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resulting in the formation of cholesterol-dependent G
M1
nanodomains
120 nm in
size (
Fig. 6.2
). Simultaneous dual-color high-resolution images revealed that the ca-
nonical raft component CD55 (a GPI-anchored protein) was recruited to regions
proximal (
<
150 nm) to CTxB-G
M1
nanodomains without physical intermixing,
but not the nonraft protein CD71. These results demonstrated the existence of
raft-based compositional connectivity at the nanoscale crucially mediated by choles-
terol. An earlier NSOM study had shown that a similar nanoscale organization of
GPI-anchored protein and adhesion receptors was essential for successful cell adhe-
sion, showing the functional relevance of preformed nanoscale platforms (
van
Zanten et al., 2009
). More conventional techniques such as FRET are unable to report
on such a spatial proximity at distances
<
10 nm. On the other extreme, diffraction-
limited techniques such as confocal microscopy would misleadingly show colocali-
zation between different components located at distances
>
300 nm. This shows that
superresolution techniques are in fact bridging the gap between 10 and 300 nm and
provide exquisite information at these important spatial scales.
Nanoscale dynamics on living cell membranes can be measured by combining
NSOM with FCS (
Manzo, van Zanten, & Garcia-Parajo, 2011
). Additionally, probe
desi
gn based on optical antennas further improves lateral resolution below 30 nm
(e.g.,
Mivelle, van Zanten, Neumann, van Hulst, & Garcia-Parajo, 2012
). For these
reasons, NSOM constitutes a valuable tool to characterize spatiotemporal details of
many biological processes occurring on the cell membrane.
<
CONCLUDING REMARKS
Over the last decade, the experimental approaches used in the study of receptor mem-
brane distribution, arrangement in clusters, association with lipid rafts, and the im-
plications on this organization to cell signaling have changed substantially. The
development of advanced imaging techniques greatly contributed to this progress.
Important features of the molecular mechanism governing cell membrane (nano-)
domain formation and their functions have been obtained using these imaging
methods. Nonetheless, this would not have been possible if biochemical approaches
to study the organization of intact cell membranes have not been available. This was
especially important in studies of protein association with lipid rafts, since for long
years, this relationship was almost exclusively assayed by testing membrane protein
resistance to solubilization by nonionic detergents. Nowadays, protein association
with lipid rafts is mainly investigated using nondestructive or at least not fully de-
structive cell methods, for example, using cholesterol depletion agents or colocali-
zation with raft markers, allowing the study of these interactions on the
compositional complex environment of the cell membrane. However, there are no
perfect methods to study cell membrane protein organization and its relation with
lipid rafts. As described above, current techniques used to investigate this problem-
atic have their intrinsic limitations, which, associated with transient nature of cell
