Biology Reference
In-Depth Information
GPCRs were assumed to exist and function as monomeric units that interact with
corresponding G proteins in a 1:1 stoichiometry. However, biochemical, structural,
and functional evidence collected during the last decade indicates that GPCRs
can form oligomers. Oligomerization can occur between identical receptor types
(homomerization) or between different receptors of the same or different GPCR
families (heteromerization). Because signaling processes mediated by GPCR
are transient and fast and usually involve multiple proteins, quantitative molecular
microscopy is mandatory to study such receptor-receptor interaction and to obtain
unprecedented detailed information. Dynamic interaction between receptors is
thought
to have a key role in regulating most cellular signal
transduction
pathways.
F¨ rster resonance energy transfer (FRET) is a technique that allows investi-
gation of molecular processes in nanometer resolution. FRET is a nonradiative
process that can occur between fluorophores when the energy is transferred
from a donor fluorophore to an acceptor fluorophore. There are different fluo-
rescence lifetime and spectral or intensity-based approaches used in microscopy
like time- and frequency-domain fluorescence lifetime techniques, acceptor
photobleaching, semiquantitative sensitized emission, FRET stoichiometry
3-cube quantification, spectral RET, and the linear unmixing FRET (lux-FRET)
(
Zeug, Woehler, Neher, & Ponimaskin, 2012
). Lux-FRET approach allows us
to measure not only the apparent FRET efficiency but also the abundance of
total receptor and total donor, as well as their ratio (
Wlodarczyk et al., 2008
).
In this chapter, we show how lux-FRET can be applied in spectroscopic and im-
aging devices based on spectral detection as well as on filter cubes and describe
how to use this method for measuring dynamic receptor-receptor interaction by
changes in FRET efficiency and also discuss various imaging protocols and anal-
ysis modes.
Using the lux-FRET technique in combination with microscopy, we have
previously characterized interaction between serotonin receptors 5-HT
1A
and 5-HT
7
at the single-cell level (
Fig. 14.1
). However, the lux-FRET method can be applied
to investigate complex protein behavior like oligomerization between any proteins
of interest. The correct interpretation of FRET measurements as well as FRET
data-based modeling represents an essential challenge in microscopy and biophysics.
14.1
THEORY
14.1.1
Basic principles of FRET
The theory of resonance energy transfer was originally developed by Theodor
F
¨
rster and, in honor of his contribution, has been named after him (
Forster,
1946, 1948, 2012
). FRET is well suited to investigate protein-protein interaction that
occurs between two molecules positioned in close proximity of each other. The fun-
damental mechanism of FRET involves a donor fluorophore in an excited electronic
