Biology Reference
In-Depth Information
It is now clear that if non-caveolae lipid rafts exist, they must be extremely small and
fleeting. The process of isolating rafts takes many orders of magnitude longer than the prob-
able raft lifetime. Perhaps the membrane components that are isolated together as lipid rafts
only share similar physical properties but never actually exist together in the intact
membrane.
Experimental Support of Rafts
There is considerable experimental support for the existence of lipid rafts, primarily phase
separations in model membranes, cholesterol depletion experiments, and multiple compo-
nent experiments linking DRM analysis, cholesterol depletion, and microscopy. Micron-sized
domains have been visualized on the surface of GUVs (giant unilamellar vesicles) using fluo-
rescent membrane lipid tags that preferentially partition into
l
o
(raft) or
l
d
(non-raft) phases
[71]
. In one report, Dietrich et al.
[72]
demonstrated phase separation into
l
o
and
l
d
domains
using fluorescence microscopy. Importantly, Triton X-100 was shown to selectively solubilize
the
l
d
non-raft domain, while not perturbing the
l
o
raft domain. Similar model membrane
studies demonstrated that phase separation could be achieved at both cold temperature
(4
C) and physiological temperature (37
C), supporting the possibility of rafts existing in
living cells.
Hammond et al.
[73]
reported a study using a complex model membrane that supported
the feasibility of lipid rafts. GUVs were made from DOPC, DOPG, SM, cholesterol, the
ganglioside GM1, and the rhodamine-labeled LAT trans-membrane peptide. This lipid
mixture formed a single phase that was poised close to a separation boundary (
Figure 13.20
,
panel a). Upon addition of the cholera toxin B subunit, a dramatic phase separation occurred
(
Figure 13.20
, panel b). The fluorescent rhodamine-peptide preferentially partitioned into the
l
d
phase. Phase transition was induced by addition of the pentavalent cholera toxin B subunit
that binds to and cross-links the GM1s. Panel (a) is before the addition of cholera toxin, while
panel (b) is after cholera toxin addition. The dark regions in panel (b) are domains in the
l
o
(raft) phase.
One of the hallmarks of lipid rafts in biological membranes is a loss of physiological func-
tion, primarily related to signal transduction or membrane trafficking, upon cholesterol
depletion
[74]
. By far the major agent used to extract cholesterol from membranes is
b
-cyclodextrin, although the polyene antibiotics filipin and nystatin (Chapter 14) can effec-
tively remove cholesterol from rafts through complexation in the membrane. One of the
strongest pieces of evidence in support of lipid rafts is a strong correlation between DRM-
associated proteins, cholesterol depletion, loss of physiological function, and microscopy.
Although it now appears that DRMs are not the same as pre-existing rafts
[75]
, a surprisingly
large number of examples of these correlations exist in the lipid raft literature.
O
-
O
-
Ca
2+
FIGURE 13.19
Calcium Oxalate.
