The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo

Methods Enzymol. 2017:587:43-54. doi: 10.1016/bs.mie.2016.09.052. Epub 2016 Nov 17.

Abstract

There is increasing evidence documenting the critical role played by autophagic and autophagy-associated processes in maintaining cell homeostasis and overall systemic health. Autophagy is considered a degradative as well as a recycling pathway that relies on encapsulated intracellular components trafficking to and fusing with degradative compartments, including lysosomes. In this chapter, we describe the use of DQ™-BSA to study autophagosome-lysosome fusion as well as a means by which to analyze hybrid autophagic pathways. Such noncanonical pathways include LC3-associated phagocytosis, better known as LAP. Both autophagosomes and LAPosomes (LC3-associated phagosomes) deliver cargo for degradation. The use of fluorescent DQ™-BSA in conjugation with autophagic makers and biomarkers of hybrid autophagy offers a reliable technique to monitor the formation of autolysosomes and LAPo-lysosomes in both fixed- and live-cell studies. This technique relies on cleavage of the self-quenched DQ™ Green- or DQ™ Red BSA protease substrates in an acidic compartment to generate a highly fluorescent product.

Keywords: Autolysosome; Cargo degradation; DQ™-BSA; Endolysosome; LC3-associated phagocytosis; Lysosome.

MeSH terms

  • Autophagosomes / metabolism
  • Autophagy*
  • Boron Compounds / chemistry
  • Cell Line
  • Epithelial Cells / metabolism
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • Humans
  • Lysosomes / metabolism
  • Microscopy, Confocal / methods
  • Microtubule-Associated Proteins / analysis
  • Microtubule-Associated Proteins / metabolism*
  • Molecular Biology / methods*
  • Serum Albumin, Bovine / chemistry
  • Serum Albumin, Bovine / metabolism*

Substances

  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • Fluorescent Dyes
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • Serum Albumin, Bovine