Biology Reference
In-Depth Information
embedment is preferred. These treatments were previously shown not to influence the
clustering behavior (
Bancroft & Stevens, 1982
). To exclude any effect of the fused
mGFP, a nice control is to express EGFR-mGFP in cells that contain endogenous EGFR
like A431 and Her14. In these cells, hetero-predimers between EGFR-mGFP and en-
dogenous EGFR will cause the anisotropy to be similar to the mGFP control.
16.3.1.3
Ligand-induced oligomerization
Cells were seeded on an 18 mm glass coverslip. The day after, cells were
serum-starved overnight in medium containing 0.5% serum. After serum starvation,
cells were treated with 8 nM EGF for 10 min at 37
C. Then, cells were fixed
with4%PFAfor 20 min at roomtemperature and autofluorescencewas quenchedwith
100 mM glycine/PBS for 10 min. Finally, the coverslips were embedded in Mowiol
and used for measurement immediately or stored at 4 or
20
C until further use.
16.3.2
Analysis
In the steady-state anisotropy experiments, cluster sizes can be determined by direct
comparison of the measured anisotropy values with the reference anisotropies found
from the reference measurements on the FKBP constructs. Here, the steady-state an-
isotropies are calculated by integrating the intensities of all four time gates for both
polarization directions. In addition, the difference in transmission between the two
polarization channels can be corrected for by multiplying
I
per
by
C
tr
. In the time-
resolved anisotropy experiments,
r
inf
values are extracted from the time-gated fluo-
rescence anisotropy images. Cluster sizes can again be obtained by comparison with
the reference measurements on FKBP constructs. The anisotropy values in the last
gates are, in good approximation, equal to
r
inf
, provided that
E
is high enough. This
condition is met for the three last gates provided that
E
>
0.5. Four-gate anisotropy
decays are created by binning the intensities
I
par
and
I
per
per gate in regions of inter-
est. In the anisotropy imaging experiments, a threshold of
I
par,inf
รพ
300
counts was applied to all images. In theory, this number of counts corresponds to
a standard deviation in the anisotropy of 0.05 (
Bader et al., 2009; Hofman et al.,
2010
). An example of cluster size images derived from homo-FRET images is shown
in
Fig. 16.6
A, where a resting cell before EGF stimulation is shown. Here, cluster
sizes are small, but a clear population of predimers is visible;
2
I
per,inf
>
40% of the EGFR
is present as dimer. After stimulating the cell with EGF for 10 min average, cluster
sizes have increased up to
>
2; and a significant fraction of the clusters are of size
>
3
(
Fig. 16.6
B).
16.4
DISCUSSION
Studying EGFR dimerization and oligomerization is important to completely under-
stand the signaling and trafficking mechanism of EGFR. EGFR dimerization has
been studied by several biochemical studies like chemical cross-linking, co-IP with
