Biology Reference
In-Depth Information
A
B
EGFR-mGFP
EGFR-mGFP
+EGF
N
AV
=1
N
AV
=2
N
AV
³
3
FIGURE 16.6
(A) Homo-FRET-based cluster size image before stimulation with EGF and (B) after
stimulation with 8 nM EGF.
Results adapted from
Hofman et al. (2010)
.
differentially labeled EGFR, and also crystallography to determine the monomer and
dimer structure. Although there is already a lot known about the dimerization of
EGFR, the mechanism of oligomerization remains obscure. Advanced microscopy
techniques were developed to study EGFR oligomerization; initially EM with
gold-labeled EGFR was used. The cluster size that can be determined by EM depends
on the labeling of EGFR, in other words the size of the gold particles and antibody
used (10 nm). Advanced fluorescence microscopy techniques were developed like
FRET-FLIM, FIDA, and N&B analysis. These techniques are able to measure small
cluster sizes and are complementary techniques. FRET-FLIM is rapid and accurate,
while FIDA is able to measure cluster size in life cells. FIDA measures the clustering
on the plasma membrane of cells and is a slowmethod; therefore, it is not suitable for
measuring EGF-induced EGFR clustering in time. EGFR will internalize before the
FIDA method is able to determine the cluster sizes on the plasma membrane. N&B
uses the same principle as fluorescence correlation spectroscopy (FCS) measure-
ments and measures the distribution and association states of molecules in live cells
(
Nagy, Claus, Jovin, & Arndt-Jovin, 2010
). This technique demonstrated the
concentration-dependent predimer formation. In contrast, FIDA is able to give infor-
mation about the distribution of cluster sizes within a single pixel. FIDA and FCS,
however, need high illumination intensities for accurate measurements, which can
cause photodamage to the proteins in the cell. To study in more detail the EGF-
induced EGFR clustering, FRET was used and more specifically the homo-FRET
method. With this method, small EGFR clusters can be accurately determined and
the cluster size distribution in one cell can be determined within single pixels.
The advantage of homo-FRET instead of hetero-FRET is that the detection of FRET
is not dependent on the concentrations of donor and acceptor, which simplifies the
sample preparation. Homo-FRET is detected by a change in anisotropy, which is a
different method than used for measuring hetero-FRET. Anisotropy is a measure for
