Biology Reference
In-Depth Information
acts as the donor molecule and YFP as the acceptor, the coupling between
them being particularly e
cient. Recent publications have detailed the
development of elegant probes for following cell signalling based on these two
approaches. Specific examples are probes for visualizing activation of Rho-
family GTPases and tyrosine phosphorylation.
Activation of Rho-family GTPases involves conversion from a GDP-bound
form to a GTP-bound one. Activated GTPases exert effects through binding
to specific effector proteins (for a historical account of Rho family signalling
see Ridley, 2001). These interactions have been visualized using FRET to
reveal spatial and temporal aspects of Rac GTPase activation in growth
factor-stimulated cells (Kraynov et al., 2000), in neutrophils undergoing
chemotaxis (Gardiner et al., 2002), and in integrin-stimulated cells (Del Pozo
et al., 2002). In all cases, the donor is GFP-Rac, whereas the acceptor is the
effector domain of PAK (p21-binding domain or PBD) conjugated with
AlexaFluor-546. The FRET signal is thought to be proportional to the
amount of GTP bound to Rac. Chemoattractant, applied by the micropipette
method, resulted in a rapid activation, as judged by an increased FRET signal
(Gardiner et al., 2002). These data suggested that, as expected, Rac was
involved in lamellipodial extension. Surprisingly, it appeared that Rac was
also activated in the retracting tail, suggesting that Rac played a role in tail
retraction.
Ting et al. (2001) have used multicoloured GFP variants to measure
tyrosine phosphorylation in live cells. Here, reporters consisted of the
contiguous fusion of CFP, a phosphotyrosine-binding domain, a consensus
substrate for the kinase under observation, and YFP. Phosphorylation of the
substrate by a specific tyrosine kinase enabled an intramolecular association
with the phosphotyrosine-binding domain. This resulted in the close
apposition of, and FRET between, the CFP and YFP modules. The FRET
signal is thought to be proportional to the extent of tyrosine phosphorylation.
One such reporter was used to provide evidence for PDGF-dependent
activation of the tyrosine kinase Abl in the regulation of cell morphology
(membrane ruing) with a possible link to paxillin via the adaptor protein
Crk.
Fluorescence lifetime imaging microscopy (FLIM)
A number of techniques have been developed that permit the analysis of
FRET interactions such as monitoring sensitized emission by the acceptor
fluorophore (as described above), monitoring donor fluorescence intensity
following acceptor photobleaching, and the fluorescence lifetime imaging of
the donor fluorophore. Techniques that observe the sensitized emission of the
acceptor molecule upon donor excitation are not independent of fluorophore
