Biology Reference
In-Depth Information
promoted, increasing signaling efficiency (
Simons & Gerl, 2010
). Indeed, several
receptors such as Fas/CD95, T-cell receptor, epidermal growth factor receptor
(EGFR), or G protein-coupled receptors translocate and oligomerize in lipid rafts
(
Allen, Halverson-Tamboli, & Rasenick, 2007; Chini & Parenti, 2004; Mollinedo
&Gajate, 2006; Suzuki, 2012
), a process that is vital for efficient signal transduction.
However, the molecular mechanisms that govern signal transduction in these
domains are still not fully understood. This is due to the highly dynamic and hetero-
geneous nature of these signaling assemblies, which make their study quite challeng-
ing using conventional optical techniques (
Munro, 2003
). Moreover, there is an
intimate link between cell cytoskeleton and the formation/inhibition of receptor olig-
omers in lipid rafts (
Kusumi et al., 2012; Simons & Gerl, 2010
), which further con-
tributes to its complexity. Several techniques have been used to investigate lipid rafts
and their participation in signal transduction. However, due to their intrinsic limita-
tions, an implicit general rule is to use complementary methods to assess membrane
protein association/interactions with lipid rafts. Traditional approaches such as cho-
lesterol depletion and colocalization with raft markers have provided important qual-
itative data regarding this relationship, whereas significant quantitative insights were
obtained from advanced optical imaging techniques, namely, photoactivated local-
ization microscopy (PALM), stimulated emission depletion (STED), and near-field
scanning optical microscopy (NSOM). A description of these methods, covering
their advantages and handicaps, is given in the succeeding text.
6.1
METHODS TO DETERMINE PROTEIN ASSOCIATION
WITH LIPID RAFTS
Historically, protein association with lipid rafts was evaluated by searching for target
protein presence in the insoluble membrane fraction obtained after gradient centri-
fugation of cell membranes lysed with cold (4
C) nonionic detergents (
Brown,
2006
). However, the different composition of the insoluble fractions and variable
yields of protein resistance to solubilization obtained by this method have led re-
searchers to progress to more robust experimental approaches. In the succeeding text
a description of the most commonly used techniques to study membrane protein as-
sociation with lipid rafts is presented.
6.1.1
Lipid raft disruption by cholesterol-depleting agents
A typical method to determine protein association with lipid rafts is to assess if target
protein membrane localization/organization is affected by lipid raft disruption in-
duced by cholesterol depletion. This can be achieved by different approaches such
as (i) depletion and removal of cell cholesterol by cyclodextrins (
Zidovetzki &
Levitan, 2007
); (ii) use of microbial cholesterol oxidases that catalyze cholesterol
to 4-cholesten-3-one; (iii) cholesterol sequestration by polyene macrolide antibi-
otics, namely, filipin, nystatin, or amphotericin (
Gimpl & Gehrig-Burger, 2007
);
