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THE PLASMA MEMBRANE, which surrounds biological cells, consists of hundreds—possibly even thousands—of different lipids that are arranged in a bi-layer. Membrane proteins embedded in the bilayer occupy a large fraction of the mem-brane surface, and the cytoskeleton, a lat-ticework of intracellular protein filaments, attaches to the inner side of the membrane. This complexity makes the plasma mem-brane and its constituent lipids difficult to study directly.

That’s where model systems come in. These stripped-down constructions allow scientists to probe the behavior of lipid membranes under carefully controlled cir-cumstances. Scientists have garnered a lot of insight about the membrane from model systems, but these ideal-ized versions of membranes may be reaching the limits of what they can reveal about biology.

Cell biologists and biophysi-cists use lipid model systems to gain a physical and chemical un-derstanding of the plasma mem-brane. Such models typically contain three components—an unsaturated lipid, a saturated lipid, and cholesterol. These three components stand in for the multitude of lipids found in the natural cell membrane. Even such simplified mixtures can answer questions about the be-havior of the lipid portion of the membrane.

With model systems “you can get at fundamental physi-cal chemical questions,” says Barbara A. Baird, a chemistry professor at Cornell University. Such questions include how the lipids organize themselves into multiple liquid phases, called domains, and under what condi-tions those phases form.

Of particular interest to re-

searchers studying membrane model sys-tems is whether membranes can spontane-ously form coexisting liquid phases in the absence of proteins. “You can understand how lipids work and then extrapolate and use that as a model of how they might work in a biological system,” Baird says.

Model systems suggest membrane lipids are in a “very peculiar state,” says Jay T. Groves, a chemistry professor at the Univer-sity of California, Berkeley. The largely linear, oily molecules, each with a polar, hydrophilic end, seem to be near a critical point in their phase diagram—a combination of composi-tion, pressure, and temperature at which two coexisting phases become identical.

The remarkable thing is that this behav-

ior occurs in lipid mixtures similar to those in cells. This suggests that cell membranes might hover around a critical point in the lipid phase diagram, where even small changes in conditions can trigger large changes in the membrane. “It’s like the cell evolved itself a solvent that doesn’t resist all the different things it would need to do,” Groves says, referring to the way lipids serve as a versatile solvent conducive to signaling and other interactions on and among cells.

Model systems are valuable because “you can really affirm with no ambiguity whatsoever that lipids and cholesterol have these physical properties as a mixture,” Groves says. “Those physical tendencies don’t go away when you put this mixture into the membrane of a cell.”

One of the key issues that can be ad-dressed with model systems is lipid phase behavior. Using three-component systems, independent groups led by biophysicists Gerald Feigenson at Cornell and Sarah Keller at the University of Washington, Se-attle, see lipid mixtures separate into coex-isting phases. Seeing such phases in model systems is a first step toward answering the question of whether such domains exist in the intact cell membrane.

In a quest for biologically rel-evant model systems, Feigenson is moving toward more complicated four-component systems—three lipids plus a “judiciously chosen protein.” He thinks such a mixture is the minimum for a model sys-tem to approximate a real system.

GIANT PLASMA membrane vesi-cles, or “blebs,” offer an even clos-er approximation. Blebs, released by cells either naturally or by labo-ratory inducement, have composi-tions similar to cell membranes. They are more complicated than synthetic model systems but sim-pler than intact cell membranes, in part because they lack connec-tion to an underyling cytoskeletal network. The group led by Baird and David Holowka at Cornell is studying the phase behavior of blebs. “I see this kind of work as a bridge between the well-defined model systems and the more com-plex biological systems,” Baird says. Nevertheless, she notes, “it’s pretty tricky business trying to relate it to a biological system and to a model system.”

SIMULATING LIFE’S ENVELOPES

Models provide clues about LIPID BEHAVIOR in cell membranes, but they may have reached their limits

CELIA HENRY ARNAUD, C&EN WASHINGTON

SCIENCE & TECHNOLOGY

LIPIDS IN FLUX This three-component unilamellar vesicle starts with a uniform distribution of lipids (top left). Slightly above (top right) and below (bottom left) the critical temperature, the lipid composition and domain boundaries fluctuate. Far below the critical temperature (bottom right), phase-separated domains appear.

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Mass spectrometric analysis shows that the lipid composition of blebs appears similar to the composition of the cell mem-brane, to the extent that the composition of the cell membrane is actually known, Baird says. Plasma membrane preparations from cells are often contaminated with lipids from the membranes surrounding internal organelles, which have differ-ent lipid compositions from the plasma membrane, she says. “The cleanest work on membranes was done on red blood cells because they don’t have those internal membranes,” Baird notes.

The work of Sarah L. Veatch, a postdoc in Baird’s group, suggests that blebs exist

in a state near a critical point on a phase diagram (ACS Chem. Biol. 2008, 3, 287). At such points, the system goes through wide fluctuations and easily switches between a single phase and multiple phases. “This kind of fluctuating system can be har-nessed to cause a rather dramatic change with the appropriate signal,” such as a changing temperature, Baird says.

Veatch studied membrane behavior by fluorescence microscopy. At 20 ºC, mi-crometer-sized domains form in the mem-branes. Extrapolating those findings to 37 ºC suggests that nanometer-sized domains should form in biological membranes at physiological temperatures.

“The idea that Sarah Veatch can take blebs and see exactly the same behavior near critical points that we routinely see in purely synthetic vesicles is hugely ex-citing,” Keller says. “It says that even our ridiculously simplified system just might really be biologically relevant.”

One of the unanswered questions about cell membrane phase behavior involves the existence of so-called lipid rafts, which are minuscule, patchlike domains that are be-lieved to be involved in protein clustering and cell signaling. These rafts are thought to consist of a more highly ordered cho-lesterol and sphingolipid-rich liquid phase (the “liquid ordered” phase) interspersed with a less ordered liquid phase (the “liq-uid disordered” phase). Although liquid-ordered phases have been seen in model systems, the evidence for their existence in natural cell membranes is hard to find. Un-

SCIENCE & TECHNOLOGY

Model membranes suggest that the lipids in membranes form coexisting liquid phases, such as the putative choles-terol- and sphingolipid-rich regions known as lipid rafts. But these domains have been hard to detect in real mem-branes, possibly because they are too small or too short-lived.

“We’re on the verge of understanding more because of the advent of new tools,” says Kenneth Jacobson of the University of North Carolina, Chapel Hill. The cell membrane is “not a crystal, but it displays some order on various length and time scales. Up to now, we haven’t really had the tools to study those things, but in the next 10 years those tools will emerge.”

Researchers hope that superresolution optical microscopy techniques like PALM (photoactivated localization microscopy), STORM (stochastic optical reconstruction microscopy),

and STED (stimulated emis-sion depletion) microscopy will give them ability to see nanometer-scale domains (C&EN, Sept. 4, 2006, page 49). A recent report suggests that they won’t hope in vain.

Using STED, Stefan W. Hell, a researcher at the Max Planck Institute for Biophysi-cal Chemistry, in Göttingen, Germany, and the inventor of STED, observed nanoscale dynamics of membrane lipids in a living cell (Nature, DOI: 10.1038/nature07596). He and his coworker Christian Eggeling found that sphingo-lipids and lipid-anchored pro-teins are trapped for 10–20 milliseconds at a time in cho-lesterol-rich regions smaller than 20 nm. These complexes could represent the first sightings of the elusive raft domains in living cells.

Mary L. Kraft, a chemical engineer at the University of Illinois, Urbana-Champaign, is developing a mass spec-trometric technique known as nanoSIMS (nanoscale

secondary ion mass spec-trometry) to look at phase behavior in cell membranes. She first developed this ap-plication of nanoSIMS while she was a postdoc in Steven G. Boxer’s group at Stanford University.

In nanoSIMS, a tightly fo-cused beam of Cs+ ions bom-bards the sample and sput-ters atomic and diatomic ions off the surface. “It’s efficient to break up and produce lots of ions from the molecules on your surface,” Kraft says. “You get a lot more pieces out” compared with conven-tional MS analysis, “so you have a lot more signal to col-lect. Therefore, you can ana-lyze much smaller regions.” However, because the lipids in a membrane are com-pletely fragmented by the nanoSIMS technique, species of interest need to be isotopi-cally labeled to be identified. Two years ago, Kraft and Box-er used nanoSIMS to observe phase separation in a two-component model membrane

(C&EN, Oct. 2, 2006, page 11; Science 2006, 313, 1948).

Now, Kraft and her col-laborators are pushing the method into cells. When they do nanoSIMS analysis of cells that were fed N-15-labeled sphingolipid precursors, they see N-15-enriched “hot spots” in the cell membrane. Kraft is in the process of confirming that these preliminary results reflect the membrane and are not artifacts of the labeling procedure. “It looks like it’s going to be very feasible” to use nanoSIMS to analyze cell membranes, she says. The spatial resolution is better than 100 nm.

Because nanoSIMS is a high-vacuum technique, Kraft sees it as complementary to electron microscopy. “Elec-tron microscopy gives you structure but not chemical composition,” she says. In contrast, through careful iso-topic labeling, nanoSIMS can provide chemical information about the structures seen with electron microscopy.

LABORATORY TECHNIQUES

New Tools Offer Look At Tiny Domains In Membranes

With model systems “you can get at fundamental physical chemical questions.”

33WWW.CEN-ONLINE.ORG FEBRUARY 9, 2009

der physiological conditions, the domains may be too small or too transient to see with conventional tools.

Model membrane systems also give re-searchers a way to study asymmetric bilay-ers, in which two layers have different com-positions, much like the outer and inner “leaflets” of the cell membrane. Asymmet-ric membranes allow researchers to address whether lipid domains in one leaflet can in-duce the formation of domains in the other, even if the lipid composition of the second leaflet doesn’t usually form domains.

One way to make these asymmetric membranes is with polymer-tethered sup-ported membranes, in which a polymer lay-er lifts a model membrane from its underly-ing substrate. The polymer supports make the asymmetric bilayers easier to prepare by giving them added stability, says Lukas K. Tamm of the University of Virginia.

“Using polymer-tethered membranes of asymmetric lipid compositions, we and others have shown that raft-mimicking domains in the bottom layer can induce corresponding domains of equal size on top,” says Christoph Naumann, a chemis-try professor at Indiana University-Purdue University, Indianapolis.

Keller and Marcus D. Collins have observed similar coupling in unsupported asymmetric planar bilayers. By tuning the lipid com-position of only one leaflet, they can induce phase separation in ei-ther both leaflets or neither leaflet (Proc. Natl. Acad. Sci. USA 2008, 105, 124).

A DANGER of model systems is in taking them too literally, says Michael Edidin, a biologist at Johns Hopkins University. Just one of the problems is that people study model systems under condi-tions in which domains are on the micro meter scale and thus easy to see. “You just don’t see domains that large in cell membranes,” he says.

Groves similarly believes that people would be wise to exercise caution in their extrapolations of behavior observed in model sys-tems to cells. The structures seen in model systems provide clues about how lipids might behave in cell membranes, but the cor-relation will not be exact. Instead, Groves believes the models show

that the lipids in membranes exist in a state that al-lows them to re-spond quickly to changes in their environment.

Edidin sounds another cautionary note about the energy of membranes. The energy needed for cell membrane processes or functions is much higher than the comparatively weak interactions between the lipid acyl chains, he says, but these higher energies are not available in model systems. “You’re talking about ATP being split to do things like en-docytosis and exocytosis,” which are much more highly energetic processes than the much weaker interactions between lipids in model membranes, such as hydrophobic effects, hydrogen bonding, and van der Waals forces.

Yet another pitfall of models is the pos-sibility of introducing artifacts with mea-

surements. For example, the intense light in fluorescence microscopes can generate free radicals that induce changes in some model membranes, Feigenson says. “It’s a problem with chemically simple mixtures, but it might not be such a problem with real cells, because they have reducing systems that will find and destroy light-induced free radicals.”

Groves doesn’t think that increasing the complexity of model systems will be useful. “Complex model systems can give you a false sense of security that you can take them more seriously” than simple

models, he says. “We have no way of knowing that the complex model system resembles the actual cell membrane any more than simpler systems.”

In fact, the time has come to move on from model systems, Groves says. “There will always be a role for model systems in stud-ies of cell membranes. However, for my own work, I think we have learned enough from them. The questions we’re asking are about the cell membrane and not about soft condensed matter physics. The model systems help us un-derstand the interesting soft con-densed matter physics of these systems, but not the cell biology” of the membrane.

That may be, but some re-searchers will continue working with model systems to further elucidate the role of lipids in cell membranes. “The models have to get more complicated, and the resolution in cells has to get bet-ter,” Edidin says, conceding how-ever that “there’s going to be some upper limit reached where you might as well look at an isolated native membrane.” ■

INDUCED STRUCTURE In a supported lipid bilayer, domains in one layer (green) can induce the formation of domains in a lipid mixture that wouldn’t usually form domains (red).

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TRIANGULATION A Gibbs triangle shows the experimental phase behavior of a ternary mixture at fixed temperature (here 23 ºC) as a function of mixture composition. This mixture is a simple model for the outer leaflet of an animal cell membrane. Three bilayer phases are possible: Ld, Lb, and Lo. Biologists are interested in the Ld + Lo region and its relationship to “lipid rafts.”

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