title: The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo.
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.
authors.affiliation: SDM, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: L S
authors.initials: LS
authors.lastname: Frost
authors.affiliation: SDM, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: A
authors.initials: A
authors.lastname: Dhingra
authors.affiliation: SDM, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: J
authors.initials: J
authors.lastname: Reyes-Reveles
authors.affiliation: SDM, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: battagli@upenn.edu.
authors.forename: K
authors.initials: K
authors.lastname: Boesze-Battaglia
mesh_terms: D000071182:Autophagosomes / Q000378:metabolism; D001343:Autophagy; D001896:Boron Compounds / Q000737:chemistry; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D012710:Serum Albumin, Bovine
keywords: Autolysosome; Cargo degradation; DQ™-BSA; Endolysosome; LC3-associated phagocytosis; Lysosome