Detection of autophagy in cell death.
Activation of autophagosomes in cell death has been described since the late 1950s as a form of cell death characterized by consumption of the bulk of the cytoplasm by lysosomal derivatives. However, it is not yet established that autophagy is a primary, causative mechanism of death rather than a response to initial problems. Methods to assess autophagic cell death are similar to those used to detect and measure autophagy, with further evidence that the affected cells are indeed dying. These methods include structural analysis using electron microscopy, examination of the activity of lysosomal enzymes, assessment of the number, size, and location of lysosomes by the uptake of fluorescent molecules; measurement of the activity of autophagy-related genes such as the cleavage and activation of LC3 by Western blotting or green fluorescent protein tagging; evaluation of the effects of inhibition of one or more lysosomal enzymes, inhibition of fusion of organelles, or inhibition of intercompartmental transfer of molecules by putatively specific inhibitors; and interference with cells in order to change the expression of components of the lysosomal system to study the effect of this change on autophagy and autophagic cell death.
Zakeri
Zahra
Z
Department of Biology, Queens College and Graduate Center of the City University of New York, Flushing, New York, USA.
Melendez
Alicia
A
Lockshin
Richard A
RA
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001343:Autophagy / Q000502:physiology*; D015153:Blotting, Western; D016923:Cell Death / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins