Image courtesy of Deborah Stalford
Divide and Conquer: Investigating the Mechanisms behind Mitosis
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 web site.
Published online October 20, 2006 10.1021/cb600414p CCC: $33.50 © 2006 by American Chemical Society
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oon after a sperm meets an egg, the single fertilized cell splits into two cells, then four, and then eight. Cell division is responsible for producing each of the trillions of cells present in every human body. During adulthood, division supplies replacements for cells lost to age, injury, and disease, but it can also form the basis for illnesses such as cancer. Despite the importance of mitosis in development and medicine, researchers have much to learn about the molecular mechanisms that regulate it. Cell biologist Rebecca Heald of the University of California, Berkeley, is striving to iron out these details. Heald’s work concentrates on the mitotic spindle, a structure that is essential for correctly distributing copied chromosomes to daughter cells. Using techniques that blend biology and chemistry, she and her colleagues are identifying molecules and proteins that play major roles in directing this dynamic cell process. Discovering Division Heald was born in 1963 in Bellefonte, PA, a town near Pennsylvania State University, where her father Emerson was a postdoctoral fellow. About a year later, she and her family moved 2 hours west to Greenville, PA, home to a small liberal arts school, Thiel College. Heald’s father, a physical chemist, taught there for more than 40 years. Some of Heald’s earliest memories are of visiting her father and his colleagues in the chemistry building. “The whole building smelled like the lab, and it’s a smell I really liked. I liked seeing all the glassware and watching people doing experiments—it seemed like a lot of fun,” she recalls. “To this day, I really love a lab. I feel really comfortable there.”
Though both of her parents were trained chemists, Heald notes that they rarely pushed her to follow in their footsteps. Rather, her mother and father encouraged Heald and her two older sisters to pursue a variety of interests in addition to science, including math, reading, and writing. By the time she graduated from high school in 1981, Heald considered math and literature to be two of her strongest subjects. However, soon after she entered Hamilton College, in Clinton, NY, she chose chemistry as her major. “I thought, I can always read in my spare time, but I can’t work in a lab in my spare time,” she says. Describing herself as “squeamish,” Heald says that she chose chemistry over biology to avoid dissecting a cat, a requirement for all biology majors at Hamilton. However, she notes that she enjoyed her biology classes, filling her schedule with selections such as microbiology and biochemistry. By the time she received her bachelor’s degree from Hamilton, Heald was almost certain that she wanted to pursue science in graduate school, although she had not decided on a field. To gain more experience to guide her decision, she wrote letters to several investigators whose work intrigued her, hoping that one might invite her to work as an assistant. Eventually, she was hired by Sarah Hitchcock-DeGregori, a muscle protein biochemist and cell biologist at Rutgers Medical School in Piscataway, NJ. For the next 2 years, Heald worked with Hitchcock-DeGregori on a project to generate mutant versions of a muscle protein called tropomyosin. The researchers expressed the proteins in bacteria, purified them, and then characterized these mutants. They observed w w w. a c s c h e m i ca l biology.org
these proteins’ ability to bind to actin and determined whether they promoted ATPase activity in conjunction with other muscle proteins. By the time Heald left HitchcockDeGregori’s laboratory, she had two published papers (1, 2) and a sense of the direction she wanted her career to take. With Hitchcock-DeGregori’s helpful mentorship, Heald decided to study cell biology at Harvard Medical School’s doctoral program. When she entered the program in 1987, Heald says that she was unsure of what focus her studies would ultimately take. However, the school used a series of laboratory rotations to provide multiple chances for students to select a mentor. After just two rotations, Heald chose to work in the laboratory of Frank McKeon. “I would choose the same lab all over again,” she says. McKeon’s work concentrated on proteins in the nuclear lamina, a network of proteins that line the nuclear envelope. One of his projects involved making mutant versions of lamin genes, transfecting these genes into cells, and then observing the effect of the mutant proteins with a microscope. The visual aspect of McKeon’s work was what drew her to join the project, says Heald. “I found looking through the microscope to be really exciting,” she adds. “A lot of my science is visually inspired, and just being able to see what something is doing is the most meaningful thing to me.” Generating new lamins harboring mutant phosphorylation sites, Heald eventually found one that wouldn’t allow the nuclear lamina to break down during mitosis, in turn preventing the nucleus from dividing once the mitotic spindle had formed (3). An image of this event, which accompanied the paper that Heald and McKeon published describing this work, was featured on the cover of the journal Cell. The image, showing a normal cell undergoing mitosis beside one with an altered lamina, “made it very clear that these sites were important www.acschemicalbiolog y.o rg
for regulation of the assembly and disassembly of the lamina,” says Heald. Positive Visualization Heald completed her doctoral degree after working with McKeon for 5 years. Encouraged by her success in studying cell division, she decided to make this process the focus of her work during her postdoctoral fellowship. Seeking a change of scenery, Heald says that she looked for postdoctoral opportunities far from home. Her first choice for continuing her studies was at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. “People warned me that it could be bad for my career, that Europe was a backwater that I’d never emerge from again, and that I’d have trouble getting a job afterwards,” says Heald. Respectfully ignoring this advice, she joined the laboratory of Eric Karsenti, a cell biologist who studies the regulation of microtubule dynamics. Heald joined Karsenti on a project to determine what factors regulate catastrophe, a process in which microtubules go from a growing to a shrinking state, and an important parameter of microtubule dynamics that is altered during spindle assembly. Her original plan, says Heald, was to fractionate Xenopus egg extracts, using this material to investigate catastrophe. Though other researchers used yeast as a model organism to identify microtubule regulatory factors, Heald appreciated the frog eggs’ robust size, which made it possible to generate extracts that could reconstitute mitotic processes and visualize them outside the cell. Heald notes that many of her early experiments in Karsenti’s laboratory ended without success, but in 1995 her work took a positive turn. At that time, she decided to pursue a new angle to investigate mitotic spindle assembly. She explains that as the mitotic spindle forms, microtubules are stabilized by interacting with chromosomes. Heald wondered
whether an enzyme on chromatin might ultimately be responsible for this activity and sought to isolate it. To more easily isolate chromatin from the egg extract, she worked with colleagues at EMBL who were coating metal beads with DNA. She reasoned that chromatin would form on beads, which would be much easier to isolate and characterize biochemically. However, she realized that it wasn’t yet known whether chromatin itself was the stabilizer or whether other material in chromosomes was responsible for this effect. After adding chromatin-coated beads to the extract, she saw that mitotic spindles could form around the beads. An image of this phenomenon earned her a second journal cover, this time in Nature (4). “This image said that you don’t need real chromosomes to build the bipolar mitotic spindle—all you need is chromatin,” she says. Heald calls this discovery a “turning point” that opened up numerous questions to steer her future research: Which motor proteins are responsible for generating the mitotic spindle? What in chromatin is responsible for allowing the mitotic spindle to grow? Heald accepted an assistant professorship in 1997 at the University of California, Berkeley, to pursue the answers to these questions. Fruitful Collaborations For almost a decade, Heald’s laboratory at Berkeley has investigated mitotic spindle regulation. As a nod to her doctoral and postdoctoral work, she and her colleagues have focused on crafting visual experiments that allow them to quickly see their results. One of Heald’s most fruitful projects at her Berkeley laboratory has been in collaboration with her Berkeley neighbor, cell biologist Karsten Weis, who studies nucleocytoplasmic transport. Previous studies had suggested that the machinery that regulates this process is also involved in controlling mitosis. Working together, their VOL.1 NO.9 • 554—556 • 2 0 0 6
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“We could see that there was a physical gradient of this protein even without the nuclear envelope or anything holding it in.”
laboratories used FRET probes to visualize proteins around mitotic chromatin in Xenopus eggs. This technique revealed that RanGTP forms a gradient that’s enriched near chromatin (5). That view challenged a general concept of how some proteins exist within cells. “In general, people think that different things are compartmentalized in cells by membranes and other structures,” Heald says. “We could see that there was a physical gradient of this protein even without the nuclear envelope or anything holding it in.” More recently, Heald and Weis collaborated with Berkeley researcher Ehud Isacoff. Using a technique called fluorescence lifetime image microscopy, Petr Kalab, a postdoctoral researcher working with all three groups, found that a similar RanGTP gradient exists in human somatic cells, a system that’s significantly smaller and more complex than Xenopus eggs (6). Heald worked on a different kind of collaborative project with the laboratory of chemist Peter Schultz, a former Berkeley researcher who now works at the Scripps Research Institute in La Jolla, CA. Heald and Schultz joined forces to screen chemical libraries for compounds that inhibit mitotic spindle assembly but don’t target microtubules. In an approach Heald describes as “low throughput,” graduate student Sarah Wignall added ~1500 individual chemicals to Xenopus egg extracts to test whether each one inhibited spindle assembly. Chemicals that passed the first test went through a second assay to determine whether they inhibited tubulin polymerization. Finally, compounds that made it through both assays were coupled to an affinity matrix and mixed with Xenopus egg extract to determine which egg proteins bound to the compound. The researchers ultimately identified one compound, called diminutol, that bound to an NADP-dependent oxidoreductase. Such a compound
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might eventually be used as a basis for anticancer therapy or as part of a genetic screen to determine what other factors are functioning in a redox pathway that could regulate spindle assembly (7 ). This September, Heald received the National Institutes of Health Director’s Pioneer Award, a grant that will guide more than half her laboratory’s efforts over the next 5 years. Heald says that she plans to use these funds to investigate the “really fascinating question” of how cells appropriately scale the sizes of their intracellular components. She and her students will use their favorite model, Xenopus laevis, along with a related species, Xenopus tropicalis, to explore this subject. X. tropicalis is about one-fifth the size of X. laevis, notes Heald. She explains that egg volume and mitotic spindle size are similarly scaled in the two species. Her laboratory has found that mixing extracts from the two species’ eggs changes the mitotic spindle’s size. “In the cytoplasm, there seems to be some readout of the size of the cell,” says Heald. “We’d like to find out how that works and what other structures in the cell are scaled like that.” Investigating this question will keep Heald occupied for years to come, but she anticipates that other questions will continually arise to pique her interest. Pursuing new lines of research with her colleagues and collaborators is one of the best parts of her job, she says. There are “still really tremendous unanswered questions, fundamental things about biology that we haven’t been able to investigate very well in the past,” she says. “There’s just so much to learn.”
2. Heald, R. W., and Hitchcock-DeGregori, S. E. (1988) The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin, J. Biol. Chem. 263, 5254–5259. 3. Heald, R., and McKeon, F. (1990) Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis, Cell 61, 579–589. 4. Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A., and Karsenti, E. (1996) Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts, Nature 382, 420–425. 5. Kalab, P., Weis, K., and Heald, R. (2002) Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts, Science 295, 2452–2456. 6. Kalab, P., Pralle, A., Isacoff, E. Y., Heald, R., and Weis, K. (2006) Analysis of a RanGTP-regulated gradient in mitotic somatic cells, Nature 440, 697–701. 7. Wignall, S. M., Gray, N. S., Chang, Y. T., Juarez, L., Jacob, R., Burlingame, A., Schultz, P. G., and Heald, R. (2004) Identification of a novel protein regulating microtubule stability through a chemical approach, Chem. Biol. 11, 135–146.
—Christen Brownlee, Science Writer
REFERENCES
1. Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) Altered actin and troponin binding of amino-terminal variants of chicken striated muscle alpha-tropomyosin expressed in Escherichia coli, J. Biol. Chem. 262, 9730–9735.
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