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

Methods Mol Biol. 2018;1682:211-219. doi: 10.1007/978-1-4939-7352-1_18.

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.

Keywords: Autophagosomes; Autophagy; Fluorescence imaging; Lysosomal dysfunction; MAP LC3.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Autophagy*
  • Green Fluorescent Proteins / analysis*
  • Green Fluorescent Proteins / genetics
  • LLC-PK1 Cells
  • Microscopy, Fluorescence / methods*
  • Microtubule-Associated Proteins / analysis*
  • Microtubule-Associated Proteins / genetics
  • Optical Imaging / methods*
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / genetics
  • Swine
  • Transfection / methods

Substances

  • Microtubule-Associated Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins