Biology Reference
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techniques, this method is not without pitfalls. As in any technique requiring
labeling, protein or raft/nonraft marker membrane localization/organization
might be influenced by their labels. Therefore, any result obtained through this
approach should always be confirmed by at
least one of
the techniques
described earlier.
6.2
OPTICAL TECHNIQUES TO STUDY RECEPTOR MEMBRANE
ORGANIZATION AND ASSOCIATION WITH LIPID RAFTS
6.2.1
Confocal fluorescence microscopy
Confocal fluorescence microscopy has been widely used to image the colocalization
between membrane receptors and lipid rafts on cell membranes (
Bournazos, Hart,
Chamberlain, Glennie, & Dransfield, 2009; Im et al., 2009
). In this case, fluorescent
molecules are used to label the target receptors and lipid rafts. Normally, cells are
fixed and stained with fluorescently labeled antibodies that bind directly to the
protein of interest or to primary antibodies that recognize the molecules of interest,
whereas lipid rafts are labeled by targeting putative raft markers (
Table 6.1
). In most
occasions, the ganglioside G
M1
is marked using fluorescently labeled pentameric
Cholera toxin B subunit (CTxB). After imaging, standard colocalization analysis
methods such as the Pearson or Manders coefficients (
Dunn, Kamocka, &
McDonald, 2011
) are normally performed to quantify the degree of spatial coloca-
lization of the signals corresponding to the target protein and lipid rafts. During
raft-mediated signaling, membrane receptors oligomerize in lipid rafts, forming
large assemblies that are easily identified by confocal microscopy. Nonetheless,
due to the dynamic nature of lipid raft-receptor assemblies, this relationship is some-
times difficult to assess. To overcome this, the molecules of interest are commonly
copatched with putative raft markers prior to cell fixation by secondary antibodies,
which enhances their interactions, thus facilitating their detection (
Hao, Mukherjee,
& Maxfield, 2001
). However, this labeling scheme should be used with caution,
since antibody copatching may lead to a false association of the target protein with
lipid rafts.
The main limitation of confocal microscopy in the study of lipid raft-associated
processes is its low optical resolution set by the diffraction of light (
200 nm in the
lateral axis). Resolving entities of a few nanometers and separated by interparticle
distances below the resolution limit is simply impossible using this technique.
Nevertheless, due to its standardization, simplicity, versatility, and minimal inva-
siveness, confocal microscopy continues to be broadly used in this type of studies,
providing relevant quantitative data about protein interactions with lipid rafts.
6.2.2
Homo-F ¨ rster resonance energy transfer microscopy
Many aspects of membrane receptor spatiotemporal organization, including insights
about the link between lipid rafts and receptor oligomerization (
Fig. 6.1
), have
been obtained using homo-FRET (F¨rster resonance energy transfer) microscopy
