Biology Reference
In-Depth Information
FRET-FLIMwas previously used successfully to determine EGFR cluster forma-
tion by
Clayton et al. (2005)
. EGFR-GFP in combination with anti-phosphotyrosine-
Alexa555 was used to label fixed and permeabilized cells. Using FRET-FLIM in
combination with image correlation spectroscopy (ICS), Clayton et al. found an av-
erage cluster size of 2.2 in the absence of EGF and an average of 3.7 in the presence
of EGF, suggesting the formation of higher-order clusters upon ligand binding.
A limitation of this method is that it assumes the cell to be stationary and therefore
only an average of the different cluster sizes in a cell can be determined instead of
determining the subcellular distribution of the different cluster sizes. To solve this,
Saffarian et al. studied EGFR clustering by a method based on fluorescence intensity
distribution analysis (FIDA) in live cells for quantifying receptor clustering on cell
membranes (
Saffarian, Li, Elson, & Pike, 2007
). Using this technique, the authors
find an average cluster size of 1.3 in unstimulated cells, which is in contrast to results
from Clayton et al. The difference between the two values might be explained by
different measurement conditions, live cells versus fixed cells, and measuring the
clustering distribution with FIDA instead of determining an average. A limitation
of the FIDA method is that intensity fluctuations might occur during the measure-
ments, which might disturb the clustering measurements. Furthermore, the FIDA
method is relatively slow and may therefore not be suitable for measuring of EGFR
clustering after EGF stimulation because of the fast internalization of EGFR. More
recently, a number and brightness (N&B) analysis technique was used to study
EGFR clustering, which demonstrated the formation of up to pentameric EGFR clus-
ters (
Sako, Minoghchi, & Yanagida, 2000
). More recently, we introduced a method
based on homo-FRET to analyze EGFR clustering. With this method, EGFR clus-
tering can be accurately measured on intact cells using only a single label
(EGFR-mGFP) (
Bader, Hofman, Voortman, en Henegouwen, & Gerritsen, 2009;
Hofman et al., 2010
).
For homo-FRET, the same fluorophore for donor and acceptor is used, which has
the advantage that the detection of FRET is not dependent on concentrations of donor
and acceptor. Another advantage of only one fluorophore is that sample preparation
is simplified. However, homo-FRET does require a different quantification method
because homo-FRET does not result in a change in the emission spectrum or in fluo-
rescence lifetime, resulting in FRET to stay unnoticed. The decreased lifetime of the
donor fluorophore in homo-FRET is compensated by the excitation by the acceptor
fluorophore. The here applied analysis of energy transfer is based upon the FRET-
induced loss of anisotropy. When fluorophores are excited with polarized light, only
the subset that is in the right orientation will be excited. In the case of large molecules
that are unable to spin on the time scale of the fluorescence lifetime, the emitted light
by this subset of fluorophores is also polarized, albeit with a slightly different angular
distribution. In the case of two fluorophores in close proximity, there is a high prob-
ability that they are in different orientations. Upon excitation of one fluorophore, the
neighboring one could be excited through FRET, resulting in the emission of light
with this different orientation. Therefore, the energy transfer between two identical
fluorophores with slightly different orientation result
in depolarization of the
