Biology Reference
In-Depth Information
However, the size of lipid rafts in biological membranes has yet to be fully determined, but it
is estimated to be very small (1 to 1,000 nm in diameter). The estimated size of biological lipid
rafts continues to decrease as imaging methodologies improve
[63]
. Many of the more recent
studies have estimated lipid raft size at the lower end of the range, ~5 nm in diameter. The
vanishingly small size of lipid rafts in biological membranes raises the possibility that even if
rafts do exist, they may not be stable enough (they are just too small) to have any biological
function. Rafts may not exist for a long enough stretch of time to attract the appropriate
signaling proteins.
Lipid rafts are of two basic types, planar rafts and invaginated, flask-shaped rafts called
caveolae
[61,64]
. Planar rafts and caveolae can be easily distinguished. Although both
have essentially the same typical raft lipid composition, planar rafts have flotillin proteins,
while caveolae have a characteristic flask-shape and caveolin proteins. Flotillins and caveo-
lins both function in recruiting signaling molecules into lipid rafts and thus provide the raft
skeletal frame. In a parallel fashion it has been proposed that cholesterol is the 'molecular
glue' that holds the raft lipids together. It has been known for decades that cholesterol has
a strong preference for sphingolipids over other phospholipids (see Chapter 10)
[65]
. Choles-
terol also has a stronger affinity for saturated acyl chains and avoids polyunsaturated chains
[66,67]
. Finally, a hallmark of lipid rafts is their dissociation upon removal of cholesterol by
b
-
cyclodextrin
[68]
.
The earliest support for lipid rafts came from cold temperature detergent extractions,
but it is not certain what these experiments actually mean. At 4
C, the non-ionic detergent
Triton X-100 (later success was also reported with Brij-98 and CHAPS) solubilized much of
the membrane, while the insoluble components, cholesterol, (predominantly) saturated
phospholipids, sphingolipids, and characteristic signaling proteins, were left behind as
lipid rafts
[69]
. The insoluble fraction is often referred to as detergent resistant membranes
(DRMs) or sometimes detergent-insoluble glycolipid-enriched complexes (DIGs). Later
advances eliminated the need for detergents, reducing possible artifacts. There are now
countless published methods to isolate lipid rafts, most of which are slight variations on
a basic theme. Below is an abbreviated outline of one of the most commonly used non-
detergent procedures, known as the 'Smart Prep' after its inventor Eric Smart of the
University of Kentucky
[70]
. The procedure is for the isolation of caveolae, a type of lipid
raft.
BASIC STEPS IN THE SMART PREP
[70]
Wash cells in 250 mM sucrose (isotonic) buffer.
Low speed centrifugation.
Homogenize cells with Dounce homogenizer (20x) and pass through a 21 gage needle
(20x) in sucrose.
Low speed centrifugation to remove debris and obtain a post nuclear supernatant.
High speed centrifuge on 30% Percoll self-generating gradient.
Band at middle is the plasma membrane.
Sonicate the plasma membrane fraction.
High speed centrifugation on Optiprep to isolate caveolae.
All steps done at 4
C.
