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.
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Acknowledgements
We thank Dr N Mizushima (Tokyo Medical and Dental University, Japan) for providing the Atg5−/− MEFs and the Tet-off Atg5 MEFs with stable expression of GFP-LC3, as well as the HeLa cells with stable expression of GFP-LC3, Dr A Ballabio (Telethon Institute of Genetics and Medicine, Italy) for providing the Flag-TFEB expression vector and the TFEB-luciferase construct, Dr DJ Kwiatkowski (Harvard University, USA) for providing the pair of TSC2 WT and KO MEFs, and Dr DM Sabatini (Massachusetts Institute of Technology, USA) for providing Torin1. We also thank Dr N Mizushima and Dr WX Ding for the insightful discussions. SHT, NDY and JZ were supported by research scholarships from NUS. This study was supported in part by research grants from Singapore National Medical Research Council (NMRC/1260/2010), Singapore Biomedical Research Council (BMRC /08/1/21/19/554) to HMS and by INSERM grants to PC.
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Supplementary information, Figure S1
Induction of autophagy by starvation and mTOR inhibitors in HeLa cells. (PDF 591 kb)
Supplementary information, Figure S2
PP242 and Torin1, but not rapamycin enhance cathepsin activity. (PDF 218 kb)
Supplementary information, Figure S3
Effects of Starvation, PP242, Torin1 and rapamycin on lysosomal function in HeLa cells. (PDF 326 kb)
Supplementary information, Figure S4
Effects of starvation, PP242 and rapamycin on lysosome in Sin1-WT and KO MEFs. (PDF 255 kb)
Supplementary information, Figure S5
Effects of Trehalose on mTORC1 activity and lysosomal function. (PDF 400 kb)
Supplementary information, Figure S6
Changes of LysoTracker staining in Atg5-deficient cells. (PDF 682 kb)
Supplementary information, Figure S7
Lysosomal activation induced by starvation or mTOR inhibitors in primary Atg5 WT/KO MEFs and Atg7 WT/KO MEFs. (PDF 531 kb)
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Zhou, J., Tan, SH., Nicolas, V. et al. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion. Cell Res 23, 508–523 (2013). https://doi.org/10.1038/cr.2013.11
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DOI: https://doi.org/10.1038/cr.2013.11
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