Despite all of the work published on lipid rafts, it is not clear whether everybody is studying the same thing or even studying what they intend to study. Lipid domains cannot be isolated in a native state and, with the exception of caveolae, are presumed to be too small and transient to be directly observed in unperturbed living cells. But the relationship between their operational definition—detergent- insoluble and cholesterol-dependent in vitro entities—and any real world counterpart is not clear.
The cold detergent procedure for preparing membranes is very convenient, and continues to be used. These analyses have perhaps reached their apex with a recent proteomic definition of raft complements (Foster et al., 2003
). But how informative are these studies? Serious caveats do apply. In vitro, the method can yield large, >1-μm2
sheets. These are generally taken to be an artifact, but may reflect aggregation of smaller, bona fide rafts. Other concerns include contamination or loss of components during extraction, and nonstandardized extraction procedures (Edidin, 2003
Still, some researchers argue for the practical value of the method. Gerrit van Meer (Utrecht University, Utrecht, Netherlands) concedes that “this is a relatively blunt tool, and not everything you see is real,” but says, “one has only to see that many proteins defined by this method are functionally involved in raft signaling…so it has been a useful tool.” Debbie Brown (State University of New York, Stony Brook, NY) believes that “detergent insolubility does reflect the affinity of certain proteins for an ordered lipid domain.” Such associations are dynamic (e.g., during signal transduction) and thus probably not a nonspecific membrane property.
Dick Anderson (University of Texas Southwestern Medical Center, Dallas TX), however, specifically blames much of the confusion regarding lipid domains on the wide usage of the extraction procedure and suggests that the procedure itself “creates the phenomenon” of rafts. “This type of cell fractionation can be very misleading, and is not necessary,” he says. He encourages others “to just give up on the detergent extraction method” in favor of other methods such as detergent-free fractionation after sonication or, in the case of caveolae, α-caveolin immunopurification.
Cholesterol dependence also defines rafts. But cholesterol depletion can alter cell morphology, exocytosis, and trafficking and, according to new work from Michael Edidin (Johns Hopkins University, Baltimore, MD), disrupt the actin cytoskeleton, which could have any number of pleiotropic effects. Sandy Schmid (Scripps Research Institute, La Jolla, CA) says that “cholesterol depletion makes cells sick.”
Others are more confident in the directness of cholesterol depletions, if they are performed judiciously. Unlike cholesterol, sphingolipids and GPI- anchored proteins are restricted to the outer leaflet of the membrane. Chris Fielding (University of California, San Francisco, CA) believes that “cholesterol is flipped slowly enough between the leaflets that careful experiments should allow relatively specific depletion of cholesterol from the exofacial leaflet,” thus specifically disrupting rafts on the outer leaflet. Arnd Pralle (University of California, Berkeley, CA) agrees, stating that “acute effects of cholesterol extraction on rafts can be observed within ten minutes, with perhaps only 50% loss of cholesterol”—a time period and decrease that should not strongly affect other cellular processes. Nevertheless, the speed with which cholesterol flips is contested and cholesterol is likely to be important for raft phenomena on the inner leaflet, so appropriate treatments are not agreed upon by researchers.