protein (red). Scale bar, 20 μm. Dot plots on the right showing the percentage of cells with nuclear TFEB or TFE3 localization. Values derived from independent experiments (dots) and their means (n = 3 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1 conditions. ns—nonsignificant (P ≥ 0.05), *P < 0.05, **P < 0.01. (G) Western blots showing levels of phosphorylation of the indicated proteins as well as total levels of TFEB, Tsg101, and HA-GST-RagC in ESCRT-I–depleted (siTsg101#2) or control (siCtrl#1) cells at 48 hpt with ectopic expression of the HA-GST-RagC protein (WT or S75L). Vinculin was used as a loading control. The bottom graph shows phosphorylation levels of Ser122 of TFEB measured by densitometry analysis of Western blotting bands, including those shown above. Values derived from independent experiments (dots) and their means (n = 3 ± SEM) are presented. Statistical significance tested by comparison to siCtrl#1. ns, nonsignificant, *P < 0.05.
- Figure S7. ESCRT-I deficiency inhibits TFEB S122 phosphorylation and activates TFEB/TFE3 factors independently of general mTORC1 signaling.
(A) Representative Western blots showing levels of phosphorylation of TFEB at Ser122 and phosphorylations of the indicated canonical mTORC1 targets in RKO cells lacking ESCRT-I due to CRISPR/Cas9–mediated TSG101 knockout (gTsg101#1 or #2) as compared to control cells (gCtrl#1 or #2, nontargeting gRNAs). The vinculin protein is shown as a gel loading control. (B) Single-plane confocal images of fixed cells lacking ESCRT-I (siTsg101#2) as compared with control cells (siCtrl#1, nontargeting siRNAs) at 48 h post transfection (48 hpt) showing mTOR localization on LAMP1-positive structures upon culture in nutrient-rich medium (EMEM) or starvation medium (EBSS) for 2 h. Cell nuclei marked with DAPI stain (blue). Scale bar, 20 μm. The dot plot on the right shows mTOR colocalization with LAMP1-positive structures in control or ESCRT-I–depleted cells calculated based on confocal images including those shown on the left. Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. ns, nonsignificant. (C) Single-plane confocal images of fixed RKO cells at 48 hpt showing intracellular distribution of TFE3 (green) with respect to LAMP1 protein (red) or DAPI-stained nuclei (blue) in ESCRT-I–depleted or control cells upon 2 h treatment with vehicle (Vh, DMSO) or 1 μM INK128. Scale bar, 100 μm. (C, D, E) Dot plots showing the percentage of cells with nuclear TFEB localization (D) or TFEB colocalization with LAMP1-positive structures (E) in control or ESCRT-I–depleted cells, calculated based on confocal images including those shown in (C). Values derived from independent experiments (dots) and their means (n = 4 ± SEM) are presented. ns, nonsignificant, **P < 0.01, ****P < 0.0001.
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