title: Measurement of Autolysosomal pH by Dual-Wavelength Ratio Imaging.
abstract: Cellular components sequestered by autophagosomes during macroautophagy must be degraded and their components recycled in order to maintain homeostasis. To this end cells orchestrate the fusion of autophagosomes with lysosomes, degradative organelles that are rich in hydrolases. Most of the lysosomal enzymes function optimally at low pH, and products of macromolecular catabolism are cotransported with protons across the autolysosomal membrane. These functions are facilitated by the ability of lysosomes to pump protons inward, acidifying their lumen. Clearly, proper homeostasis of the luminal pH is crucial for autolysosomal function. We describe a method for the measurement of the absolute pH of individual autolysosomes in live cells. This technique involves measurement of the fluorescence of a pH-sensitive probe initially delivered to lysosomes and subsequently determined to have reached autolysosomes. By measuring the fluorescence at two separate wavelengths and calculating their ratio, potential artifacts introduced by photobleaching or by changes in autolysosome size, shape, or positioning are minimized. Combining such ratio determinations with an in situ calibration procedure enables absolute measurements of pH, which are superior to the qualitative estimates obtained with fluorescent weak bases such as LysoTracker.
authors.affiliation: Ryerson University, Toronto, ON, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Saric
authors.affiliation: Hospital for Sick Children, Toronto, ON, Canada; Keenan Research Centre, St. Michael's Hospital, Toronto, ON, Canada. Electronic address: sergio.grinstein@sickkids.ca.
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
authors.affiliation: Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: S A
authors.initials: SA
authors.lastname: Freeman
mesh_terms: D001343:Autophagy; D002264:Carboxylic Acids / Q000737:chemistry; D003911:Dextrans / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000737:chemistry* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods; D061848:Optical Imaging / Q000379:methods*; D010588:Phagosomes / Q000737:chemistry* / Q000648:ultrastructure; D012984:Software
chemical_list: D002264:Carboxylic Acids; D003911:Dextrans; D005456:Fluorescent Dyes; C436864:Oregon Green 488 carboxylic acid
keywords: Autophagolysosome; Autophagosome; Fluorescent dextran; LC3; Lysosome; Oregon Green 488; Ratio imaging