From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 192

A AKAR3

3 ns

T = 0s

H-89

Forskolin

1.72

1.7

1.68

1.66

10 m m

1 ns

1.64

T = 500s T = 1500s

1.62

1.6

1.58

1.56

1.54

Time (s)

1.92

0 500 1000 1500 2000

B AKAR4 3 ns

H-89 T = 0s

Forskolin

2.14

2.12

2.1

2.08

10 m m 1 ns

2.06

T = 500s T = 1500s

2.04

2.02

2

1.98

1.96

Time (s)

1.94

0 500 1000 1500 2000

Figure 5.21 U2OS cells transfected with AKAR3 (A) or AKAR4 (B). PKA was activated
using an adenylate cyclase activator (forskolin) and then inhibited with H-89. Lifetime
measurements 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. Images represent intensity (top left) and phase lifetime (others) at
specific times.

of Dicosoma genus in 1999. 38 Although red-shifted FP variants would
undoubtedly result in lower phototoxicity upon biological sample illumination
(less energy), they are not commonly employed for sensing application. The
only example is the ERK biosensor EKAR 42 in its green-red version which
does not show a large dynamic range (unpublished results). However, a
green-red FRET pair has proven its usefulness in the detection of molecular
interaction by FRET-FLIM. Besides, even in their optimized versions, the in-
trinsic fluorescence properties of RFPs (oligomerization, quantum yield,
brighteness, etc.) do not stand a chance when compared with their yellow
counterparts. Indeed, the historical FRET pair is cyan-yellow based and has
received much attention directed toward its optimization. Ongoing efforts
in developing red-shifted variants will most likely yield adequate acceptance
and will prove themselves useful in multisensing approaches.