Autophagy relies on a tightly regulated and interconnected
cascade of protein complexes encoded by more than 30 ATG
(autophagy-related) genes [10–13]. Focusing on the AMPK-
dependent induction of autophagy, AMPK on the one hand inhi-
bits mTORC1 through phosphorylation of its regulatory subunit
Raptor at Ser792 [14] and on the other hand through an activating
phosphorylation of Unc-51-like autophagy activating kinase
1 (ULK1/ATG1) at several sites including the most prominent
Ser555 [15, 16]. The active ULK1 complex initiates the autophagy
cascade resulting in the formation of an isolation membrane, which
expands to engulf cargo. During the process of expanding this
autophagic membrane is marked with the microtubule-associated
protein 1 light chain 3 (LC3/ATG8) by conversion of the cyto-
solic, soluble LC3-I form into the lipidated, autophagosome-
incorporated LC3-II. To lipidate LC3, it is first proteolytically
cleaved by ATG4 to obtain a processed form (LC3-I) that is cova-
lently modified with phosphatidylethanolamine (PE) by an
ubiquitin-like conjugation system resulting in LC3-II
[17–19]. The membrane association is documented by the change
of the subcellular localization of LC3 from a more diffuse to a
dot-like cytoplasmic distribution [20, 21]. The incorporated
LC3-II is also involved in the recognition and recruitment of
cargo, a process mediated through selective autophagy receptors
like p62/SQSTM1 [22]. While p62 is degraded together with the
autophagic cargo, LC3 is recycled to the cytoplasm and freed of its
lipidation [18, 19, 23, 24].
Based on the knowledge of the autophagic cascade, several
techniques have been established to measure general as well as
AMPK-dependent induction of autophagy and the autophagic
flux employing immunoblotting and immunofluorescence analysis
that will be described in detail hereafter [23, 25]. The most com-
mon readout for autophagy is the characteristic conversion of
LC3-I into lipidated LC3-II. Alternatively, phospho-specific anti-
bodies can visualize the AMPK-dependent phosphorylation of
ULK1 and Raptor and thus are a readout specific for AMPK activ-
ity. Monitoring autophagy only by measuring steady-state amounts
of LC3-II can result in an incorrect interpretation of the autophagy
status [26]; therefore the integrity of the autophagic flux has to be
assessed, typically by using inhibitors of the flux. Bafilomycin A1
inhibits the fusion of the autophagosome with the lysosome and
thus allows estimations of the autophagosomal flux [27]. Moreover,
a double-tagged mCherry-EGFP-LC3 construct has been gener-
ated that differentiates between autophagosomes and autolyso-
somes due to the differential sensitivity of mCherry and EGFP to
the acidic conditions in lysosomes [28–30]. Furthermore, knock-
down and knockout approaches can be used to define the function-
ality of factors associated with the autophagic process. In particular,
the knockdown of AMPKα1/2 can provide evidence for a key role374 Sarah Krieg et al.