Seeking Accurate Traffic Reports

JENNIFER LIPPINCOTT-SCHWARTZ

Courtesy of Jennifer Lippincott-SchwartzOn the desk in Jennifer Lippincott-Schwartz's office sits a small box labeled "Mr. Wizard's Golgi Model Kit." Along one edge it reads, "Fun for whole lab! No biochemistry, photobleaching, or batteries required." A gift from a former postdoc, the homemade kit is stuffed with sugar packets and other cafeteria-pilfered goodies that Lippincott-Schwartz likes using to demonstrate how the Golgi apparatus – the flattened stack of membranes responsible for sorting and packaging cellular proteins – assembles and operates.Lippincott-Schwartz, head of the Organelle Biology Unit at the National Institutes of Health, is a big fan of visual aids. She grew up in a house where the periodic table was displayed prominently on the kitchen wall. This passion for looking at things and seeing how they work now drives her investigation of the ins and outs of membrane trafficking.Using green fluorescent protein (GFP) and its derivatives to tag various cellular proteins, Lippincott-Schwartz and her colleagues track the movements of molecules through the Golgi, the endoplasmic reticulum (ER), and other membranous compartments in living cells. The work has drawn her into a diverse array of biological arenas, including Drosophila development, poliovirus infection, even prion protein aggregation, and into more than a few heated scientific debates."Jennifer enjoys a good punch up," says Ben Nichols, a former postdoc, now at the MRC Laboratory of Molecular Biology in Cambridge, UK. "She certainly doesn't shy away from controversy," adds Peter Walters, a membrane biologist at the University of California, San Francisco."Some people think I'm trying to be heretical," says Lippincott-Schwartz. "But I'm not. It's just that we're using techniques that haven't been used before to visualize molecules. And it just so happens that a lot of times the way people thought about these systems has not been completely correct. So when we test the basic predictions of some of these models, they just don't hold up."Consider, for example, the current models that attempt to explain how newly synthesized proteins wind their way from the ER, through the Golgi, to the plasma membrane. According to the maturation model, proteins destined for export are embedded in the cisternae, the membrane sacs that make up the Golgi. As the cisternae mature, they move toward the plasma membrane, carrying the cargo proteins in conveyor-belt fashion along with them. In the vesicular trafficking model, on the other hand, the cargo is loaded into vesicles that ferry it in an orderly fashion from one cisterna to the next.To put these models to the test, Lippincott-Schwartz tracked the travels of three different cargo proteins tagged with GFP. Not only could she monitor these proteins as they passed through the Golgi, but by measuring the fluorescence in a quantitative manner, she could also determine how many molecules came in, and how quickly they left.For all three types of cargo, she found, the kinetics of transport were "completely inconsistent with the two classic models." Instead the data suggested a third possibility, a situation Lippincott-Schwartz calls "rapid equilibration." In this model, proteins enter the Golgi and mix rapidly with the cargo already in the system. Because the molecules are so thoroughly shuffled throughout the Golgi, each protein has the same chance of being exported as any other protein, regardless of when it entered the system. Unlike the setup in the classic models, the order of entry has nothing to do with the order of exit."There's going to be howls," she predicts, when colleagues hear her idea. Presentation of the data at a recent meeting in Barcelona "got people riled up," says Lippincott-Schwartz. Half the attendees applauded her story and half were "stunned and unhappy." Part of the problem, she says, lies in the math. "People are dubious because they're not used to thinking this way, [that is,] kinetically," she says. Lippincott-Schwartz grew comfortable with this quantitative, mathematical mindset only after spending "hours and hours with biophysicists, discussing what the models would predict."After performing additional experiments in which she could see where GFP-tagged molecules congregate within the Golgi, and the duration of their stay, Lippincott-Schwartz proposed that cargo proteins diffuse in and out of discrete export domains within the cisternal membranes. This rapid partitioning allows the cargo to swiftly mix, and explains why every protein has an equal chance of being exported at any given time. Cargo molecules that happen to be present when a transport vesicle buds from an export domain exit without a fuss."This is so much fun I can't stand it," says Lippincott-Schwartz. "I love this stuff!" Her love of peering inside cells and putting together stories stretches back to her grad school days, when she worked with Doug Fambrough at Johns Hopkins University. She used fluorescence imaging and electron microscopy to characterize the pathways that lead to and from lysosomes, the little bags of digestive enzymes that cells use to degrade proteins. She pursued microscopic techniques because she "wanted to see what these cells looked like, how the whole cell was put together," she says. "Then I really felt I was getting somewhere in terms of understanding the way the system works."She continued her investigations of membrane trafficking as a postdoctoral fellow with Rick Klausner at NIH. It was in Klausner's lab that Lippincott-Schwartz first watched the Golgi disappear. She was searching for ways to block the egress of proteins from the ER when she stumbled across brefeldin A, a drug that causes the Golgi to disassemble completely. The observation revealed that the Golgi is not a stable cellular structure, like the ER, but a dynamic system that can be built up and broken down as needed. "That was a huge development that some people still have problems with," says Lippincott-Schwartz.For the young investigators in her lab, Lippincott-Schwartz's willingness to question dogma and pursue unusual observations is inspirational. Nihal Altan-Bonnet, a postdoc who is now looking for a faculty position, plans to adopt her mentor's philosophy. "Jennifer taught me to not stick to the safe questions, to go for what's interesting," she says. "I definitely wouldn't want to do science any other way."Altan-Bonnet was drawn to Lippincott-Schwartz after hearing her give a talk at Rockefeller University. "She showed these phenomenal movies with molecules zipping around inside cells. It totally blew my mind," she says. "The techniques really bring everything to life. They make biology more exciting."And perhaps easier to decipher. "Cell biologists often have their noses pressed up against the glass or can't see the woods for the trees or whatever metaphor you want," says Nichols. But using GFP to track protein movement in living cells "allows you to look at the systems operating in an integrated way to get a handle on how things are organized." And it yields images that are stunning, and informative. "When you get a beautiful picture, it's beautiful in effect because it's a correct representation of what's going on," says Lippincott-Schwartz.But "she's not just looking at pretty pictures," notes Walter. "Jennifer makes a major effort to come up with the numbers. She gets a lot of credit for pioneering the use of GFP combined with quantitative analysis," which is the sort of thing that systems biology "claims to do," he adds.The one limitation: fluorescence microscopy can't distinguish structures smaller than the wavelength of light. "A lot of questions don't need that kind of resolution," notes Lippincott-Schwartz, "and you can always reframe your questions so that they can be addressed by the techniques that you have."That approach certainly keeps her busy. With a dozen or so people in the lab, all working on a broad range of problems, Lippincott-Schwartz worries that she might be spreading herself too thin. But she's loath to rein in anyone's intellectual curiosity, and with good reason. She recalls hosting a scientist on sabbatical from India. He proposed working on glyceraldehyde dehydrogenase (GAP-DH), one of the key enzymes in the glycolytic pathway. "When this guy came in I thought, 'I can't imagine what we'll get out of this,"' she says.But the visitor wound up demonstrating that GAP-DH can prevent aggregation of proteins, including prions and huntingtin, suggesting that this housekeeping enzyme might also act as a chaperone. "There's no way I would have done this project, except that he wanted to work on it, and now it's this amazing story," says Lippincott-Schwartz. "This teaches me that science is unpredictable in terms of where important concepts will come from. You've got to just be open. And ready."And being at NIH doesn't hurt. At a university, where most research requires grant writing, she says, "there's no way I'd ever be funded for this kind of work to start off with. But here at NIH we're free to do what we want to. This is an environment where you're encouraged to do high-risk research."It's in that environment that she's likely to stay. "We've been trying to recruit her for 20 years," laughs Walter. "But her husband works in the State Department, on issues of the Middle East." Since that situation is not likely to be resolved anytime soon, Lippincott-Schwartz will probably be staying put, which is just how she'd like it."I've been traveling too much lately," she laments. To maintain her productivity, she says, she needs to sit still and spend more time in the lab writing papers, advising her charges, and letting the proteins do all the running for a change.

Seeking Accurate Traffic Reports

On the desk in Jennifer Lippincott-Schwartz's office sits a small box labeled "Mr. Wizard's Golgi Model Kit."

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