Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 262

5.1. Multicolor imaging Multisensing, multiplex, or

multiparameter imaging
Multicolor imaging approaches have already been well described for study-
ing protein/protein interactions. However, multiplex analysis of kinase ac-
tivities through multicolor imaging constitutes a challenging objective, as it
implies using different sets of fluorescent probes, which are sufficiently dis-
tinct with respect to their spectral properties and which allow imaging the
complex dynamic patterns of kinases from different signaling pathways.
Multisensing, multiplex, or multiparameter imaging consists in devising
means of probing several targets using different fluorescent probes or biosen-
sors simultaneously by measuring different excitation and emission wave-
lengths. This strategy is extremely useful for fundamental research, as it
potentially allows the following of several signaling pathways in parallel
and in real time, to gain insight into their hierarchy of activation and study
the dynamics of their interconnections or their independent behavior.
Obviously, from a diagnostics perspective, the multisensing approach allows
a more detailed readout of the biomarker status. Indeed, most pathologies
are characterized by the concomitant activation of multiple kinases from
different signaling pathways. Moreover, in drug discovery strategies,
multisensing allows gaining a larger set of information concerning the
specificity of pharmacological inhibitors to characterize the underlying dif-
ferences in potency, off-target effects, and genetic backgrounds.
The main challenge, however, consists in combining different tools into
one single experimental design and finding suitable means to acquire, dis-
criminate, and quantify their signals. This proves extremely challenging
from a technological perspective when using FRET-based biosensors, as
it requires the use of spectrally distinct FRET pairs for multiparameter
live-cell fluorescence imaging. 160,161 Notwithstanding, this strategy has
been successfully developed for imaging of dual FRET-based caspase-3
biosensors 162 for simultaneous imaging of cAMP and cGMP in single
cells, thanks to the sapphire/RFP (red fluorescent protein) and CFP/YFP
set of FRET biosensors, respectively, 163 and for monitoring three calcium-
dependent signaling events, thanks to a cytosolic CamKII a -CFP/YFP
biosensor, a membrane-bound PKC-CFP/YFP sensor, and a translocating
annexin A4-mOrange/mCherry FRET biosensor. 161 Likewise, multiplex
strategies have been developed using nongenetic biosensors coupled to
synthetic probes with different spectral properties. For example, Lawrence
and collaborators developed a set of orthogonal fluorescent peptide biosensors
whose combination allows

—

for simultaneous multicolor monitoring of