A Sensitive and Quantitative Technique for Detecting Autophagic Events Based on Lysosomal Delivery

Chem Biol. 2011 Aug 26;18(8):1042-52. doi: 10.1016/j.chembiol.2011.05.013.

Abstract

We sought to develop a sensitive and quantitative technique capable of monitoring the entire flux of autophagy involving fusion of lysosomal membranes. We observed the accumulation inside lysosomal compartments of Keima, a coral-derived acid-stable fluorescent protein that emits different-colored signals at acidic and neutral pHs. The cumulative fluorescent readout can be used to quantify autophagy at a single time point. Remarkably, the technique led us to characterize an autophagy pathway in Atg5-deficient cells, in which conventional LC3-based autophagosome probes are ineffective. Due to the large Stokes shift of Keima, this autophagy probe can be visualized in conjunction with other green-emitting fluorophores. We examined mitophagy as a selective autophagic process; time-lapse imaging of mitochondria-targeted Keima and GFP-Parkin allowed us to observe simultaneously Parkin recruitment to and autophagic degradation of mitochondria after membrane depolarization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anthozoa / chemistry
  • Anthozoa / genetics
  • Autophagy*
  • Autophagy-Related Protein 5
  • Cell Line
  • Gene Deletion
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / genetics
  • Lysosomes / metabolism*
  • Mice
  • Microscopy, Confocal / methods*
  • Microtubule-Associated Proteins / genetics
  • Mitochondria / metabolism
  • Mutagenesis, Site-Directed
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / genetics

Substances

  • Atg5 protein, mouse
  • Autophagy-Related Protein 5
  • Luminescent Proteins
  • Microtubule-Associated Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins