Autophagy Monitoring Assay II: Imaging Autophagy Induction in LLC-PK1 Cells Using GFP-LC3 Protein Fusion Construct
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 wordsAutophagy MAP LC3 Fluorescence imaging Autophagosomes Lysosomal dysfunction
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.
- 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
- 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.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
- 8.Melendez A, Levine B (2009) Autophagy in C. elegans. WormBook:1–26. doi: 10.1895/wormbook.1.147.1