Utilizing flow cytometry to monitor autophagy in living mammalian cells.
Autophagy is a major intracellular catabolic pathway that takes part in diverse biological events including response to amino acid starvation, protein and organelle turnover, development, aging, pathogen infection and cell death. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, was shown to selectively incorporate into autophagosome, thus serving as a unique bona fide marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming, labor-intensive and require much experience for accurate interpretation. Here we took advantage of the Fluorescence Activated Cell Sorter (FACS) to quantify the turnover of GFP-LC3 as an assay to measure autophagic activity in living mammalian cells. We showed that during induction of autophagy by rapamycin, tunicamycin or starvation to amino acids, fluorescence intensity of GFP-LC3 is reduced in a time-dependent manner. This decrease occurred specifically in wild type LC3, but not in mutant LC3(G120A), and was inhibited by autophagic or lysosomal inhibitors, indicating that this signal is specific to selective autophagy-mediated delivery of LC3 into lysosomes. By utilizing this assay, we tested the minimal nutrient requirement for the autophagic process and determined its induction by deprivation of specific single amino acids. We conclude that this approach can be successfully applied to different cell-lines as a reliable and simple method to quantify autophagic activity in living mammalian cells.
Shvets
Elena
E
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Fass
Ephraim
E
Elazar
Zvulun
Z
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D016466:CHO Cells; D002469:Cell Separation; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism
D015415:Biomarkers; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins