Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates.
Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.
a Istituto di Fisica; Università Cattolica del Sacro Cuore ; Rome , Italy.
Giuseppe
G
Maulucci
a Istituto di Fisica; Università Cattolica del Sacro Cuore ; Rome , Italy.
Michela
M
Chiarpotto
a Istituto di Fisica; Università Cattolica del Sacro Cuore ; Rome , Italy.
Massimiliano
M
Papi
b Istituto di Patologia Generale; Università Cattolica del Sacro Cuore ; Rome , Italy.
Daniela
D
Samengo
b Istituto di Patologia Generale; Università Cattolica del Sacro Cuore ; Rome , Italy.
Giovambattista
G
Pani
a Istituto di Fisica; Università Cattolica del Sacro Cuore ; Rome , Italy.
Marco
M
De Spirito
D001343:Autophagy / Q000502:physiology*; D002738:Chloroquine / Q000494:pharmacology; D003952:Diagnostic Imaging / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000473:pathology*
D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins; D002738:Chloroquine
3-methyladenine; LysoSensor; autophagic flux; autophagy; cathepsin B; chloroquine; fluorescence microscopy; mRFP-GFP-LC3; mechanistic target of rapamycin (serine/threonine kinase); microtubule-associated protein 1 light chain 3; pH ratiometric sensor; pHrodo red; phosphatidylinositol 3-kinase catalytic subunit type 3; rapamycin; starvation