bio sphere
Where the lipids are W
hen Christoph Thiele started investigating lipid biology, he noticed that the field lacked appropriate labels. The most commonly used fluorescent lipid tags—nitrobenzoxadiazole (NBD) and BODIPY—do not behave like natural lipids. “That was the starting point for me when I was setting up my group,” he says. “We wanted to have a tool that was simply more reliable in reflecting where endogenous lipids really are.” Thiele and his colleagues at the Max Planck Institute of Molecular Cell Biology and Genetics (Germany), therefore, developed fluorescent polyene lipid tags that are more similar to natural lipids (Nat. Methods 2005, 2, 39– 45). The polyene tags developed by Thiele’s group are lipids that contain a conjugated double bond system of at least five double bonds. Although naturally occurring lipids can have several double bonds, they are always isolated. Thiele says that conjugated double bonds allow the molecule to fluoresce. After synthesizing polyene tags, Thiele and his colleagues studied their properties in several assays. In an in vitro test, polyene-labeled sphingomyelin (SM) preferred the liquid-ordered, raftlike phase of a liposome, whereas BODIPY-labeled SM localized to the liquid-disordered phase. The researchers then tested the tags in living mammalian cells by feeding labeled precursors for SM to the cells. Both polyenelabeled SM and untagged SM were in lipid raft fractions of a sucrose gradient, but NBD- and BODIPY-tagged SM were present in different fractions. By altering the lengths and the chemistries of the polyenes, Thiele and his colleagues could change the tags’ specificities for different classes of lipids. Shorter polyene-labeled precursors added to cells tagged phosphatidylcholine (PC), and longer ones tended to label phosphati© 2005 AMERICAN CHEMICAL SOCIETY
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Fluorescent polyene lipids in (a) kidney cells, (b) adipocytes, and (c) muscle cells. (Adapted with permission. Copyright 2005 Nature Publishing Group.)
dylethanolamine and phosphatidylinositol (PI). When the researchers added radiolabeled natural precursors of the same lengths to cells, the distribution was the same. Since this work was published, Thiele has synthesized additional precursors that specifically label a single lipid. “Some are not yet accessible; we have problems with PI [specificity], for example, because the chemistry is much more complex,” he says. The researchers also examined the subcellular localizations of polyenetagged lipids in living cells. Because the tags are very sensitive to photobleaching, one of the biggest challenges Thiele faced was finding an appropriate fluorescence microscope for his studies. Members of his team tested several of them in the microscopy facility at their institute. An expert initially advised Thiele against two-photon imaging because it could
damage the fluorophores, but the team eventually tried this type of setup anyway. “We didn’t have really major hopes, and then it was a breakthrough,” he says. Colocalization studies with a marker protein indicated that polyene-tagged PC localized to the nuclear envelope and endoplasmic reticulum (ER) in fibroblast cells. This result was expected on the basis of biochemical studies by other groups. No one has been able to image ether lipids before now, says Thiele, but his tags enabled the visualization of polyenelabeled ether lipids in the ER and in lipid droplets. “There are other localizations, and we are studying where [ether lipids] are and how they actually get there,” he says. Researchers in the field are interested in these new tags. Burton Litman at the National Institutes of Health says, “I think it’s a very important advance [because the polyene tags] eliminate the bulky probes that have been used in the past, [such as] NBD and BODIPY, which certainly perturb the physical properties of membranes.” He says that the polyenes appear to be more similar to natural lipids in many respects and that they could be used as markers for lipid rafts. “One of the limiting factors is going to be . . . the size of the raft,” says Litman, because rafts may be too small to accumulate enough polyene-labeled lipids to produce a strong signal. Barbara Baird at Cornell University says that the new tags appear to be very promising but warns that more characterization is necessary. She says that partition coefficients should be determined and that the tags should be compared to additional labels, such as Cy dyes. “What will be important is that these get into the hands of a lot of researchers so that they can be tested,” says Baird. a —Katie Cottingham
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