Biomedical Engineering Reference
In-Depth Information
Chapter 12
Assessing Mammalian Autophagy
Sharon A. Tooze , Hannah C. Dooley , Harold B. J. Jefferies ,
Justin Joachim , Delphine Judith , Christopher A. Lamb ,
Minoo Razi , and Martina Wirth
Abstract
Autophagy (self-eating) is a highly conserved, vesicular pathway that cells use to eat pieces of themselves,
including damaged organelles, protein aggregates or invading pathogens, for self-preservation and survival
(Choi et al., N Engl J Med 368:651-662, 2013; Lamb et al., Nat Rev Mol Cell Biol 14:759-774, 2013).
Autophagy can be delineated into three major vesicular compartments (the phagophore, autophagosome,
autolysosome,
see
Fig. 1). The initial stages of the pathway involve the formation of phagophores (also
called isolation membranes), which are open, cup-shaped membranes that expand and sequester the cyto-
solic components, including organelles and aggregated proteins or intracellular pathogens. Closure of the
phagophore creates an autophagosome, which is a double-membrane vesicle. Fusion of the autophago-
some with the lysosome, to form an autolysosome, delivers the content of the autophagosome into the
lysosomal lumen and allows degradation to occur.
Autophagy is a dynamic process that is initiated within 15 min of amino acid starvation in cell culture
systems (Köchl et al., Traffi c 7:129-145, 2006) and is likely to occur as rapidly in vivo (Mizushima et al.,
J Cell Biol 152:657-668, 2001). To initiate studies on the formation of the autophagosomes, and traf-
fi cking to and from the autophagic pathway, an ideal starting approach is to do a morphological analysis
in fi xed cells. Additional validation of the morphological data can be obtained using simple Western blot
analysis. Here we describe the most commonly used morphological technique to study autophagy, in
particular, using the most reliable marker, microtubule-associated protein 1A/1B-light chain 3 (LC3). In
addition, we describe a second immunofl uorescence assay to determine if autophagy is being induced,
using an antibody to WD repeat domain, phosphoinositide interacting 2 (WIPI2), an effector of the
phosphatidylinositol (3)-phosphate (PI3P) produced during autophagosome formation.
Key words
Autophagy, Autophagosome, LC3, Atg8, Phagophore, Autolysosome
1
Introduction
When cells are subjected to stress, either from internal and exter-
nal origin or pathologies that cause stress (e.g., ROS produced by
damaged mitochondria), they respond in many ways, including
the induction of autophagy [
1
]. If the reader is considering the
assays in this chapter, they have most likely had some indications
