Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion

Abstract

Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

Figure 1

Induction of autophagy by starvation and mTOR inhibitors in MEFs. (A) MEFs with stable expression of GFP-LC3 were treated with EBSS, rapamycin, PP242 or Torin1 (all at 1 μM) with or without CQ (50 μM) for 3 h. At the end of treatment, cell lysate was collected and subject to immunoblotting. (B) MEFs with stable expression of GFP-LC3 were treated as described in panel (A). Scale bar, 10 μm. (C and D) MEFs with stable expression of GFP-LC3 were treated as indicated in Panel (A), and total GFP intensity were measured by flow cytometry. Typical histograms were shown in panel (C) and the quantification data in panel (D). Data are presented as mean ± SD from two independent experiments (each in duplicate) (^**P< 0.01, Student's t-test).

Figure 2

Activation of lysosomal function in cells under starvation or treated with Torin1 and PP242, but not in cells treated with rapamycin. (A) MEFs were treated with EBSS, rapamycin, PP242 or Torin1 (all at 1 μM) for 3 h. Cells were then stained with LysoTracker Red DND-99 (50 nM) or LysoSensor Yellow/Blue DND-160 (5 μM) for 15 min. Scale bar, 50 μm. (B) MEFs were treated with EBSS, rapamycin, PP242 or Torin1 (all at 1 μM) as indicated. Cells were then loaded with Magic Red Cathepsin B or L reagent for 15 min. Fluorescence intensity of 10 000 cells per sample was measured by flow cytometry. (C and D) MEFs were treated with EBSS, PP242 (1 μM) or Torin1 (1 μM) with or without BA (50 nM) or CQ (50 μM) for 3 h. The fluorescence intensity of LysoTracker and LysoSensor was checked as described in panel (A). Scale bar, 50 μm. (E) MEFs were treated as indicated in panel (C-D), and cells were then loaded with Magic Red Cathepsin B reagent and determined as described in panel (B). (F) Rate of degradation of long-lived proteins in MEFs following the same treatment as in panel (A). Data are presented as mean ± SD from three independent experiments (^*P< 0.05, Student's t test).

Figure 3

Activation of lysosomal function is correlated to the suppression of mTORC1. (A) MEFs were treated with EBSS, rapamycin, PP242 or Torin1 (all at 1 μM) for the indicated times. (B) TSC2-WT and TSC2-KO MEFs were treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 3 h. (C) TSC2-WT and TSC2-KO MEFs were treated as indicated in panel (B), and cathepsin B enzyme activity was measured as described in Figure 2B. (D) MEFs were incubated in full medium, EBSS, or full medium with PP242 (1 μM) for 2 h, followed by the addition of IGF-1 (200 nM), or Leucine (Leu, 0.2 mg/ml) or IGF1+Leu for another 2 h. (E) MEFs were treated as indicated in panel (D), and cathepsin B enzyme activity was measured as described in Figure 2B. (F) MEFs were treated with EBSS, rapamycin, PP242 or Torin1 (all at 1 μM) in the absence or presence of CHX (10 μM) for 3 h. (G) MEFs were treated as described in panel (F) and cathepsin B enzyme activity was measured as described in Figure 2B. Cell lysate was collected and subject to immunoblotting at the end of above treatment.

Figure 4

mTORC1 activity, rather than its lysosomal localization, is critical for its inhibitory effect on lysosomes. (A) MEFs were treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 3 h, then lysosome fractions were collected as described in Materials and Methods. LAMP1 was used as a marker for the lysosome fraction. (B) MEFs were treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 3 h, and cells were coimmunostained for LAMP2 (red) and mTOR (green). Scale bar, 10 μm. (C) HET293T cells were transfected with pcDNA or the dominant negative RagB^GDP-RagD^GTP heterodimer for 48 h, then exposed to EBSS or PP242 (1 μM) for 3 h. (D) HET293T cells with the same transfection as described in panel (C), and then coimmunostained for LAMP2 (red) and mTOR (green). Scale bar, 10 μm. (E) HET293T cells with the same transfection and treatment as described in panel (C), and cathepsin B enzyme activity was measured as described in Figure 2B. Cell lysate was collected and subject to immunoblotting at the end of above treatment.

Figure 5

Lysosomal activation induced by starvation or mTOR inhibitors is Atg5 or autophagosome dependent. (A) Atg5-WT and Atg5-KO MEFs were treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 3 h. (B) Atg5-WT and Atg5-KO cells were treated as in panel (A), and the cathepsin B enzyme activity was measured as described in Figure 2B. (C) Atg5 Tet-off inducible MEF cells (m5-7) were pretreated with or without doxycycline (DOX) for 4 days, then treated as indicated in panel (A). (D) Cells as described in panel (C) were treated with EBSS or PP242 (1 μM) for 3 h, and the cathepsin B enzyme activity was then measured as described in Figure 2B. Cell lysate was collected and subject to immunoblotting at the end of above treatment.

Figure 6

TFEB activation is necessary but not sufficient for upregulation of lysosomal function following mTORC1 suppression. (A) MEFs were first transiently transfected with a TFEB luciferase reporter vector together with Renilla luciferase vector for 48 h. Cells were then treated with EBSS or PP242 (1 μM) for 4 h. Data were presented as mean ± SD from two independent experiments (each in triplicates) (^**P< 0.01 comparing to the control, Student's t test). (B) MEFs were first transiently transfected with the Flag-tagged TFEB for 48 h, and then treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 2 h. At the end of treatment, cell lysate was collected and subject to immunoblotting. (C) MEFs were treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 4 h, and the mRNA levels were measured by RT-qPCR. Values are expressed as fold increase compared to the control group. Data are presented as mean ± SD from two independent experiments (each in triplicates) (^*P< 0.05, ^**P < 0.01 comparing to their respective group in the control cells, Student's t test). (D) TFEB mRNA levels measured by qPCR. Data were presented as mean ± SD from two independent experiments (each with duplicates) (^**P< 0.01, Student's t test). (E) After transfection with TFEB siRNA or scrambled siRNA for 48 h, cells were treated with EBSS or PP242 (1 μM) for 3 h, then cathepsin B and L activities were determined as in Figure 2B. (F) Atg5-WT and Atg5-KO MEFs were transfected with TFEB luciferase reporter vector together with Renilla luciferase vector for 48 h, cells were then treated in EBSS for 4 h. The luciferase activity was determined and presented as in panel (A). (G) Atg5-WT and Atg5-KO MEFs were first transiently transfected with the Flag-tagged TFEB for 48 h, and then treated with EBSS for 2 h. Cell lysates were collected and subjected to cytoplasm and nucleus subfraction, as described in panel (B).

Figure 7

Activation of lysosome function depends on autophagosome-lysosome fusion. (A) MEFs with stable expression of GFP-LC3 were transfected with scrambled or Rab7 siRNA for 48 h. (B) MEFs with or without Rab7 KD were treated with EBSS for 3 h, then processed for LAMP-2 immunostaining (red) to observe the colocalization with GFP-LC3 (green). Scale bar, 10 μm. (C) Following the same treatments as in panel (B), cathepsin B activity was measured as described in Figure 2B. (D) MEFs were pre-treated with vinblastine (20 μM) or thapsigargin (3 μM) for 2 h, followed by EBSS for another 3 h. (E) MEFs with stable expression of GFP-LC3 were subjected to the same treatment as in panel (D) and then processed for LAMP-2 immunostaining (red) and its colocalization with GFP-LC3 (green) was examined. Scale bar, 10 μm. (F) MEFs were treated as indicated in panel (D), and cathepsin B activity was determined as described in Figure 2B. Data are presented as mean ± SD from two independent experiments (^**P< 0.01, Student's t-test). (G) Illustration showing the mechanisms mediating the activation of lysosomal function in autophagy involving the mTORC1-TFEB signaling axis and autophagsome-lysosome fusion. Cell lysate was collected and subject to immunoblotting at the end of above treatment.