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Analytical Chemistry

New Tools Offer Look At Tiny Domains In Membranes

by Celia Henry Arnaud
February 9, 2009 | A version of this story appeared in Volume 87, Issue 6

Model membranes suggest that the lipids in membranes form coexisting liquid phases, such as the putative cholesterol- and sphingolipid-rich regions known as lipid rafts. But these domains have been hard to detect in real membranes, 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 emission 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 Biophysical Chemistry, in G??ttingen, 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 sphingolipids and lipid-anchored proteins are trapped for 10–20 milliseconds at a time in cholesterol-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 spectrometric technique known as nanoSIMS (nanoscale secondary ion mass spectrometry) to look at phase behavior in cell membranes. She first developed this application of nanoSIMS while she was a postdoc in Steven G. Boxer's group at Stanford University.

In nanoSIMS, a tightly focused beam of Cs+ ions bombards the sample and sputters 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 conventional MS analysis, "so you have a lot more signal to collect. Therefore, you can analyze much smaller regions." However, because the lipids in a membrane are completely fragmented by the nanoSIMS technique, species of interest need to be isotopically labeled to be identified. Two years ago, Kraft and Boxer 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 collaborators 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. "Electron microscopy gives you structure but not chemical composition," she says. In contrast, through careful isotopic labeling, nanoSIMS can provide chemical information about the structures seen with electron microscopy.


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