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the depolarization of the linear polarized excitation beam. To be able to convert the
obtained anisotropy values into cluster sizes, we use reference proteins like EGFR-
FKBP-mGFP constructs, which form clusters of known sizes when AP20187 is
added. With this method, cluster sizes of EGFR in the order of N
¼
1, N
¼
2, or
N
3 can be determined. A conventional fluorescence microscope can be converted
in a homo-FRET-detecting system when a set of polarizers is integrated and suffi-
cient signal is generated. The limitation of this method is that it is up till now only
able to distinguish monomers, dimers, and oligomers. This method is not able to
measure the precise amount of molecules in higher-order clusters. Homo-FRET
was used to study the predimer formation of EGFR and showed to be receptor
concentration-independent by homo-FRETmeasurements. This is in contrast to what
was found by the N&B analyses ( Hofman et al., 2010; Nagy et al., 2010 ). This dif-
ference might be explained by the different sample preparations as homo-FRET was
measured in fixed cells and N&B analysis in live cells. We note that the N&Bmethod
does not include the immobile fraction of the receptors, which is known to exist. An-
other explanation for the difference could be that the N&B analysis cannot distinguish
the cluster size distribution within a single pixel and homo-FRET can. It might be that
the amount of predimers changes upon receptor concentration but that there are still
predimers formed when there are low EGFR concentrations present. This is just one
example of contradicting results of EGFR clusteringmeasurements. Combining these
different methods is required to overcome these contradictions. Most probably,
the most ideal way to measure clustering is by combining FRET studies with high-
resolution microscopy methods like FIDA, N&B, EM, or others because in that situ-
ation, you measure both the larger cluster organizations and the smaller subclusters
using homo-FRET. The super resolution microscopy technique STED would be a
good possibility to combine with FRET because the resolution of STED can be up
to 20 nm, which is slightly higher than homo-FRET, which makes it possible when
the two methods are combined to measure small clusters simultaneously with larger
clusters. STEDcan also be applied in life cells and gives clustering information about a
whole cell, which is not the case with, for example, single-molecule tracking where
only a set of clusters can be analyzed ( Pellett et al., 2011 ).
In this chapter, we have described the homo-FRET method for measuring EGFR
clustering and compared them with other experimental techniques. This method can
equally be applied for other proteins, provided their functioning is not affected by the
fusion to mGFP. Essential are the reference proteins, which are mGFP fused to FKBP
for cytoplasmic proteins and FKBP fused to a receptor can be used as reference for
the analysis of receptor clustering. Homo-FRET has the advantage of simple sample
preparation and accurate cluster size determination. However, homo-FRET is not
able to detect larger cluster organization.
Acknowledgments
The authorswish to thankDr. ErikG.HofmanandDr.ArjanN. Bader for their fruitful discussions.
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