Gateways to Collaboration
Photo courtesy of Ehud Isacoff
Profiles provide insights into the lives, backgrounds, career paths, and futures of scientists who serve as Experts on ACS Chemical Biology’s online Ask the Expert feature. Readers are encouraged to submit questions to the Experts at www. acschemicalbiology.org. The editors will post the most interesting exchanges on the website.
Published online February 17, 2006 10.1021/cb0600048 CCC: $33.50 © 2006 by American Chemical Society
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s the name of this new journal implies, science is becoming more and more interdisciplinary. Researchers from what were once separate fields are increasingly combining their knowledge and talents to tackle problems that those from any one discipline would be hard-pressed to achieve on their own. Three investigators from the University of California at Berkeley epitomize this ever more popular style of collaboration: biologists Ehud Isacoff and Richard Kramer, and chemist Dirk Trauner. For the past 5 years, these researchers have mingled their unique ideas and diverse skill sets, culminating most recently in an article on a redesigned glutamate receptor that responds to light (1 ). Ehud Isacoff Born in 1959 in Darmstadt, Germany, Isacoff initially thought that the idea of entering a scientific field had been solely his own. In hindsight, however, he realized that he was likely influenced by his father, who had been a high school electronics teacher in Israel. “As in previous generations, it’s a case of keeping to the family business. My father studied electronics of wires, and I study electronics of brains,” says Isacoff. He remembers first becoming interested in neuroscience as a student at McGill University in Montreal, Canada. In his third year at the school, Isacoff enrolled in a lab course in which he took neural recordings of Acatina, a terrestrial slug whose cousin Aplysia is frequently used as a model organism by neuroscientists. Against instructions issued by the class teaching assistant, he and a few of his classmates used up reams of expensive chart paper one evening recording the slugs’ neural responses. The next morning, Isacoff recalls, “I woke up in the early hours, and
when I opened my eyes, I saw the chart paper grid on the walls. No matter how much I rubbed my eyes, it was still there.” “Neuroscience had been indelibly imprinted on my brain,” he adds. Isacoff continued on to a Ph.D. program at McGill under the mentorship of neuro scientist Richard Birks. Birks had been working for years measuring the secretion of the neurotransmitter acetylcholine that occurs during the “fight or flight” response. He used a smoke-barrel kymograph, an instrument now seen only in museums, to measure the acetylcholine that leached out of neurons and into blood vessels by examining how the neurotransmitter lowered blood pressure in lab animals. Isacoff extended this work by determining how changes in acetylcholine altered the electrical activity of responding neurons. Isacoff remembers the six years he spent working in Birks’ lab as an idyllic time, full of hard work and regular periods of pursuing nonscientific interests. “I had the world’s best Ph.D. experience,” he says. However, there were some drawbacks. He felt some frustration with the indirect methods he was using to measure what was taking place in the neurons that were releasing acetylcholine. That led Isacoff to take a different direction for his future research. “What I really wanted to start doing was working at the molecular level,” he says. He picked up some new molecular biology skills at a Cold Spring Harbor short course on Drosophila genetics two summers before he completed his doctoral degree in 1988. One of his instructors there was Lily Jan, a well-known neurobiologist at University of California at San Francisco. Impressed with Isacoff’s interest and initiative, Jan invited him to join her lab as a postdoctoral fellow. Jan and her colleagues had recently cloned the first potassium channel, w w w. a c s c h e m i ca l biology.org
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any of which could have been the source of an unknown signal. After Scheiffele identified a candidate signaling protein in dendrites, the researchers together purified the protein and incorporated it into an artificial bilayer. This protein-lipid complex was coated onto glass beads, which were then applied to axons. “It’s incredible,” says Isacoff. “The axon thinks it’s just been contacted by a dendrite and develops a transmitter release site. It’s not aware that it’s been contacted only by a bead.” Isacoff continues to advance this project and others, including those on which he collaborates with Kramer and Trauner. Richard Kramer Born in 1956, Kramer grew up near New York City. He remembers a favorite teacher turning him on to biology in a science class. The class encouraged his natural love for animals to grow, which in turn inspired the rest of his career. “I came at this job from being interested in animal behavior, then how the nervous system controls behavior, then working my way down to getting interested in the nutsand-bolts mechanisms of how neurons work,” he says. He began trying to understand these mechanisms as an undergraduate at the State University of New York in Albany. There, like Isacoff, Kramer began studying slugs. With the cast-off projects and equipment of a graduate student who was headed to medical school, Kramer spent a summer between his junior and senior year in a lab recording electrical feedback from Aplysia’s neurons. “It was a unique opportunity for an undergraduate to be doing independent research,” he says. He also remembers the experience as lending insight into the academic lifestyle, which he was keen to join. “The professor I was working for would have extravagant
Photo courtesy of Richard Kramer
an ion channel present in all neurons and many other cells throughout the body. Isacoff immediately chose two related projects on which to focus: determining whether the channel is made of more than one subunit, and exploring the mechanisms for how the channel’s gates open and close. By the time he finished his fellowship four years later, Isacoff had completed both projects. He accepted a job at University of California at Berkeley, where he started his own lab in February 1993. Isacoff has continued to work on a number of different projects with the help of graduate students, postdoctoral fellows and collaborators. For example, he and then postdoctoral fellow Lidia Mannuzzu and Lawrence Berkeley National Laboratory staff scientist Mario Moronne developed a novel way detect the movements within ion channels when they open and close. The three researchers monitored the movement of various amino acids in the channel protein by tagging them with fluorophores, such as rhodamine. By electrically stimulating the membranes of cells, which caused the ion channels to respond, Isacoff and his colleagues watched to see how the fluorescence of the probes changed. Their work provided the first real-time measure of protein motion in the channel’s voltage sensor (2 ). Recently, Isacoff’s team devised a new way to investigate how a synapse forms between two neurons (3 ). He and his colleagues worked with Peter Scheiffele, a former postdoctoral fellow in a neighboring lab at Berkeley, who now runs his own lab at Columbia University in New York. The researchers wanted to identify the minimum protein signal during synapse formation that dendrites, the projections that conduct electricity into one neuron, send to axons, the projections that conduct electricity away from another neuron. Understanding what forms a connection between compatible dendrites and axons is tricky because both cells contain a host of proteins, says Isacoff,
parties at his house now and then, and he traveled around the world. It seemed like an attractive lifestyle,” Kramer notes. When he finished his undergraduate degree in 1978, Kramer was intent on heading to the West Coast for graduate school. He applied, and was accepted, to the University of California at Berkeley. There, he worked under the mentorship of Robert Zucker, a neurophysiologist who also studied Aplysia. But Kramer had his own project in mind. He became interested in nerve cells that act as oscillators, firing rapidly for short bursts, then pausing before beginning another firing cycle. “If you took apart your watch, you could figure out what makes the hand go around at constant intervals. But how do the cells do it? What makes them keep time?” Kramer wondered. He set out to answer this question using cultured neurons from Aplysia. He found that the neurons operated in a negative feedback loop that involves calcium acting on ion channels in cells (4, 5 ). At the time, he says, few researchers were directly studying how ion channels were controlled. The techniques for measuring the behavior of single ion channels had just been invented, and not yet applied to real living neurons. Ion channels were “much more of a vague concept as opposed to an understood entity,” he adds. Instead, scientists spoke mainly of “ion currents” because the only way to visualize a channel at the time was to monitor the flow of ions that passed through it. Nonetheless, the project was his first intensive exposure to studying the mechanisms behind ion channels. By the time Kramer completed his Ph.D., he was intent on studying ion channels further. For his postdoctoral fellowship, he headed to Brandeis University in Waltham, Mass., where neuroscientist Irwin Levitan was taking some of the first recordings of the activity of how single ion channels were controlled by post-translational modifications such as phosphorylation. VOL.1 NO.1 • ACS CHEMICAL BIOLO GY
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“If you express these sensors in appropriate cells, in a way we’re creating artificial senses,” Trauner elaborates.
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Kramer found that the dimeric cGMP stimulates ion channels thousands of times more potently than that of single cGMP molecule. The study, published in 1998 (7 ), was his first foray into chemical biology. In 2000, Kramer took his knowledge of ion channels and the retina to start a new lab at the University of California at Berkeley. He continues to study how light information is received and processed in the retina, along with his continuing collaboration with Isacoff and Trauner. Dirk Trauner Trauner was born in Linz, Austria, in 1967. “I wasn’t one of those kids who grew up with a chemistry set,” he recalls. Instead, Trauner says, he became interested in science through a biology class in high school. By the time he began earning his undergraduate degree at the University of Vienna, he had decided to study genetics. The field was a “hot topic,” Trauner says, when he was applying to schools in the late 1980s. However, his interests abruptly shifted after taking an organic chemistry class during his sophomore year. He tried his hand at total chemical synthesis for the first time, making a natural product called ferulic acid that’s found in many plants. “At that point, I knew I wanted to switch to chemistry,” he remembers. He turned his studies first to biochemistry, then later to synthetic organic chemistry. In 1994, Trauner completed his undergraduate degree and moved to Berlin, Germany, to begin a Ph.D. program under the mentorship of synthetic chemist Johann Mulzer. His thesis centered on the total synthesis of the opiod analgesic drug morphine. His aim was not to derive a method to produce more morphine, but to demonstrate novel chemical reactions that are important in crafting the drug from scratch (8 ). Working on the project was
Photo courtesy of Dirk Trauner
Soon after arriving at Levitan’s lab, Kramer read a paper showing evidence that olfactory neurons were directly activated by cyclic adenosine monophosphate (cAMP), an important biological second messenger. The finding represented a new mechanism for neuronal response. Kramer wondered whether the membranes of olfactory neurons, which have ion channels that respond to cAMP, could function as sensors for cAMP levels in other cells. To test this hypothesis, he devised a new technique that involved taking a portion of olfactory neuron membrane and inserting it into other cell membranes. Once successful, he named the new technique “patch cramming,” a play on the “patch clamp” technique that neuroscientists use to stimulate and measure electrical responses from individual neurons (6 ). After more than three years at Levitan’s lab, Kramer took a second postdoctoral fellowship in the lab of Steve Siegelbaum at Columbia University. He was soon invited to work with Richard Axel, one of Siegelbaum’s colleagues, to clone the ion channel in olfactory neurons that responds to cAMP. On successfully completing this project, Kramer started his own lab at the University of Miami School of Medicine. There, he became interested in photoreceptor cells, the rod and cone cells in the eye that gather information on an image. Ion channels that respond to cyclic guanosine monophosphate (cGMP) also played a fundamental role in generating the photoreceptor’s response to light. Kramer wondered how these ion channels would respond to chemically dimerized cGMP. He collaborated with chemist Jeff Karpen to synthesize the new molecule. “Individual cGMP molecules are diffusing around like flies, and the probability of them landing on their target is low,” he says. “But if you tie two flies together, when one binds to its target, the other is held in close proximity. The probability of finding a neighboring target is much higher.”
Trauner’s first taste of studying a chemical important to neuroscience, a topic he had been interested in since dissecting human brains as an undergraduate. After four years and two moves with Mulzer, first to Frankfurt, Germany, and then to Vienna, Trauner completed his doctoral degree. He chose to head to the United States for his postdoctoral fellowship. In 1998, he began working with bioorganic chemist Samuel J. Danishefsky at Memorial Sloan Kettering Hospital in New York. There, he derived a method to synthesize halichlorine, an alkaloid molecule that interferes with communication between cells and has anti-cancer properties (9 ). Previously, the molecule was derived only from sponges, a method that’s an expensive and inefficient method, says Trauner. Thus, he adds, his successful synthesis “attracted attention from many labs. Halichlorin was a celebrity target for total synthesis in those days.” At the end of his postdoctoral fellowship, Trauner accepted a faculty position at the University of California at Berkeley. “An offer at Berkeley you can’t refuse,” he quips. In July of 2000, he moved to California and set up his lab. He took with him some ideas for performing the total synthesis of several organic compounds involved in immunology and cancer research. However, he says, he became intrigued in ion channels when their structures were first elucidated in the late 1990s. “Synthetic chemists always become interested when a structure is known,” he says. “We get really excited because structures are three-dimensional objects that you can rotate, think about their function, and contemplate ‘how can one reengineer this?’ As a chemist, you think that there should be opportunities to do something to soup up these molecular machines and manipulate them.” w w w. a c s c h e m i ca l biology.org
The Collaboration Based on Trauner’s interest in ion channels and his skill in synthesizing organic molecules, Isacoff and Kramer approached him soon after he arrived at Berkeley to propose combining their efforts. The three eventually hit on an ideal way to showcase all of their talents: designing an ion channel that responds simply to light, something no ion channel in animals does. “We all came up with this idea at the same time of trying to use particular kinds of chemical elements that change their shape when you shine light on them as a way of inducing functional change in an ion channel,” says Isacoff. To prove that such engineering would be possible, the researchers agreed that their first proof of principle experiment would focus on the potassium channel, a geometrically simple channel that’s present on cells throughout the body. Inserting a specific ligand, or plug, inhibits the flow of ions through the doughnut-shaped channel. Isacoff, Kramer, and Trauner devised a plan to control the plug’s placement: Trauner designed an azobenzene molecule, which lengthens in long wavelengths of light but contracts with shorter wavelengths. He affixed one end of the molecule to the ligand and another to the channel. “The azobenzene is like a fishing line that can be spooled back in or sent out with a bait. What’s swallowing the bait is what it’s attached to: the ion channel,” says Kramer. “You’ve tethered the ligand onto the target it belongs to.” After the design was completed and fitted onto live neurons, Isacoff and Kramer tested it in their own labs, with Isacoff taking on the majority of the molecular biology work and Kramer taking on the bulk of the electrical recordings. In the end, the project was successful (10 ). Ready for a fresh challenge, the researchers transferred their design to a new channel: one controlled by the neurotransmitter glutamate. Unlike the simple round www.acschemicalbiolog y.o rg
pore of a potassium channel, the three scientists needed a design compatible with the clam-shell shape of the glutamate receptor. Trauner created a string of mole cules with azobenzene at its center to act as a tether tying glutamate to its receptor. “The question became, can the design strategies that worked with the potassium channel be transferred to something more complex geometrically in the glutamate receptor? Yes, it works—and it’s very satis fying,” says Isacoff. He, Kramer, Trauner, and their colleagues published their results recently (1 ). The three researchers have many ideas of what to pursue next. Trauner says that he and his collaborators are interested in working on new designs for ion channels that respond to other types of stimuli. “It’s potentially limitless to think of signals to which ion channels could respond: light, magnetic fields, chemicals, or changes in temperatures,” he elaborates. “If you express these sensors in appropriate cells, in a way we’re creating artificial senses.” Kramer suggests that the new technique that he, Isacoff, and Trauner have created could make an ideal way to explore or enhance the senses that already exist. For example, the ability to stimulate individual light-sensitive neurons may aid neuro scientists in understanding how neurons are wired throughout the brain. It could also give researchers the means to replace light-sensitive cells that no longer work, such as those destroyed by macular degeneration, a leading cause of blindness. Isacoff, Kramer, and Trauner continue to meet with each other and the graduate students of all three labs at least once a week to compare notes and bounce ideas around. Rather than shuttling compounds and cells between each other’s labs, Trauner says, the three researchers simply send each other’s students across Berkeley’s small campus. Though their collaboration is usually unhindered, Kramer notes that the three
have had their share of rough patches. “Like any ménage a trois, things get a little complicated and don’t always run so smoothly,” he says. “Since there’s three of us, there are more ideas that we can possibly do, so we have to prioritize and wrestle with one another to see who is going to do what.” “It’s sometimes heated,” agrees Isacoff. But with the number of ideas and successful accomplishments growing between the three scientists, he says, “It’s been a marvelous collaboration that will continue for some time.” “It’s the classical example of a meaningful collaboration,” adds Trauner. “We’re bringing together so many techniques that are impossible to master in one lab. [Isacoff and Kramer] have learned a bit of chemistry, and I’ve learned a bit of biology. We’re bridging the huge cultural divide between chemists and biologists.” —Christen Brownlee, Science Writer
REFERENCES
1. Volgraf, M., Gorostiza, P., Numano, R., Kramer, R.H., Isacoff, E.Y., and Trauner D. (2006) Allosteric control of an ionotropic glutamate receptor with an optical switch, Nat Chem Biol. 1, 47–52. 2. Mannuzzu, L.M., Moronne, M.M., and Isacoff, E.Y. (1996) Direct physical measure of conformational rearrangement underlying potassium channel gating, Science 271, 213–216. 3. Dean, C., Scholl, F.G., Choih, J., DeMaria, S., Berger, J., Isacoff, E., Scheiffele, P. (2003) Neurexin mediates the assembly of presynaptic terminals, Nat Neurosci. 6, 708–716. 4 . Kramer, R.H. and Zucker, R.S. (1985) Calciumdependent inward current in Aplysia bursting pacemaker neurons, J Physiol. 362, 107–130. 5. Kramer, R.H. and Zucker, R.S. (1985) Calciuminduced inactivation of calcium current causes the inter-burst hyperpolarization of Aplysia bursting neurons, J Physiol. 362,131–160. 6. Kramer, R.H. (1990) Patch cramming: monitoring intracellular messengers in intact cells with membrane patches containing detector ion channels, Neuron. 4, 335–341. 7. Kramer, R.H. and Karpen, J.W. (1998) Spanning binding sites on allosteric proteins with polymerlinked ligand dimers, Nature. 395, 710–713. 8. Mulzer, J., Dürner, G., and Trauner, D. (1996) Formal Total Synthesis of (-)-Morphine by Cuprate Conjugate Addition, Angew. Chem. Int. Ed. Engl. 35, 2830–2832. 9. Trauner, D., Schwarz, J.B., and Danishefsky, S.J. (1999) Total Synthesis of (+)-Halichlorine, an Inhibitor of VCAM-1 Expression, Angew. Chem. Int. Ed. Engl. 38, 3542–3545. 10. Banghart, M., Borges, K., Isacoff, E., Trauner, D., and Kramer, R.H. (2004) Light-activated ion channels for remote control of neuronal firing, Nat Neurosci. 7, 1381–1386. VOL.1 NO.1 • ACS CHEMICAL BIOLO GY
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