Biology Reference
In-Depth Information
FIGURE 13.20
Fluorescence imaging of micron-sized domains on the surface of GUVs
[73]
. The model bilayer
membranes were composed of DOPC, DOPG, SM, cholesterol, the ganglioside GM1, and a rhodamine-labeled LAT
trans-membrane peptide. This lipid mixture formed a single phase (Panel a) that was poised close to a separation
boundary. Upon addition of the cholera toxin B subunit, a dramatic phase separation occurred (Panel b). The
fluorescent rhodamine-peptide preferentially partitioned into the
l
d
phase.
Questions Concerning Rafts
Many unanswered questions concerning the nature and even existence of lipid rafts
abound
[61]
. The major trouble concerns the extremely small apparent size of lipid
rafts in biological membranes (perhaps as small as 5nm and still shrinking) while
lipid rafts in model membranes can be in the micron range. How small is too small for
lipid rafts to have any biological significance? GPI-anchored proteins are routinely found
in DRMs, indicating that they are common raft components, yet by fluorescence resonance
energy transfer (FRET) GPIs are shown to be evenly distributed on the cell surface or are
present in at most very small (nanoscale) clusters containing only a few proteins. Also,
lateral diffusion measurements using fluorescence recovery after photobleaching (FRAP)
show that lipid raft components (GPI-anchored proteins, other raft proteins, and raft
lipids) do not diffuse as a single large unit in the plasma membrane. If raft components
diffuse at different rates, how can they be found together in a raft? It remains to be shown
if lateral membrane diffusion rates are sufficient to allow proteins time to diffuse into
lipid rafts. The preponderance of lipid raft isolations into DRM fractions use cold
temperatures (4
C) and non-ionic detergents, both of which can produce misleading arti-
facts. Using model membranes it is hard to access the potential importance of lipid asym-
metry and cytoskeletal involvement in rafts. Also, it is not certain if intracellular rafts even
exist.
Reputed raft proteins present another group of problems. Since raft lipids exist in the
l
o
state
where they are extended, membrane lipid raft domains should be thicker than the surrounding
non-raft
l
d
state regions. Indeed this has been confirmed by atomic force microscopy in model
