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the production of phosphatidic acid by phospholipase D2 (PLD2) has been
demonstrated to be required for EGFR clustering ( Ariotti et al., 2010 ). Besides recep-
tor oligomerization and downstream signaling toward cell proliferation, activation of
EGF receptors results in a rapid internalization mainly via clathrin-mediated
endocytosis. The intracellular transport trajectory finally ends up in lysosomes where
the ligand-induced signaling is terminated by degradation of both ligand and recep-
tors ( Goh, Huang, Kim, Gygi, & Sorkin, 2010; Sorkin & Goh, 2009 ).
For studying the clustering of EGF receptors, different experimental techniques
have been developed. Dimerization of EGFR, for example, can be determined using
chemical cross-linking and detection by SDS-PAGE, by co-IP with differentially
tagged EGFR, or by fluorescence complementation ( Moriki, Maruyama, &
Maruyama, 2001; Yu, Sharma, Takahashi, Iwamoto, & Mekada, 2002; Zhu, Iaria,
Orchard, Walker, & Burgess, 2003 ). Studying receptor clustering is more difficult
and demands other experimental strategies. Receptor clustering was initially inves-
tigated using electron microscopy (EM), which can be used at high resolution, en-
abling visualization of gold-labeled EGFR ( van Belzen et al., 1988 ). Analysis of
cluster formation can be done by determining particle distances, either by hand or
automatically using Ripley's function ( Hancock & Prior, 2005 ). However, limita-
tions exist with respect to determining cluster size, which is dependent on the size
of the gold particle and the antibody ( 10 nm) ( Hancock & Prior, 2005 ). Another
approach for determining receptor clustering involves fluorescence light micros-
copy. However, conventional microscopy has a resolution limit of
200 nm, which
makes this technique not very suitable for measuring the formation of small receptor
clusters. To overcome this resolution limit problem, more advanced light microscope
techniques were developed, which do provide information about small cluster sizes.
One of the first examples was described by Gadella and coworkers and included
time-resolved fluorescence microscopy based on F¨rster resonance energy transfer
(FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM) ( Gadella &
Jovin, 1995 ).
FRET is based on the energy transfer between a donor and an acceptor fluorophore.
For FRET to occur, the donor emission spectrum and acceptor excitation spectrum
need sufficient spectral overlap and the two fluorophores should be within a distance
of
10 nm. Because energy transfer only occurs when fluorophores are within 10 nm
of each other, the detection of FRET can be used to study receptor dimerization or
small cluster formation. There are different methods to detect FRET, for example,
by measuring changes in the donor/acceptor emission intensity ratio, changes in fluo-
rescence lifetime (FLIM), or differences in anisotropy. The donor/acceptor ratio is
determined by measuring the emission of both fluorophores. However, this donor/
acceptor ratio is dependent on the concentrations of both fluorophores. With FLIM,
the time that the donor fluorophore is in its excited state is measured. This lifetime
can be determined by measuring the fluorescence decay of the donor probe after ex-
citation by using a short laser pulse. The fluorescence lifetime changes upon its envi-
ronment and also when energy transfer occurs. This method is less dependent on local
concentrations of both probes and is therefore a more robust way to detect FRET
( Chen, Mills, & Periasamy, 2003; Hofman et al., 2008 ).
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