From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 202

Mutant

Forskolin

3 ns

2.2

2.15

2.1

2.05

AKAR3

2

mutant

1.95

AKAR3

1.9

0 200 400

Time (s)

600 800 1000

1 ns

Figure 5.25 U2OS cells transfected with AKAR3 or AKAR3 inactive mutant. PKA was
activated using an adenylate cyclase activator (forskolin, 12.5 mM). Lifetimemeasurements
were performed in the frequency domain as described in Section 4 . Graphs represent the
average phase fluorescence lifetime measured for the entire cell as a function of time.
Experiments with either biosensor were performed separately, but are represented on
the same graph for clarity. Images represent phase lifetime before (left panel) and after
(right panel) induction.

unfortunately, no positive control for FRETmeasurements in living cells (all
sensors are in folded position), but a strong induction using activating agents
can be envisaged as an alternative. Molecular strategies for such a positive
control should be envisioned in the near future, as both control biosensors
could provide a complete sensing range.

7.3. Biosensor dynamic range and reproducibility
The dynamic range of a biosensor is a critical parameter to consider, and in-
creasing this range is often the purpose of biosensor optimization. The dy-
namic range depends directly on the FRET efficiency and can be increased
by switching fluorophores, improving linkers, and enhancing kinase binding
to the biosensor (with the insertion of a docking domain, for example), as
described earlier (see Section 6 ). Possessing the appropriate pharmacology
allowing maximum stimulation and inhibition is essential for estimating this
dynamic range. More than just control, the revealed amplitude (as well as
other criteria such as sensitivity) directs the choice of the biosensor to use.
As another example, the EKAR 42 shows a relatively low dynamic range,
both for ratiometric or lifetime-based measures, compared to a cAMP bio-
sensor ( T Epac VV ) recently published. 73 Indeed, cells expressing EKAR ex-
hibit a 20% average DR / R and 120 20 ps average D t, while cells

expressing T Epac VV give a mean DR / R of more than 80% and a mean D t