cell growth, proliferation, autophagy, polarity, and transcription. So
far, over 50 proteins have been identified as AMPK substrates
[8]. Notably, these substrates are present in various subcellular
compartments (Table1), suggesting a role for spatial regulation
of AMPK activity.
The signaling dynamics of AMPK have been studied extensively
with biochemical assays [1, 2]. However, the experimental methods
used to assess the signaling dynamics of AMPK are limited because
of the daunting task of visualizing these processes at the subcellular
compartment level in living cells in real time. Therefore, the spatio-
temporal dynamics of AMPK signaling have remained mostly
unknown until the recent advent of imaging techniques based on
Fo ̈rster Resonance Energy Transfer (FRET).
FRET is a physical process by which energy transfers from an
excited donor fluorophore to an acceptor fluorophore through
non-radiative dipole-dipole coupling [9]. Since the efficiency of
the energy transfer depends on the donor-acceptor distance
and/or their relative orientation, FRET has been widely used to
monitor protein-protein interactions and protein conformational
changes. Among many applications, genetically encoded FRET-
based biosensors have been exploited to visualize various kinase
dynamics in living cells. These kinase activity reporters (KARs)
consist of a pair of fluorophores that have sufficient spectral overlap,
an optimized substrate motif of the target kinases, and a
phosphopeptide-binding forkhead-associated (FHA1) domain.
Phosphorylation of the substrate motif increases its affinity toward
FHA1 domain, bringing donor and acceptor fluorophores such
that FRET. Based on these principles, three FRET-based AMPK
biosensors have been developed: AMPKAR, ABKAR, and BimAB-
KAR (Fig.1).
The first generation of the biosensor called AMPKAR is a
unimolecular FRET biosensor [10]. The structural layout is similar
to many other KARs. It consists of ECFP and circularly permuted
variants of Venus cpV E172 as a fluorophore FRET pair, bracketing
an FHA1 domain and an AMPK substrate motif. When AMPK is
activated, AMPKAR undergoes conformational changes that lead
to an increase of the FRET signal. The use of AMPKAR thus allows
for visualization of AMPK dynamics in either cytoplasmic or
nuclear compartment under different experimental conditions;
however, there still exists a need to monitor AMPK activity with
subcellular resolution to capture spatially differentiated dynamics.
ABKAR, a second-generation AMPK biosensor, lived up to this
demand [11–13]. It was achieved by having the donor fluorophore,
ECFP, in AMPKAR replaced with Cerulean 3, a brighter version of
ECFP, resulting in an approximately two-fold larger dynamic range
relative to the original AMPKAR. Additional refinements were256 Takafumi Miyamoto et al.