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Autophagy Monitoring Assay II: Imaging Autophagy Induction in LLC-PK1 Cells Using GFP-LC3 Protein Fusion Construct

  • Pavan P. AdiseshaiahEmail author
  • Sarah L. Skoczen
  • Jamie C. Rodriguez
  • Timothy M. Potter
  • Krishna Kota
  • Stephan T. Stern
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1682)

Abstract

Autophagy is a catabolic process involved in the degradation and recycling of long-lived proteins and damaged organelles for maintenance of cellular homeostasis, and it has also been proposed as a type II cell death pathway. The cytoplasmic components targeted for catabolism are enclosed in a double-membrane autophagosome that merges with lysosomes, to form autophagosomes, and are finally degraded by lysosomal enzymes. There is substantial evidence that several nanomaterials can cause autophagy and lysosomal dysfunction, either by prevention of autophagolysosome formation, biopersistence or inhibition of lysosomal enzymes. Such effects have emerged as a potential mechanism of cellular toxicity, which is also associated with various pathological conditions. In this chapter, we describe a method to monitor autophagy by fusion of the modifier protein MAP LC3 with green fluorescent protein (GFP; GFP-LC3). This method enables imaging of autophagosome formation in real time by fluorescence microscopy without perturbing the MAP LC3 protein function and the process of autophagy. With the GFP-LC3 protein fusion construct, a longitudinal study of autophagy can be performed in cells after treatment with nanomaterials.

Key words

Autophagy MAP LC3 Fluorescence imaging Autophagosomes Lysosomal dysfunction 

Notes

Acknowledgment

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

References

  1. 1.
    Guimaraes RS, Delorme-Axford E, Klionsky DJ, Reggiori F (2015) Assays for the biochemical and ultrastructural measurement of selective and nonselective types of autophagy in the yeast Saccharomyces cerevisiae. Methods 75:141–150. doi: 10.1016/j.ymeth.2014.11.023 CrossRefPubMedGoogle Scholar
  2. 2.
    The Nobel Prize in Physiology or Medicine 2016. Nobel Media AB 2014. http://www.nobelprize.org/nobel_prizes/medicine/laureates/2016/. Accessed 14 Oct 2016
  3. 3.
    Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290(5497):1717–1721CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Johnson-Lyles DN, Peifley K, Lockett S, Neun BW, Hansen M, Clogston J, Stern ST, McNeil SE (2010) Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction. Toxicol Appl Pharmacol 248(3):249–258. doi: 10.1016/j.taap.2010.08.008 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Adiseshaiah PP, Clogston JD, McLeland CB, Rodriguez J, Potter TM, Neun BW, Skoczen SL, Shanmugavelandy SS, Kester M, Stern ST, McNeil SE (2013) Synergistic combination therapy with nanoliposomal C6-ceramide and vinblastine is associated with autophagy dysfunction in hepatocarcinoma and colorectal cancer models. Cancer Lett 337(2):254–265. doi: 10.1016/j.canlet.2013.04.034 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Stern ST, Adiseshaiah PP, Crist RM (2012) Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol 9:20. doi: 10.1186/1743-8977-9-20 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    McLeland CB, Rodriguez J, Stern ST (2011) Autophagy monitoring assay: qualitative analysis of MAP LC3-I to II conversion by immunoblot. Methods Mol Biol 697:199–206. doi: 10.1007/978-1-60327-198-1_21 CrossRefPubMedGoogle Scholar
  8. 8.
    Melendez A, Levine B (2009) Autophagy in C. elegans. WormBook:1–26. doi: 10.1895/wormbook.1.147.1
  9. 9.
    Potter TM, Stern ST (2011) Evaluation of cytotoxicity of nanoparticulate materials in porcine kidney cells and human hepatocarcinoma cells. Methods Mol Biol 697:157–165. doi: 10.1007/978-1-60327-198-1_16 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Pavan P. Adiseshaiah
    • 1
    Email author
  • Sarah L. Skoczen
    • 1
  • Jamie C. Rodriguez
    • 1
  • Timothy M. Potter
    • 1
  • Krishna Kota
    • 2
  • Stephan T. Stern
    • 1
  1. 1.Cancer Research Technology Program, Nanotechnology Characterization LaboratoryLeidos Biomedical Research, Inc., Frederick National Laboratory for Cancer ResearchFrederickUSA
  2. 2.Perkin Elmer, Inc.WalthamUSA

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