From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 205

9. OUTLOOK AND PERSPECTIVE
9.1. What about multisensing?
In the prospect of detecting several parameters simultaneously in the same
cell, the idea of using multiple probes is becoming more and more attractive.
As the number of available tools is increasing, monitoring several modulators
of the same pathway provides opportunities for the understanding of cell re-
action dynamics. The principal challenge of these experiments is combining
the well-chosen fluorophore pairs with the appropriate technique. Several
parameters have to be considered and are well described and illustrated in
the excellent review by Carlson and Campbell. 169
Piljic and Schultz 170 have presented such an example of multisensing
applied to the monitoring of several effectors involved in the regulation
of calcium chloride conductance. It was achieved in an epithelial cell line
using simultaneously four biosensors, including three FRET-based ones.
These refined experiments were performed by ratiometric imaging, taking
advantage of (i) the spectral shift between the two FRET pairs (namely
CFP/YFP and mOrange/mCherry) and (ii) the subcellular targeting of
some of these sensors to spatially discriminate signals from each reporter.
Taking into account the complexity, dynamics, and stress reactivity of
the cellular environment, measurements of molecular dynamics could re-
quire the combination of fluorescence analysis techniques (such as fluores-
cence lifetime, spectrum or intensity fluctuations analysis) and a multimodal
microscopy method suitable for the different complexity degrees of the liv-
ing matter: cells, tissues, and whole organism. So far, biosensor recording has
been achieved using mainly ratiometric techniques; however, FRET-FLIM
could emerge as an interesting alternative.
9.2. Kinase activity measurements in living animals
Studying kinase activity profiles in living tissue or animals provides new
insights on cell behavior in the intact biological context. A few publications
describe the use of FRET-based biosensors in living animals. For example,
ATP levels in a transgenic worm ( Caenorhabditis elegans expressing
ATeam 171 ), cAMP levels in the fruit flies (transgenic Drosophila melanogaster
expressing GFP-PKA 172,173 ), as well as Rac GTPase activity in zebrafish
(pRaichu RacFRET mRNA microinjected in Danio rerio embryos 174 )
have already been performed.