Biology Reference
In-Depth Information
2006; Le Lay et al., 2009
). Compared to cyclodextrins, this treatment is less harm-
ful, since no cholesterol is extracted from the cells, only converted. Nonetheless,
cholesterol present on the membranes of living cells is usually a poor subtract for
cholesterol oxidase and thus this treatment is not as efficient in disrupting rafts as
that with cyclodextrin.
6.1.2
Colocalization with putative lipid raft markers
A direct detection of target protein interactions with lipid rafts on intact cell mem-
branes can be done by studying its membrane localization and comparing it with that
of putative raft markers. This colocalization approach overcomes the intrinsic lim-
itations of the invasive methods described earlier. The identification of the protein of
interest and putative raft markers is done by specifically labeling with antibodies,
chemicals, or fusion proteins, which are chosen according to the experimental tech-
nique applied. Putative raft markers are selected among the class of molecules found
in high percentages in the low-density fractions resulting from membrane solubili-
zation studies (
Simons & Gerl, 2010
). As a control, target protein membrane local-
ization is usually also compared to that of a nonraft marker. In
Table 6.1
is a list of
putative raft and nonraft markers commonly used in this type of studies. Recently,
important aspects of the molecular mechanisms governing lipid raft formation
and targeting membrane protein to these domains have been unraveled by combining
this approach with fluorescence imaging of cell-derived giant plasma membrane ves-
icles (
Levental, Lingwood, Grzybek, Coskun, & Simons, 2010; Lingwood, Ries,
Schwille, & Simons, 2008
). These structures maintain the compositional complexity
and protein content of the cell membrane, not exhibiting the complexity introduced
by the cell's cytoskeleton and intracellular proteins, which are not present in these
vesicles.
Although valuable information about protein localization with lipid rafts can be
gathered by this approach, especially when combined with advanced imaging
Table 6.1
Lipid Raft or Nonlipid Raft Markers Commonly Used in Colocalization
Studies
Marker
Lipid Raft Associated
Ganglioside (G
M1
) (labeled with cholera toxin B)
Yes
GPI-anchored proteins (PLAP, CD59, DAF, etc.)
Yes
Caveolin-1
Yes
Flotillin
Yes
EGFR
Yes
Transferrin receptor (TfR)
No
Calnexin
No
Unsaturated glycerophospholipids (DOPC, DOPE, etc.)
No
