Quantitation of autophagy by luciferase release assay.
Autophagy is a cellular process that has been defined and analyzed almost entirely by qualitative measures. In no small part, this is attributable to the absence of robust quantitative assays that can easily and reliably permit the progress of key steps in autophagy to be assessed. We have recently developed a cell-based assay that specifically measures proteolytic cleavage of a tripartite sensor protein by the autophagy protease ATG4B. Activation of ATG4B results in release of Gaussia luciferase from cells that can be non-invasively harvested from cellular supernatants. Here, we compare this technique to existing methods and propose that this type of assay will be suitable for genome-wide functional screens and in vivo analysis of autophagy.
Ketteler
Robin
R
Center for Computational and Integrative Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114, USA.
Seed
Brian
B
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D033342:Copepoda; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D051379:Mice; D020123:Sirolimus / Q000378:metabolism
D000900:Anti-Bacterial Agents; D000071183:Autophagy-Related Proteins; D008156:Luciferases; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases; D020123:Sirolimus