The Tiniest of Life's Rafts

GETTING TECHNICAL

The Tsien study used fluorescence resonant energy transfer (FRET) to demonstrate protein interactions, by mapping energy transfer between labeled molecules. "What FRET measures is the distance between the so-called donor and acceptor," says Anne Kenworthy, assistant professor of molecular physiology and biophysics at Vanderbilt University in Nashville, who coauthored a seminal paper on FRET in 1998.³ "But it has a very sharp cutoff. So if you get to distances above around 100 Ångstroms, then there's no FRET signal anymore." By comparing experimental FRET values to those predicted for a random distribution, scientists can infer molecular organization.

To track lipid aggregation, the investigators needed a fluorescent molecule that didn't aggregate on its own. The GFP from Aequorea has the weakest propensity to congregate, but in a two-dimensional space such as the plasma membrane, they dimerize readily, says David Zacharias, first author from the Tsien group, and now an assistant professor of neuroscience at the University of Florida, Gainesville. To prevent GFP dimerization, Zacharias eliminated the inherent stickiness of the fluorescent tag via site-directed mutagenesis. Thus, any clustering of fluorescence was determined by the lipid modification of GFP, not GFP itself. "One important technical aspect [the paper] gets cited for is the monomeric GFPs," says Zacharias.

The methodology has "huge potential," although it has yet to advance knowledge of raft function, says Richard Anderson, professor of cell biology at the University of Texas Southwestern Medical Center at Dallas. "I don't think they're giving better data, just different data." Anderson laments that technology can obfuscate biology, especially when techniques require special expertise. "If I have one complaint about this field, it's that no one verifies that their technique meets the criteria that other techniques have already established." Kenworthy agrees, saying that it's not clear how clusters detected by FRET are correlated with molecules found in detergent-resistant membrane fractions.

BIOLOGICAL CONTRIBUTIONS

By attaching GFP to lipids rather than proteins, Zacharias' experiments favor a primary role for lipids in raft formation. "It was the lipid modifications themselves that contained the targeting information," says Kenworthy. Simons says this should dispel a lingering contention that raft proteins are in the driver's seat.

© 2002 AAAS

Researchers created myristoylated and palmitoylated (MyrPalm), geranylgeranylated (GerGer), tandemly palmitoylated (PalmPalm), and caveolin fusion constructs and expressed them in MDCK cells. Arrows denote representative sites for data collection.

Zacharias, who was a postdoctoral fellow in Tsien's lab when he did the studies, confesses "a serious naïveté" regarding the probes he chose to study. Nonetheless, that innocence led to two major contributions with regard to characterizing lipid rafts. Because the lipid chains he used were inner-leaflet probes, "it was the first physical evidence in live cells that you get raft formation in the inner leaflet," he says.

More surprising was the finding that a prenyl group can target proteins to the plasma membrane. "This is perhaps the most contentious point of the whole paper," says Zacharias, because prenylation had been thought to target proteins exclusively to endomembranes, such as endoplasmic reticulum and Golgi.

This new role for prenylation jolted the field in the way that breeds doubt. But subsequent work has confirmed the existence of prenylated protein rafts, says Kenworthy. "Now we have a lot more information about that, in particular for the Ras family of proteins. So that's one area where new work has been done, using other techniques," including electron microscopy, functional assays of Ras signaling, and fluorescence recovery after photobleaching (FRAP).

Another unusual aspect of the prenylated protein clusters was that they were not disrupted by cholesterol depletion. Because classic lipid rafts are enriched in cholesterol, "If you deplete cholesterol, the clusters disperse," says Kenworthy. The prenylated proteins Zacharias found were in the cholesterol independent domains, she says, indicative of a truly different type of raft.

Criticisms of the Zacharias paper center on how well experimental data fit predictions for clustering because the variability was huge. But many researchers believe rafts themselves are transient, with lipid and protein components constantly moving in and out. "That would definitely explain some of that scatter," says Jennifer Lippincott-Schwartz, head of the section on organelle biology at the National Institute of Child Health and Human Development.

FROM FRET TO FRAP

As evidence accumulates for a wide variety of lipidraft structures, caveolae, once the granddaddy of membrane domains, may be "the exception to the rule," says Lippincott-Schwartz, because of their discrete size and shape. And still, after decades of study, caveolae function has yet to be sorted out.

Van Deurs wanted to address the longstanding question of whether caveolae are involved in endocytosis. It is a question, van Deurs says, that is inspired in part by the "nice shape that they have," referring to the flask-shaped indentation of the plasma membrane. "If caveolae are equivalent to clathrin-coated pits, then they must be dynamic structures," says van Deurs. The movement of clathrin-coated pits is evidenced by clathrin's short half-life on the plasma membrane, which is about one minute.

To study caveolae mobility, van Deurs tagged caveolin with GFP and then used FRAP. After photo-bleaching a small portion of the cell surface to wipe out the fluorescent signal, they monitored the area for renewed fluorescence. A large recovery would be consistent with the tagged caveolin moving into the bleached area, but this is not what occurred. "It turned out that they have very little mobility," says van Deurs. "So they cannot be involved in endocytosis in the normal definition of the word," although van Deurs acknowledges that special circumstances, such as stimulation by SP-40 virus, would cause caveolae to internalize.

So while the van Deurs study characterized caveolae as more anchored than previously thought, the Tsien study has pushed the boundaries of what lipid rafts can be in the other direction, that is, smaller and more transient. The enormous appeal of linking functional molecules across the plasma membrane initially propelled lipid raft research. But structural evidence, characterized in molecular detail with fluorescence technology, has failed most preconceived notions of these membrane organelles and forced a paradigm shift in thinking about function of these elusive structures.

Jill U. Adams juadams@the-scientist.com