Document Detail


Utilizing flow cytometry to monitor autophagy in living mammalian cells.
MedLine Citation:
PMID:  18376137     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Elena Shvets; Ephraim Fass; Zvulun Elazar
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-03-20
Journal Detail:
Title:  Autophagy     Volume:  4     ISSN:  1554-8635     ISO Abbreviation:  Autophagy     Publication Date:  2008 Jul 
Date Detail:
Created Date:  2008-06-23     Completed Date:  2008-08-19     Revised Date:  2011-06-30    
Medline Journal Info:
Nlm Unique ID:  101265188     Medline TA:  Autophagy     Country:  United States    
Other Details:
Languages:  eng     Pagination:  621-8     Citation Subset:  IM    
Affiliation:
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
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MeSH Terms
Descriptor/Qualifier:
Animals
Autophagy / physiology*
Biological Markers / metabolism
CHO Cells
Cell Separation*
Cricetinae
Cricetulus
Flow Cytometry*
Green Fluorescent Proteins / metabolism
Hela Cells
Humans
Microtubule-Associated Proteins / metabolism
Phagosomes / metabolism
Chemical
Reg. No./Substance:
0/Biological Markers; 0/Microtubule-Associated Proteins; 0/light chain 3, human; 147336-22-9/Green Fluorescent Proteins

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