Image-based genome-wide siRNA screen identifies selective autophagy factors (original) (raw)

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References

  1. Levine, B., Mizushima, N. & Virgin, H. W. Autophagy in immunity and inflammation. Nature 469, 323–335 (2011)
    Article ADS CAS Google Scholar
  2. Noda, N. N., Ohsumi, Y. & Inagaki, F. Atg8-family interacting motif crucial for selective autophagy. FEBS Lett. 584, 1379–1385 (2010)
    Article CAS Google Scholar
  3. Wild, P. et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333, 228–233 (2011)
    Article ADS CAS Google Scholar
  4. Komatsu, M. & Ichimura, Y. Selective autophagy regulates various cellular functions. Genes Cells 15, 923–933 (2010)
    Article CAS Google Scholar
  5. Lipinski, M. M. et al. A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions. Dev. Cell 18, 1041–1052 (2010)
    Article CAS Google Scholar
  6. Orvedahl, A. O. et al. Autophagy protects against Sindbis virus infection of the central nervous system. Cell Host Microbe 7, 115–127 (2010)
    Article CAS Google Scholar
  7. Monastyrska, I., Rieter, E., Klionsky, D. J. & Reggiori, F. Multiple roles of the cytoskeleton in autophagy. Biol. Rev. Camb. Philos. Soc. 84, 431–448 (2009)
    Article Google Scholar
  8. Lee, J. Y. et al. HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy. EMBO J. 29, 969–980 (2010)
    Article CAS Google Scholar
  9. Longatti, A. & Tooze, S. A. Vesicular trafficking and autophagosome formation. Cell Death Differ. 16, 956–965 (2009)
    Article CAS Google Scholar
  10. Nair, U. et al. SNARE proteins are required for macroautophagy. Cell 146, 290–302 (2011)
    Article CAS Google Scholar
  11. Behrends, C., Sowa, M. E., Gygi, S. P. & Harper, J. W. Network organization of the human autophagy system. Nature 466, 68–76 (2010)
    Article ADS CAS Google Scholar
  12. Mizushima, N. The role of the Atg1/ULK1 complex in autophagy regulation. Curr. Opin. Cell Biol. 22, 132–139 (2010)
    Article CAS Google Scholar
  13. Narendra, D., Tanaka, A., Suen, D. F. & Youle, R. J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803 (2008)
    Article CAS Google Scholar
  14. Pagliarini, D. J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008)
    Article CAS Google Scholar
  15. Xing, L., Zhang, M. & Chen, D. Smurf control in bone cells. J. Cell. Biochem. 110, 554–563 (2010)
    Article CAS Google Scholar
  16. Talloczy, Z. et al. Regulation of starvation- and virus-induced autophagy by the eIF2α kinase signaling pathway. Proc. Natl Acad. Sci. USA 99, 190–195 (2002)
    Article ADS CAS Google Scholar
  17. Talloczy, Z., Virgin, H. W. I. V. & Levine, B. PKR-dependent xenophagic degradation of herpes simplex virus type 1. Autophagy 2, 24–29 (2006)
    Article CAS Google Scholar
  18. Orvedahl, A. et al. HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe 1, 23–35 (2007)
    Article CAS Google Scholar
  19. Geisler, S. et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nature Cell Biol. 12, 119–131 (2010)
    Article CAS Google Scholar
  20. Narendra, D., Kane, L. A., Hauser, D. N., Fearnley, I. M. & Youle, R. J. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both. Autophagy 6, 1090–1106 (2010)
    Article CAS Google Scholar
  21. Yamashita, M. et al. Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation. Cell 121, 101–113 (2005)
    Article CAS Google Scholar
  22. Cho, W. & Stahelin, R. V. Membrane binding and subcellular targeting of C2 domains. Biochim. Biophys. Acta 1761, 838–849 (2006)
    Article CAS Google Scholar
  23. Lu, K. et al. Pivotal role of the C2 domain of the Smurf1 ubiquitin ligase in substrate selection. J. Biol. Chem. 286, 16861–16870 (2011)
    Article CAS Google Scholar
  24. Singh, R. et al. Autophagy regulates lipid metabolism. Nature 458, 1131–1135 (2009)
    Article ADS CAS Google Scholar
  25. Mizushima, N. & Levine, B. Autophagy in mammalian development and differentiation. Nature Cell Biol. 12, 823–830 (2010)
    Article CAS Google Scholar

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Acknowledgements

We thank M. Vishwanath, S. Wei and B. Posner for assistance with high-throughput siRNA screening; W. Sun for information technology support; K. Scudder for assistance with image analysis algorithms; A. Diehl for expert medical illustration; V. Stollar, M. McDonald, R. Kuhn and R. Youle for helpful discussions and providing reagents; A. Bugde for assistance in the UTSW Live Cell Imaging Facility; and L. Mueller and T. Januszewski for assistance with electron microscopy. This work was supported by NIH grants AI109617 (B.L.), CA84254 (B.L.), UL1 RR024982 (G.X., Y.X.), AI062773 (R.J.X.), DK83756 (R.J.X.), DK086502 (R.J.X.) and DK043351 (R.J.X. and A.N.); NSF grant DMS-0907562 (G.X.); and the Center for Cancer Research, National Cancer Institute Intramural Research Program (Y.E.Z.).

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Author notes

  1. Anthony Orvedahl and Rhea Sumpter Jr: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Anthony Orvedahl, Rhea Sumpter Jr., Zhongju Zou, Qihua Sun & Beth Levine
  2. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Anthony Orvedahl & Beth Levine
  3. Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Rhea Sumpter Jr., Zhongju Zou, Qihua Sun & Beth Levine
  4. Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Guanghua Xiao, Christian V. Forst & Yang Xie
  5. Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, 02114, Massachusetts, USA
    Aylwin Ng & Ramnik J. Xavier
  6. Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, 02114, Massachusetts, USA
    Aylwin Ng & Ramnik J. Xavier
  7. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, 02142, Massachusetts, USA
    Aylwin Ng & Ramnik J. Xavier
  8. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Zhongju Zou & Beth Levine
  9. Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA
    Yi Tang & Ying E. Zhang
  10. Center for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
    Masahiro Narimatsu & Jeffrey L. Wrana
  11. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Christopher Gilpin & Katherine Luby-Phelps
  12. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Michael Roth
  13. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
    Michael Roth, Yang Xie & Beth Levine
  14. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada
    Jeffrey L. Wrana

Authors

  1. Anthony Orvedahl
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  2. Rhea Sumpter Jr.
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  3. Guanghua Xiao
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  4. Aylwin Ng
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  5. Zhongju Zou
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  6. Yi Tang
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  7. Masahiro Narimatsu
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  8. Christopher Gilpin
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  9. Qihua Sun
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  10. Michael Roth
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  11. Christian V. Forst
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  12. Jeffrey L. Wrana
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  13. Ying E. Zhang
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  14. Katherine Luby-Phelps
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  15. Ramnik J. Xavier
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  16. Yang Xie
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  17. Beth Levine
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Contributions

A.O., R.S., M.N., M.R., J.L.W., Y.E.Z., K.L.-P., C.G. and B.L. designed the experiments. A.O., R.S., Z.Z. Q.S. and Y.T. performed the experiments. G.X., A.N., C.V.F., R.J.X. and Y.X. performed statistical and bioinformatic analyses. A.O., R.S. and B.L. wrote the manuscript. G.X. and A.N. contributed equally to the manuscript.

Corresponding author

Correspondence toBeth Levine.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Methods, Supplementary References and Supplementary Figures 1-12 with legends. The original file posted online was corrupted and has been replaced on 23 November 2011. (PDF 8705 kb)

Supplementary Table 1

This table lists the primary data for the virus capsid/autophagosome colocalization screen. Shown are the z-scores for each replicate for each gene in the Dharmacon siRNA library. “NA” denotes insufficient numbers of green or red puncta per cell or total number of cells per well for analysis. (XLS 4656 kb)

Supplementary Table 2

This table lists the data for the virus capsid/autophagosome colocalization confirmation screen, using a customized library (from Dharmacon) composed of individual siRNAs from the pool of 4 siRNAs targeting each gene that scored “positive” in the primary co-localization screen. Genes with p-values of <0.05 for 2 or more individual siRNAs were considered confirmed colocalization hits. “NA” denotes insufficient numbers of green or red dots per cell or total number of cells per well for analysis (XLS 127 kb)

Supplementary Table 3

This table lists the results for each individual siRNA from a pool of 4 targeting each gene that scored positive in the primary screen for viral capsid/autophagosome colocalization, with respect to whether they scored positive in the confirmation screen of viral capsid/ autophagosome colocalization (C) screen, the secondary screen for survival of virus-infected cells (S) and the secondary screen for Parkin-mediated mitophagy (M). siRNA sequences are listed in column J. For each siRNA, this table also lists the number of 7-8mer miRNA seed sequences (positions 2-8 on mature miRNA) contained in each siRNA oligo (column K), the identity of such seed sequences (columns L-0), and the specific miRNAs that contain the seed sequences (columns P-S). The confirmed siRNAs in each screen are not enriched for siRNAs containing miRNA seed sequences (P=0.95 for colocalization screen; P=0.71 for cell survival screen; and P=0.97 for mitophagy screen) (XLS 200 kb)

Supplementary Table 4

This table lists the predicted targets (identified using TargetScan) for each miRNA seed sequence listed in Supplementary Table 3 (MS Excel spreadsheet, 302 KB). (XLS 302 kb)

Supplementary Table 5

This table lists the molecular function and biological process categories from Panther and Gene Ontology, and protein class and pathway assignments from Panther for the siRNA hits in the viral capsid/ autophagosome colocalization screen. Clusters listed correspond to graphical representation in Supplementary Figure 3a (MS Excel spreadsheet, 36 KB). (XLS 36 kb)

Supplementary Table 6

This table lists the data from the cell survival screen, using a using a customized library (from Dharmacon) composed of individual siRNAs from the pool of 4 siRNAs targeting each gene that scored “positive” in the primary colocalization screen. Genes with p-values of <0.05 for 2 or more individual siRNAs were considered to be confirmed cell survival factors during viral infection (MS Excel spreadsheet, 36 KB). (XLS 127 kb)

Supplementary Table 7

This table lists the data from the mitophagy screen, using a customized library (from Dharmacon) composed of individual siRNAs from the pool of 4 siRNAs targeting each gene that scored “positive” in the primary colocalization screen. Genes with p-values of <0.05 for 2 or more individual siRNAs were considered to be confirmed mitophagy factors. (XLS 127 kb)

Supplementary Table 8

This table includes the data in Figure 2a of the main text, with additional details for each gene including Locus ID, Gene Accession numbers, and Gene Annotations from Panther Molecular Function (MF), Panther Biological Process (BP), and UniProt. (XLS 112 kb)

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Orvedahl, A., Sumpter, R., Xiao, G. et al. Image-based genome-wide siRNA screen identifies selective autophagy factors.Nature 480, 113–117 (2011). https://doi.org/10.1038/nature10546

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