title: Quantitative visualization of autophagy induction by mTOR inhibitors.
abstract: Autophagy is a catabolic pathway that degrades bulk cytosol in lysosomal compartments enabling amino acids and fatty acids to be recycled. One of the key regulators of autophagy is the mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase which suppresses the initiation of the autophagic process when nutrients, growth factors, and energy are available. Inhibition of mTOR, e.g., by small molecules such as rapamycin, results in activation of autophagy. To quantify autophagy induction by mTOR inhibitors, we use an mCherry-GFP-LC3 reporter which is amenable to retroviral delivery into mammalian cells, stable expression, and analysis by fluorescence microscopy. Here, we describe our imaging protocol and image recognition algorithm to visualize and measure changes in the autophagic pathway.
authors: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, MA, USA.
authors: Beat
authors: B
authors: Nyfeler
authors: Philip
authors: P
authors: Bergman
authors: Christopher J
authors: CJ
authors: Wilson
authors: Leon O
authors: LO
authors: Murphy
mesh_terms: D000595:Amino Acid Sequence; D001343:Autophagy / Q000187:drug effects*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
chemical_list: C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D009025:Morpholines; D011743:Pyrimidines; D049452:Green Fluorescent Proteins; C541932:Ku 0063794; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus