Key regulators of the cell division cycle

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Lee Hartwell

Fred Hutchinson Cancer Research Center

Yoshio Masui

University of Toronto

Paul Nurse

Imperial Cancer Research Fund

For pioneering genetic and molecular studies that revealed the universal machinery for regulating cell division in all eukaryotic organisms, from yeasts to frogs to humans.

Lee Hartwell is a yeast man—a connoisseur of Saccharomyces cerevisiae, or budding yeast, that are essential for brewing beer and baking bread. But Hartwell sees neither brew nor bread in these simple organisms. Rather, he sees something even closer to the core of life itself. By studying yeast, Hartwell has seen the cell cycle up close and has identified genes that are crucial to controlling the intricate program of instructions by which a cell grows, rests, and divides to replicate itself.

Paul Nurse is also a yeast man—a scientist whose work with fission yeast, or Schizosaccharomyces pombe, also revealed previously unknown things about how genes regulate the lives of cells. Among Hartwell’s discoveries in budding yeast is a gene called CDC28 that cells need in order to progress through their various stages of division. Nurse and his colleagues discovered a nearly identical gene in fission yeast, cdc2 , that performs a similar regulatory role. Then Nurse not only showed that the CDC28 and cdc2 genes make proteins that are functionally like one another; he also identified the first protein in humans whose role in the cell is analogous to the role of the proteins in yeast.

Meanwhile, Yoshio Masui, whose experimental work has focused on frogs, discovered a protein, called maturation promoting factor (MPF), in the cytoplasm of cells that control cell division in the fertilized eggs of frogs.

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Each of these discoveries—occurring in the late 1960s and early 1970s—is important in and of itself. But more important still was the observation that Masui’s MPF contains a protein that is like the yeast proteins discovered by Hartwell and Nurse. That a similar protein exists in humans brings the research full circle. If ever there was a set of stories that offer compelling evidence of how organisms are related to each other, from the lowly yeast to the higher frog, these are the stories. Ultimately, the implications of this research to human medicine would become clear.

Hartwell’s early decision to focus on budding yeast was an easy one. “At the time it was the only eukaryotic cell [or cell with a nucleus] for which the basic genetics had been worked out. So it was a good starting point and I’ve always seen yeast as a model organism for human biology,” Hartwell says. As budding yeast pass through various stages of development, the bud protruding from the organism changes in size, giving scientists a remarkable and quite visible way to follow the outward signs of the cell cycle.

At the University of Washington Department of Genetics, Hartwell discovered many of the genes that are essential to the development of yeast, whose function is to operate successively to assure that the cell reproduces according to an orderly system in which step A must be completed before the cell moves on to step B, and so on. Hartwell also discovered “checkpoint genes” that are called upon to function when the essential genes do not function properly. In the normal course of the cell cycle of any organism, errors are likely to occur as information encoded in DNA is passed along. Sometimes the DNA is damaged or some essential process, such as assembling the machinery to distribute chromosomes, is delayed.

The checkpoints are transient stops that enable the cell to monitor whether it has accurately accomplished everything it needs to do, and to make necessary repairs, before continuing its progression through the cell cycle. Like the intricate steps in a complex dance, the healthy development of the cell, which ends with cell division and the creation of daughter cells, depends on good choreography.

For a cell, errors that go undetected and, therefore, unrepaired, are not to be desired. But for a geneticist like Hartwell, errors that result in mutant cells are a gold mine of information. Learning when and why the cell cycle goes awry (often leading to the uncontrolled growth that is characteristic of malignancy) is the centerpiece of Hartwell’s work.

The conviction that the development of cells (including human cells) could be discerned from yeast was “a fairly risky assumption,” Hartwell says, looking back on the early days of his career in the 1960s. In retrospect it is clear the risk was well worth taking. Now, after 30 years with yeast, Hartwell is committed to the application of knowledge that he and his many disciples have acquired. Last year, he became president and director of the Fred Hutchinson Cancer Research Center in Seattle, where he is spearheading a drug discovery program based on detailed knowledge of the cell cycle.

Paul Nurse, who is director-general and head of the Cell Cycle laboratory at the Imperial Cancer Research Fund in London, is also concentrating on the application of basic science to human disease after a long, productive career as a yeast geneticist. Unlike Hartwell, who took what might be called a “holistic” approach to studying everything he could about budding yeast, Nurse quickly zeroed in on what he believed to be the most important regulatory pathway in fission yeast—like Hartwell’s organism, but a yeast that is elongated and divides in the middle, like links of sausage.

Nurse focused his attention on a mutant form of fission yeast that makes very small cells (Nurse named them “Wee”), which go through certain stages of the cell cycle with unusual speed—at times dividing in half the time other yeast require. Nurse discovered three different proteins, each encoded by separate genes, that control the rate at which the cell enters the M (for mitosis) phase of the cycle—the point in the cycle when cells divide to create two daughter cells. Each of these proteins acts on the important cdc2 protein that Nurse recognized as the key protein regulating this phase of the cell cycle.

The essence of science is not only to make important observations but also to find connections among discoveries to achieve a synthesis of ideas. This, clearly, is one of Nurse’s principal contributions, for it was he who saw that he and Hartwell and Masui, working in different types of yeast and in the frog, had each found equivalent proteins that are key to cell division. His subsequent identification of a similar protein in human beings clinched his achievement as a synthesizer.

As a young man, Yoshio Masui took a sabbatical from Konan University in Japan to study enzymes that control development in a laboratory at Yale, where his work on frog oocytes began. Initial studies showed that division of a cell that will give rise to an egg (an oocyte) could be stimulated by the hormone progesterone, but it only worked when the surface of the eggs were exposed to progesterone—not when the hormone was injected directly into the oocytes.

Masui concluded that progesterone acting on the egg’s surface must affect something in the cytoplasm of the cell that, in turn, stimulates cell division. He set out to find that “something,” which turned out to be an activity in the cytoplasm that he called MPF, without knowing precisely what it was. After his sabbatical at Yale, Masui continued his research at the University of Toronto and found that MPF is a protein.

Ultimately, Masui and his students developed techniques for preparing highly concentrated extracts of egg cytoplasm which then made it possible to analyze cell cycle processes biochemically, and to purify MPF. From there came the observation that Masui’s MPF from frog eggs was analogous to the CDC28 and cdc2 proteins in yeast. It took about 15 years to make the journey that began with the identification of MPF as an agent that could stimulate the cell cycle to the discovery of its molecular composition. Masui remained on the faculty of the University of Toronto for most of his professional life until his retirement in 1997.

Key Publications of Lee Hartwell

Paulovich, A.G. and Hartwell, L.H. (1995) A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell 82: 841–847.

Hartwell, L.H., and Kastan, M.B. (1994) Cell cycle control and cancer. Science 266: 1821–1828.

Weinert, T.A., Kiser, G.L., and Hartwell, L.H. (1994) Mitotic checkpoint genes in budding yeast and the dependence of mitosis no DNA replication and repair. Genes-Dev. 8: 652–665.

Weinert, T.A. and Hartwell, L.H. (1993) Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics 134: 63–80.

Hartwell, L. (1992) Defects in a cell cycle checkpoint may be responsible for the genome instability of cancer cells. Cell 71: 543–546.

Hartwell, L.H., Culotti, J., and Reid, B. (1970) Genetic control of the cell-division cycle in yeast. I. Detection of mutants. Proc. Natl. Acad. Sci. USA 66: 352–359.

Key Publications of Yoshio Masui

Masui, Y. (1967) Relative roles of the pituitary, follicle cells, and progesterone in the induction of maturation in Rana pipiens. J. Exp. Zool. 166: 365–376.

Masui, Y. and Markert, C.L. (1971) Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177: 129–146.

Wasserman, W.J. and Masui, Y. (1975) Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes. Exptl. Cell Res. 91: 381–388.

Wasserman, W.J. and Masui, Y. (1976) A cytoplasmic factor promoting oocyte maturation: its extraction and preliminary characterization. Science 191: 1266–1268.

Lohka, M.J. and Masui, Y. (1983) Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220: 719–721.

Lohka, M.J. and Masui, Y. (1984) Effects of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs. Dev. Biol. 103: 434–442.

Key Publications of Paul Nurse

Nurse, P. (1975) Genetic control of cell size at cell division in yeast. Nature 256: 547–551.

Nurse, P. and Thuriaux, P. (1980) Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe. Genetics 96: 627–637.

Beach, D., Durkacz, B., and Nurse, P. (1982) Functionally homologous cell cycle control genes in fission yeast and budding yeast. Nature 327: 31–35.

Russell, P. and Nurse, P. (1986) cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45: 145–153.

Lee, M.G. and Nurse, P. (1987) Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2+. Nature 327: 31–35.

Enoch, T. and Nurse, P. (1990) Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell 60: 665–673.

Award presentation by Ira Herskowitz

One of the simplest but also one of the most profound questions to ask about biology is “How do you make a cell?” By that I mean, “How does one cell produce two cells?” A human skin cell divides to produce two cells like itself in about ten hours. A bacterial cell produces two bacterial cells in about 30 minutes, and a yeast cell produces two yeast cells in about ninety minutes.

In an intellectual feat as exciting as the athleticism displayed by the dynamic duo of Sosa and McGwire, Watson and Crick explained how the genetic instructions of the original cell are copied so that each of the daughter cells receives a precise replica of the original genetic information—a DNA double helix. That’s why each of the daughter cells looks like the original cell: they both get the identical genetic information.

As breathtaking as Watson and Crick’s explanation was, it didn’t answer another fundamental question about cells: how do the daughter cells manage to receive the new set of instructions? Do the newly replicated DNA molecules, the chromosomes, just float into the daughter cells or get distributed randomly to these cells? No, of course not. Cells have precise machinery to distribute the genetic information so that each cell gets exactly one copy of each chromosome.

So, now we can see some of the richness of a deep problem in biology: How do cells choreograph not only copying their genetic information but distributing this information? The cells had better do the copying before the distribution, or there will be a mess. How do cells know in what order to carry out such processes? Studies by Lee Hartwell, Paul Nurse, and Yoshio Masui identified the molecular machinery and the logic of how this machinery works to program the sophisticated events involved in duplicating cells.

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Here’s another fundamental question: What tells a cell when to divide? Many cells in our body aren’t dividing; they are just sitting around in a resting state, waiting for a signal to divide. What are these triggers? And what about cells that become deranged, so that they think they are getting a stimulatory signal when they are supposed to be resting? These are cancer cells. What is the molecular basis for their derangement? Once again, the studies by Masui, Nurse, and Hartwell identified the molecular machinery that lets us understand many of the derangements that we see in cancer.

Beginning in the 1800s, with observations of cells by microscopy, and continuing into the mid-1950s, it became clear that the cell cycle has observable phases: S—when the chromosomes are duplicated; M—when the chromosomes are distributed, and two gap phases between them. Some of the first clues about what governs the cell cycle came from experiments in the late 1960s when nuclei from one stage of the cell cycle were injected into the cytoplasm of cells in other stages or when cells in different stages of the cell cycle were fused with each other. These experiments revealed that there was something in the cytoplasm of the cell, some factor, controlling the behavior of the nucleus. Would it be possible to get one’s hands on such molecules? That was the Holy Grail of the new cell biology. Our awardees worked on three different organisms—two different kinds of yeast and on frog oocytes, exploiting specific features of these experimental systems to find these cell cycle regulatory molecules, the Grail. It was a journey that took them and others in the field more than fifteen years, but what they found was enormously satisfying: All of their work converged to identify a particular piece of machinery, a protein kinase, that is central to regulating the cell cycle.

Lee Hartwell was exposed to microbiology and the power of genetic analysis at an early and impressionable age. As an undergraduate student at Caltech in Pasadena, he worked in Robert Edgar’s laboratory, which pioneered the strategy of how to identify every gene in an organism. They used an organism that had only around 150 genes, a virus that grows in bacteria, and exploited a genuinely elegant strategy, the use of conditionally lethal mutants. These are mutants that can grow under some conditions, for example, at low temperature, but not under other conditions, for example, at high temperature. While a beginning assistant professor at the University of California, Irvine, Hartwell decided that he wanted to know how a eukaryotic cell works. So he chose the simplest possible eukaryotic organism, in this case a eukaryote with just a single cell, yeast, and proceeded to identify yeast genes essential for its growth, employing the same strategy that had been used in the Edgar laboratory—finding mutants that are temperature sensitive for growth. He found hundreds of mutants and dozens of interesting genes this way. A few years later, after Hartwell had moved to the University of Washington, he realized that some of these mutants had the striking property that they got stuck at a particular position in the cell cycle when they were incubated at high temperature. He could readily discern the step at which they got stuck because he was studying budding yeast, an organism whose daughter cells form by a process of budding: Cells with a small bud are in an early stage of the cell cycle, and cells with a large bud are in a later stage of the cell cycle. Studies of mutants like these led him to identify dozens of genes, including one, CDC28, that proved to be particularly interesting, and to define a small set of processes that were carried out in a particular order: Cells don’t move on to step 2 until they complete step 1. Later work led to an understanding of how cells monitor the completion of these steps—they have molecular “checkpoints,” where this monitoring takes place.

Paul Nurse had also become smitten by microbes during his studies as an undergraduate in Birmingham and began working on a different yeast, fission yeast, to learn about the cell cycle. Like Hartwell, Nurse isolated many temperature-sensitive mutants, which defined a variety of steps during the cell cycle of this organism. But his great insight was to appreciate the novelty and value of another type of mutant: not one that gets stuck in the cell cycle but rather one that speeds through the cell cycle inappropriately. These mutants had the interesting phenotype that the cells are small or, as he described them using local lingo, Wee. In the normal process of cell division, a cell gets longer and longer until it reaches a critical size, and then the cell pinches off to form two cells. Nurse reasoned that the Wee mutants are altered in this important decision-making step in the cell cycle: they pinch off to form two cells prematurely. He had six or so Wee mutants and sought to figure out which genes were altered in these mutants. The first five all affected a gene he called wee1. Considerable legend surrounds the sixth Wee mutant. This mutant was on a plate that got contaminated by some volunteer microorganism like bacteria or a mold, and rather than purify mutant #6 away from the contaminant and carry out the analysis to find that it, too, was a mutation in the wee1 gene, Nurse tossed it into the garbage. Something gnawed at Paul and he returned to the lab. Sifting through the garbage, he recovered the plate, and using standard microbiological procedures, he separated the mutant yeast from the contaminant. Much to his delight, this mutant did not have a mutation in the wee1 gene. Rather, it had a special alteration in the cdc2 gene, a gene that was already known because other types of mutations in it caused the cell cycle to become stuck. Discovering this special mutation in the cdc2 gene induced Nurse wisely to focus on this gene and how it is regulated. Focusing on the cdc2 gene proved to be just the right thing to do: In later studies, Nurse showed that it is so important that not only does budding yeast have a version of it (none other than Hartwell’s CDC28 gene), but Paul fished out a human version as well.

Yoshio Masui’s early work at Kyoto University in Japan was on development of amphibians—his PhD thesis was on “Studies of the Effects of Lithium Upon Morphogenesis of Amphibian Embryos.” Although frogs were excellent for classical studies of development, Masui sought to learn more about the molecules involved, and so he took a sabbatical from his position at Konan University. He came to Clement Markert’s laboratory at Yale to learn about enzymes involved in development and spent six months studying particular enzymes from penguin embryos. He soon recognized that penguins were not an exceptionally propitious experimental system and, anyway, Markert told him he “should choose something less expensive to experiment with.” And so he returned to amphibians. The key feature of frogs that Masui exploited was that their oocytes—progenitors of eggs—are naturally arrested at one stage of development. Further development of the oocyte, the process of “maturation,” was triggered by addition of a hormone such as progesterone. In a series of remarkable experiments, Masui discovered that the cytoplasm of oocytes treated with progesterone contained an activity, “maturation promoting factor,” that induces maturation in untreated oocytes. He had demonstrated an activity that could drive the cell cycle in these cells. To people who sought a molecular understanding of cell-cycle regulation, MPF was truly a Holy Grail. What was its molecular nature? What did it do? What controlled it? For a variety of reasons, it took more than 15 years before the molecular identity of MPF was identified, which occurred in a flurry of activity in the late 1980s. Astonishingly, a key ingredient of maturation promoting factor was, you guessed it—the frog version of cdc2 protein.

And so resulted one of the most remarkable convergences in modern science, a truly unifying discovery, in which it was seen that yeasts, frogs, and humans share universal machinery for regulating cell division.

Interview with Lee Hartwell and Rochelle (Shelly) Esposito

Nature Medicine Essay

In an October 1998 interview with Dr. Rochelle (Shelly) Esposito, a professor of molecular genetics and cell biology and chair of the Committee on Genetics at the University of Chicago, Dr. Leland (Lee) Hartwell discusses how he developed a fascination with science and cellular research. He talks about his work and its value to medicine, his teaching philosophy, and what genetic developments he believes will occur in the not-so-distant future.

Part 1: From a Non-academic Beginning to the Halls of Cal Tech
After early work studying mammalian cell biology, Dr. Hartwell sought a new focus. Frustrated with the prevailing models framing mammalian cell research, he realized yeast cells presented the ideal environment for investigating mutant cells and DNA replication.

Esposito: On behalf of all of us who have been impacted by your work, I want to convey our warmest congratulations on this wonderful honor and award. We’re really pleased and delighted, and it’s very richly deserved. I’d like to ask you, can tell us what this award means to you and what you feel it’s recognizing?

Hartwell: Yes. It’s, of course, very exciting and rewarding to me. It’s always terrific in your career to feel like you’ve made a contribution, and that’s the gift the Lasker committee is giving me. I think they’re recognizing the whole field of cell cycle research and yeast genetics. I feel fortunate to have been in on some of the very early phases of that work, but it’s really the work from hundreds of labs and thousands of students that have brought it to the stage where it’s deserves this kind of recognition.

Esposito: Can you tell me what, from your perspective, are the most significant contributions that you think your lab has made to this field?

Hartwell: I think it was perhaps in seeing that a study of cell division, even though it was motivated by an interest in human cells and medical problems like cancer, needed to be explored in a much simpler system using genetics as a powerful tool. It was in attempting to place the groundwork there, that’s probably one of the major things we contributed at the beginning.

Esposito: What I’d like to do in the next series of questions is address how this all came about. Maybe on a personal note you could begin by telling us a little bit about your family background and how you first got interested in science? I know this is going back a bit, but it’s always interesting, I think, to others, to know what the motivating forces are that drive us to the discoveries that we make.

Hartwell: I came from a family that was very nonacademic, so I didn’t recognize at an early stage the clear interest that I had in science as a child. Looking back, it’s easy to see now. I always collected bugs and took things apart and spent time at the library trying to learn things about radios and astronomy and various stuff like that without noticing that my peers weren’t spending their time the same way.

I don’t think I would have ever gotten the opportunity to spend my career in science if it hadn’t been for a few key teachers along the way. [There were] a couple in high school that recognized my interest and ability in math and physics, and gave me extra work and encouragement, and a counselor I had in junior college—I went to Glendale Junior College for a year—who also took an interest in me and got me to interview with a professor from Cal Tech who was visiting. It was through that interview that I eventually ended up at Cal Tech and discovered a whole fabulous world of science that I really didn’t even know existed.

Esposito: I know that your undergraduate career at Cal Tech was a transforming experience for you. Can you tell us what the atmosphere there was like and how it really set the stage for the future directions that you wound up taking?

Hartwell: Cal Tech was just an unbelievable sort of fairy tale place for me. You were spending your time thinking about really interesting scientific issues, and the faculty there treated the undergraduates like they were colleagues rather than students. It just gave me a sense of involvement and the possibility of participating in science, sort of an invitation, I guess I would say, that I look back on and just cherish those years. A particularly influential individual to me was Bob Edgar, who worked in Max Delbruck’s group and, of course, did all the work with Bill Wood on phage morphogenesis. But I can still remember the afternoon when I was walking by Bob’s office, and he stepped out and pulled me in and explained to me a neat idea he had for determining how all the genes and phage function. To think that an undergraduate would warrant that kind of collegiality just made it a very, very special place.

Esposito: It sounds like a wonderful environment. You were an undergraduate working in his lab. How does that come about?

Hartwell: The neat thing about Cal Tech was that they really encouraged people to do research, and so as an undergraduate I did research the whole time I was there—I mean both during school and in summers. There were so few undergraduates compared to faculty that you could essentially work for anybody there you wanted. They were always happy to have a student come in and work. I felt pretty much like it was a graduate level experience because I did so much research as an undergraduate.

Esposito: Did you work in several different labs, or did you find your way to Edgar’s group early on?

Hartwell: I worked in several different labs. I worked for Hildegard Lamfrom for a while, who was trying to figure out what messenger RNA was. I worked in Renato Dulbecco’s group with Howard Temin for a while and then in Delbruck’s group.

Esposito: Did your interest in the cell cycle originate at Cal Tech or later when you went to do post-doctoral work?

Hartwell: It didn’t really come until I was doing post-doctoral work. As an undergraduate I guess I was interested in phage and then as a graduate student I was interested in gene regulation. Then when I was thinking about becoming a post-doc, it seemed like the appropriate thing to do was to pick a problem that you were going to spend your career working on, something that wasn’t understood yet. I decided to work on cancer and cell division, growth control sorts of things. I went to work with Renato Dulbecco and came to a tremendous interest in the problems there. I had another wonderful experience with a colleague, Marguerite Vogt, who is just a delightful scientist who is always interested in each new theory about cancer. She is a person who has developed a sort of artistic love for cell culture and what cells are. That was a big experience for me, too.

Esposito: I guess when you went to do your post-doctoral work you really had in mind by your choice that you wanted to work in cancer and maybe cell cycle and cell division. Did this stem from your studies on gene regulation when you were at MIT? Also, I kind of transited through your graduate work without asking how you wound up at MIT, and what were the problems that were being addressed when you were there? What were your thoughts and the influences on you at the time you were there?

Hartwell: At MIT, that was a pretty neat experience, too. The reason that I ended up there was because I went to Bob Edgar and said, where should I go to graduate school? He said MIT, and so I did. One of the things they encouraged undergraduates to do at Cal Tech was to read in areas that you wanted to study in. And so every quarter I took a reading course under a faculty member’s direction. I read a lot about gene regulation, so I knew when I went to graduate school I wanted to study gene regulation. And so when I went to MIT I knew I wanted to work with Boris Magasanik and was fortunate enough to be able to do so. I think the main thing I took away from that experience was how Boris just let you find your own way.

He would come by every afternoon and ask you how your experiments were going, so it sort of kept you at this feverish pace for having results every day. But he never told you what to do. He’d always just asked you how things were going, and he would make suggestions, but he made it clear that you were plotting your own course. That was very comfortable for me having already had so much research experience. But I think it also really helped me develop that sense of: I was the master of my research, and I had to find my own way.

Esposito: I had a similar experience. I wonder if when you look back and compare the way you were trained and the climate in which science was done to the way it is now, whether there was, in reality, more freedom to explore and follow your own instincts. What’s your thought on that?

Hartwell: My experience and my colleagues’ is that there is tremendous anxiety about getting grants and keeping your career going, so that there is much greater, often, direction of students to fill necessary goals, to get the right experiments done for the next grant and the paper out and that sort of thing. I never experienced that kind of pressure myself, and I must say I never put my students under it. I think I work with my students the same way I experienced science, where they’re in charge of what they do, and we hope for the best. Fortunately we have been lucky enough that that has worked. I do think the climate is very, very different now and not one that I would thrive in if I was a student and feeling like I was “doing project 17 on somebody’s RO1 grant.”

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Part 2: A “turning point” Conversation Leads to a Shift in Research Focus

After early work studying mammalian cell biology, Dr. Hartwell sought a new focus. Frustrated with the prevailing models framing mammalian cell research, he realized yeast cells presented the ideal environment for investigating mutant cells and DNA replication.

Esposito: Tell me about when you first decided to make the switch into working with yeast. When did that happen? Why did it happen? What was your goal in switching from mammalian cells to yeast when you left Dulbecco’s lab?

Hartwell: It’s one of those things where I have learned over the years that often the most important generalization we can draw is what not to work on, what not to be interested in, and, thereby, what to focus on. Also, to have times when you know that you’re puzzled and frustrated and take time to let that be until some idea matures. I left Dulbecco’s lab actually quite frustrated with the whole problem I was supposed to work on. I had actually gotten a grant to work on growth control and mammalian cells, but I was frustrated with the techniques and experimental paradigms that were available at that time to approach the problem.

I had moved to the University of California, Irvine, and had several months while I had ordered equipment and was waiting for a few things to come in before I could get a lab up and running. I had some time to think and spend some time in the library and talk to some of my colleagues. It was a really turning point sort of conversation, I remember, with Dan Wolf, who was also a young assistant professor there, where I expressed this frustration. He said, “Why don’t you work on a eukaryotic cell where you can do genetics?”

I thought, “Great idea, why didn’t I think of that?” I went back to the library and just looked for something like that, and came across a lot papers on yeast, and it seemed like an organism that fulfilled all of those needs. I hadn’t had any experience with that organism, but I called up some of the people in the field, Herschel Roman being a prominent name (you were in Herschel’s lab then), and I just started asking about the organism and how do you work with it? And Herschel in his very generous way invited me up to talk and see how things were done. So, that’s really how I got started.

Esposito: At that time yeast was really in its infancy. Did you find the transition easy to make?

Hartwell: It was very sophisticated as a genetics organism. But most of the people working on it were asking questions about recombination and mutation and things like that. It wasn’t being used very much for cell biology. An exception was Don Williamson, who had done some work on cell division in yeast, and that was a stimulus to me. As far as working with the organism and trying to do what I wanted to do, I was actually rather fortunate in that I treated it like it was bacteria, and for the most part, that got me through. It was only…it took a few years before we really began to appreciate the differences that made it more relevant from a sort of cell cycle point of view.

Esposito: When you first started, what was your goal in isolating temperature-sensitive mutants?

Hartwell: It was modeled exactly after what Bob Edgar had done with T4. He said, “Let’s find out what all the genes do. We’ll do that by isolating mutations in all the genes and then seeing what goes wrong.” In order to do that, he used temperature sensitive and amber mutants. So, temperature sensitive was really the only way to approach essential genes in a cell. It was natural from that paradigm to just isolate a lot of temperature sensitives and then use a different variety of techniques to figure out what might be the primary defect in different mutants.

Esposito: As I recall, your original goal was to study DNA replication and what was involved in triggering the S phase itself. Is that correct?

Hartwell: That’s right. That’s what I was focused on coming out of Dulbecco’s lab. First initial experiments were to isolate a bunch of temperature-sensitive mutants, shift them to the restrictive temperature, and look at the RNA/DNA synthesis, and look for mutants where DNA synthesis shut off before RNA or protein. We found a few mutants like that. Along the way we found a lot of interesting phenotypes having to do with protein and RNA synthesis, so we spent a few years chasing those down also, because I had colleagues—Calvin McLaughlin, who was interested in protein synthesis, and John Warner, who was interested in ribosomes—that we were able to collaborate with.

But you’re right. The real passion for me was DNA replication, and we didn’t find very many mutants that looked like they were DNA replication mutants. It was only later when we began to look at cells by photo microscopy that we really were able to get into the cell cycle as a global problem in a productive way.

Esposito: I wanted to ask you two questions related to that. The first is that, at the time at which the temperature-sensitive mutants were being isolated, going from isolating the mutant to actually finding the function that was defective was a daunting task. How did you feel about that? Did you feel that you would be able to do this? Did you have ideas about how to do this? What was your approach there?

Hartwell: The only real paradigm was to have a mutant and be able to make extracts where the process of interest occurred in vitro and then show that the mutant was defective in that, and then use the mutant extract to purify the wild type function. That was the paradigm. That sort of thing had been done in DNA replication and bacterial systems, so I guess that was our thinking. But once we got into the cell cycle and beyond DNA replication to a lot of other processes, it seemed pretty hopeless to actually ever be able to get mitosis to go in vitro, and spindle pole duplication, and things like that. I think we were…it wasn’t at all clear that there was a path to really get the kind of answers that you wanted.

Esposito: Let’s go back to the original idea of obtaining cell cycle mutants and ask how that idea originated. Who was involved? How did the idea of using the bud as a time marker for the stage of division come about?

Hartwell: That’s one of these wonderful stories of serendipity, where I had an undergraduate working in the laboratory, Brian Reid. My experience of working as an undergraduate in laboratories was they always gave you some real wild project that you could just pursue on your own and have fun with. The problem that I had suggested Brian work on was looking to see if he could find in yeast the phenomenon of cortical inheritance that had been described in paramecium by Tracy Sonneborn, a phenomenon where surgically he took a piece of the cell wall and turned it around, and that pattern was inherited. We had yeast mutants that formed odd shapes at the high temperature. Brian was to take these mutants and see if, once the shape of the cell changed, that odd shape would be inherited when the cells were shifted down to the permissive temperature.

It didn’t take him very long to discover it wasn’t inherited, and we said, “Let’s look at how the cell shifts from an odd shape to a normal shape. Let’s look at the intervening divisions.” So, to do that, he needed to look through the microscope, and he actually started taking photographs of the cells. I think it was one of these “the lights going on” experiences, when we sat around looking at these photographs, and you could see the bud and the small buds and the big buds and realized there was a tremendous amount of cell cycle information staring us in the face.

Esposito: Then you just started collecting mutants that were defective?

Hartwell: Yes, so then he started looking through just mutants at random. And about 10% of the mutants made sense from the point of view that the cells were originally asynchronous in the cycle, but had a certain stage of the cycle where they became defective and then became morphologically synchronous at the high temperature. That was the foot in the door. We just had a wonderful four or five years when Joe Culotti and Lynna Hereford and Brian Reid and John Pringle and I just studied cell cycle mutants.

Esposito: So within that first group the major conceptual advances of termination points, execution points and start pathways all emerged and seemed to follow each other very quickly.

Hartwell: Yes, it was in the first five years that much of the future questions that my lab followed for many decades were formulated–as a result of just looking at these mutants.

Esposito: How did the work on mating in yeast, which you spent quite a bit of time also studying in parallel to the cell cycle, how did that fit into your interest in cell division controls?

Hartwell: That came about very early as well because Wolfgang Duntze in Germany had reported that mating pheromone stopped cell growth. We were interested in whether it affected the cells at a particular stage in the cycle, and actually he came out and visited and brought some of his mating factor with him, and we did experiments just like we did on TS mutants. Sure enough, the mating factor arrested the cells in G1, and, that then led to looking at, in normal mating mixtures, did cells mate in G1? And yes, they did, and that led to looking for the other mating factor that must be complementary in inhibiting the other cell type in G1. Then we got interested in exactly where in G1 this mating factor was acting. The mating factor work really grew all out of the cell cycle and its role in controlling cell division.

Esposito: In it’s own way it also was quite significant, because it really laid the groundwork for understanding signal transaction pathways.

Hartwell: Yes, we isolated a lot of temperature-sensitive sterile mutants, mutants that couldn’t mate, many of whom turned out to be components in the signal transduction pathway. Vivian McKay had isolated some of the mutants prior to that. Ours were a little more useful in some sense because they were conditional so you could do genetics on them at one temperature and then study their defects in mating at a different temperature. We got interested in the receptor, but again, that’s an example of many, many labs contributing to something that eventually turned out to be a major story. I just want to acknowledge Bob Edgar again.

It’s funny, when I was an assistant professor at UC, Irvine, after about a year, and I had all these TS mutants. He [Edgar] came through and visited, and one of the things he said to me was very memorable. He said, “Well, these are really interesting mutants. You’re not going to be able to work on all of them. You’ll learn a lot more about them if you share them with other people. “That was just so prophetic, because we did and indeed, we learned about them from other people’s work.

Part 3: Methodical Research Opens an Important Door; What’s next in Genetic Research

From investigating termination and execution points in yeast, Dr. Hartwell’s work shifted to the dynamics of signal transaction pathways and dysfunctions in cellular mating cycles. Here, Dr. Hartwell explains why yeast systems will help in deciphering the human genome and previews his next avenues of research as well as what he views as the next major issues in genetics.

Esposito: Let me jump forward a number of years. After the original very important period in the early 70s, when the whole cell cycle paradigm started to emerge, most of the activity that followed involved studying the mating process. Then, only later, did the checkpoint idea start to emerge. Could you give us some insight into how that idea developed which ultimately provided a critical key to understanding how the cell cycle proceeded?

Hartwell: That was just another good example of turning a student loose on whatever they were interested in. Ted Weinert, who had joined the laboratory as a post-doctoral fellow, said, “You know, I’m interested in regulation. I want to study regulation in the cell cycle.” The more we talked, I recalled what seemed like an obvious case of regulation where when you irradiate cells, they arrest in the cell cycle, so as a result of DNA damage, cells arrest. He started looking at that phenomenon, and it was out of that the checkpoint idea came, because he discovered there were mutants that didn’t arrest the cell cycle in response to DNA damage. So they identified the components responsible for that checkpoint.

That initial work on radiation response solved a major paradox for us in the cell cycle because we had found that almost any mutation that affected some component of the cell cycle even the polymerase or the spindle or anything arrested the cells. And as the paradigm was developing, at that same period of the cyclin-dependent kinases, it was becoming clear that there was no need for that. That if the cyclin-dependent kinase were triggering the events in the cell cycle, with a defect in the event itself, like the spindle or DNA replication, there was no reason to expect it to stop the progression of the CDK cycle. In fact, in embryonic cells, it didn’t. So, there was a real paradox there of why all the cells stopped. And it was this discovery of checkpoints that eventually explained that.

Esposito: Using a simple model system to get at the cell cycle proved to be a really important insight in your work. Do you think that yeast as a model system is going to continue to play a central role in understanding pathways of gene function in higher systems as we move to an era where the human genome itself will be sequenced very shortly? Do you think there will be a shift to working with higher systems themselves?

Hartwell: I think the model systems like yeast are going to continue to lead the way for a very long time. I found myself over and over again when I start thinking about a problem, a problem that may exist in higher systems, like cancer or something, that eventually, as I begin to think about what the fundamental issues are, I can always approach the fundamental issues in a system like yeast, and I always go back there. We’re beginning to see now, as the genome of yeast is complete, and people are beginning to use whole genome approaches to problems, that there is just a renaissance. That even though we may know a lot about what individual genes do, we know almost nothing about the complexity of how these pathways interact. And issues of evolution, and issues of robustness, and all kinds of things that characterize living cells. My feeling is there is so much left yet to learn about something as complex as a yeast cell with 6,000 different proteins in it, that fundamental issues of biology will continue to be fruitfully explored in those systems where there’s so much power.

Esposito: What excites you most now? What research directions are you planning to pursue?

Hartwell: I’ve gotten very interested in issues of the system, the complexity of the cell. The point at which I decided to try to think about that again comes from looking at humans. As I look at human biology and compare model systems, the thing that strikes me that is so different about human systems is the fact that they’re outbred, and you have all this genetic variability. No two organisms are the same. Yet, everything we deal with in the model systems is isogeneic, genetically inbred. That seems to me like a really fundamental issue. We’re just starting to think about what does genetic diversity, natural variation, mean to biology? How can we explore some of those fundamental questions in yeast?

Esposito: Why did you take on the directorship of the Fred Hutchinsen Cancer Research Center? What are you trying to accomplish, and how does that interface with your own research interests now?

Hartwell: There are two answers to that question. And I’m not sure which is really the strongest motivating one. The simple scientific answer is the yeast cell cycle work and our interest in the instability of the genome in cancer cells, and what controls the stability of the genome? The fundamental issue in cancer, many of the fundamental issues, can be studied in yeast, and I think that new technologies of genomics that are really ready to make an impact can allow us to have a real impact on human cancer. That’s the scientific answer, that we’re “ready,” to learn things from the models in a real human venue.

But I suspect an emotional answer is more correct. That is, I’m at a time in my life when I’m beginning to realize that people, and them trying to accomplish things and working together is more interesting than science. I like the aspect of being more involved with dealing with people in an institution and peoples’ careers and things, people that are trying to accomplish something with their lives and interact. I just find the human element, I think, of playing the game of science more fun than the science.

Esposito: In some ways it’s also dealing with complex interactions but at a different level!

Hartwell: Yes, we are, after all, biological systems!

Esposito: I want to bring the interview to a close by asking you what you think the key problems are going to be in genetics over the next decade or so.

Hartwell: I think it’s the incredible complexity of living systems. Most of our thinking is in terms of a gene or a couple of proteins interacting, or a simple pathway. Cells are just so incredibly complex and our methods for looking at them are so removed from what’s really going on. The way that we decipher a pathway, which is most of what’s going on in genetics now is to look at terminal states of a cell where something has been altered. We’re going to be developing methods for looking in real time, at space and time, at things that are going on in a cell at a molecular level, and just the complexity of everything that’s interacting with one another. It’s exciting that now there are an increasing number of technologies being developed that are beginning to allow us to approach things at the level of complexity of thousands of elements rather than just a single one.

Esposito: Congratulations again. I wish lots of luck in your future work. You’ve really been an inspiration to all of us.

Hartwell: Thank you. By the way, I want to mention that I got my original yeast strain from you. You played a big role in this whole thing.

Esposito: It was an honor to give it to you!

Yoshio Masui Interviewed by Joan Ruderman

Nature Medicine Essay

Dr. Joan Ruderman, a professor of cell biology at Harvard Medical School, interviewed Dr. Yoshio Masui, whose research on cell division in frog oocytes led him to discover the mechanism that drives the division of all plant and animal cells; August 31, 1998.

Part 1: Starting with the Materials at Hand
As a junior high school student in Kyoto at the end of WW II, Dr. Masui was chosen for a special science class that offered him a good education and the opportunity to pursue his boyhood curiosity about living things using the research material he knew best: frogs.

Ruderman: I am very glad to have this time to talk to you about your life and your scientific journey.

Masui: Oh, thank you, yes.

Ruderman: You know, so much of your life has been devoted to explorations using amphibian eggs, even from your earliest days as a student.

Masui: Yes.

Ruderman: I wanted to know what attracted you to biology as a young student?

Masui: Well, when I was around twelve years old, in grade six or so, I was subscribing to children’s science magazines. There were some stories about animals and medicine. Particularly, I was impressed by the history of medicine, very easy child-oriented stories about Pasteur and so forth. That fascinated me first, then I just started collecting frogs and that sort of thing to see their heart beating. It was a child’s hobby or a kind of play, first of all, and perhaps I was attracted to living things in nature. But I was not interested in a collection of insects, that sort of thing.

Ruderman: So you were not a naturalist?

Masui: No, no. I had no sort of inclinations to natural history kinds of things in my life. Next I was attracted by chemistry. So, when I went to the university, I chose natural science courses. But I wasn’t sure which way I should go, if I should go into chemistry, physics, or then biology.

I was also interested in physics, so it was a little bit difficult to chose. But then one of my high school senior [teachers] was a zoologist, and one night I talked with him in his office. He told me how interesting zoology was. But that still…that was not “real.” It was an attraction, yet I was still thinking which way I should go.

And then, one day I talked with my friend, who is now a real mathematician. But anyway, I was talking with him. I found that, you see, the homework…I could not finish it in one week. He finished it in two hours.

[both laughing]

You see?

Ruderman: You got a message.

Masui: Yes, yes. So, that is the final thing that made me decide to go into biology or zoology, because, “if I go with these guys, I cannot do anything.”

Ruderman: Well, we’re all very glad you made this decision. That your homework took a long time.

Masui: Yes. I was so, so interested in medicine, but my father said that…you see, my sort of a character, is not sociable in a sense…that medical doctors need that kind of social skills, which I lacked. So my father recommended that maybe I’d better go into some pure science. So, I accepted his advice.

My father was not graduated from a university, so he said to me, “I don’t know anything about education, about the universities.” He said I could choose anything that I liked, but I should take all the responsibility myself.

He gave me quite a bit of freedom in a sense, so that I finally went to zoology, knowing that zoology is not a job that is well paid.

Ruderman: Yes. We are not known for our high salaries.

Masui: Right, right. So, I made up my mind that I could do something teaching in high school—that I wouldn’t mind to be a high school teacher, a biology teacher. I thought, at the worst, “perhaps, I can be a high school teacher and then, using the extra time, summer time or holidays, I can do small experiments, using a dissecting microscope and glass needles that you can get in a high school student lab.”

Ruderman: Right. So it was feasible to do this kind of experimentation.

Masui: I knew a little bit about classical experimental embryology from reading—some Spemann kind of work. I could do that in a high school.

Ruderman: I was very surprised to hear you say that as young as twelve years old, you started collecting frogs and looking at them.

Masui: Yes, yes.

Ruderman: So you’ve been with amphibians for a very long time. We’ll get to the penguins later; I want to ask you about that. But I want to ask you about collecting materials. So you were living in Kyoto at that time. And you were collecting frogs?

Masui: This is a perhaps a most important factor for me to have decided coming to biology. My teacher was a very dedicated biologist, who was teaching me in high school as sort of a teacher. But he also was using his extra time working with diatoms, using an electron microscope to look at diatom structure.

Ruderman: Yes.

Masui: That was in junior high. He encouraged everybody to do something on a small project. My class was a little bit special, because during the war…Japan was just about to be defeated in 1945…when I entered junior high, that was 1944 or ’43….At that time the Japanese government belatedly realized that science is important for winning a war.

So, now we have to raise the next generation as scientists. They started a special class in Tokyo, Kyoto and Hiroshima—the three towns had special classes. I was then recruited to that class.

And in that special science class I was exempted from all sorts of duties or work. Other students had to do something like helping farmers or factory workers. But I was exempted.

Also, during the war they prohibited learning English. But I was also exempted from that rule. So I had sort of a good chance to learn English as well as science…

Ruderman: Wow.

Masui: …rather than going to a factory or farmland to help. That was very lucky. That is a very important factor for me. Otherwise, I couldn’t have a good education.

Ruderman: Yes, I understand. And in those early days, did you have the opportunity to work with other material, like starfish eggs or fish eggs, which were at the time and still are very popular and useful material for many Japanese scientists?

Masui: Well, you see, Kyoto is sort of, as you know, surrounded by hills. There is no outlet to the sea or anything, only the big lake nearby. So I had no chance at all to do anything with marine animals.

Ruderman: So then what happened?

Masui: Well, the only materials I could get were insects and frogs and that sort of stuff. But, you see, there are a lot of things about physiology in the children’s magazines or junior high class books. They mostly picked up the materials from frog physiology and development of frogs, you know, tadpoles, that sort of stuff. So I think frogs became the most familiar materials.

Ruderman: Well it’s very wonderful experimental material. Large enough so you can see it under a simple microscope, and they’re very hardy. You can culture them in all sorts of circumstances.

Masui: Right, right. Particularly in Kyoto. At that time it was still a suburban area not well developed. So, still there were a lot of farmlands around. It was a walking distance to collect frogs in the rice field, so it was a very easy material for children to play with.

Ruderman: You were a high school teacher for several years, while you were working on your PhD research.

Masui: Yes, yes. I was finishing in 1953, or I think actually up to ’54. I was working on my Master’s thesis. I wasn’t sure about what job I could get. There was assistant professor, a very, very good professor, Dr. Takaya. He was an experimental embryologist. I really admired him when I was an undergraduate. Then he moved to Konan University, so I asked to be his assistant, research assistant.

As soon as I got the Master’s, I moved to Konan University as a research assistant for him. The real job that I was working at that time was a high school teacher, because Konan University had also Konan High School.

Ruderman: Yes.

Masui: I was teaching eight to twelve hours a week in high school, and I taught high school students a general sort of biology. Meanwhile, I was working in his lab, as a research assistant on one hand, but also I did PhD research afterwards.

Several years later I submitted my PhD thesis to Kyoto University, where I graduated from, and I got that degree.

Ruderman: So did you like teaching high school?

Masui: Well, no. [both laugh] Not much. And perhaps I was not a very good teacher. I didn’t like…somehow, I am not a good teacher per se, but my enthusiasm about a subject I am teaching… somehow that sort of impressed the students.

Ruderman: I have to disagree with you. I remember when you were teaching in the embryology course. I think it was in 1983?

Masui: Yes.

Ruderman: You were a very good teacher then.

Masui: Yes, it’s something I like. I just naturally, I kind of, sort of totally [get] involved in a subject.

Ruderman: Well, you know, you gave several wonderful lectures, and the auditorium was packed. People stayed on and took lots of notes. It was a wonderful time. So I think you should not be so modest.

Masui: Yes, thank you. You see, I was a little while a lecturer at Yale, because Markert told me that I should start practicing teaching. So he gave me a chance to teach Yale students. At that time my English was…well, even now [it’s] terrible…but anyway it was really, really terrible in 1969.

I was teaching, but that was team teaching, Frank Ruddle and Ian Sussex and myself. So anyway, Ian Sussex told me that…I said that I’m sorry my teaching was terrible and students perhaps had a lot of troubles to understand.

He told me, “Oh, no, no. That’s all right because of the enthusiasm that you showed. [You are] the most enthusiastic instructor, and that impressed the students.” That made up my deficiencies in English and lecturing.

So, I think my strength is a kind of enthusiasm about the sort of subjects I’m teaching, you see.

More Info

Part 2: Opportunity in the United States
After his PhD Dr. Masui took a sabbatical from Konan University to work in the Yale University lab of Dr. Clement Markert, where he began his work on frog oocytes, but not after a learning experience with penguins.

Ruderman: So, how was it that you came to end up at Yale?

Masui: Well, you see, it was the early sixties, and just after I finished and submitted the PhD, and I got the degree, I was looking for somewhere I should go for my sabbatical. At that time I was really interested in differentiation, rather than cell division.

Ruderman: Right. Your thesis was on embryonic induction and lithium, I think. Right?

Masui: Yes. Right, right. Lithium chloride. My thesis conclusion is that lithium chloride has something to do with mesodermalization of whole embryos and vegetalization. I was looking for somebody from whom I could learn something in differentiation. Then I happened to come across a book edited by Glass and McElroy, “The Chemical Basis of Development.” In one of the chapters, Clement Markert wrote a very impressive sort of an approach. It was mostly conceptual, rather than experimental.

In that chapter he formulated gene expression in development, that is, differential gene activation. That is the sort of concept he proposed. I was really interested in this idea, and I was thinking about the chance to go to his lab.

By the way, at that time, 1962 I suppose, the late professor at Michigan, George Nace, came to Japan on his sabbatical because he was born in Japan and his Japanese was very proficient. He was sort of a Navy intelligence officer during the war. He interrogated Japanese prisoners of war. So he liked Japan, and he came to Japan on his sabbatical.

He happened to be living near my Konan University, you see.

Ruderman: Oh, how wonderful.

Masui: Yes. So, one day, he happened to visit me. He stayed with me about a month or two…we were working and collecting frogs and that sort of thing. We went to the field with him, together and with students, collecting frogs and bringing them back.

He showed me how to induce ovulation in vitro. I didn’t know oocyte maturation, anything. But anyway he showed me that. He just cut out the ovary and put in the pituitary suspension to induce ovulation as well as maturation.

Ruderman: Oh, that must have been so exciting.

Masui: Yes, I was amazed because a hormone directly acted on the ovary, and he showed me that.

Then I was working, and he said, “By the way, Masui..,” he always called me “Masui-kun,” because it’s the Japanese way.

Ruderman: Yes.

Masui: He asked me if I was interested in going to the United States to study something. In fact, he was asked by Markert that he should find somebody in Japan, a post-doc. He asked me if I was interested in going to Markert’s lab. Well, of course [I was], because then I was interested in his article. So he said, “Okay, fine. Let me write a letter for you.” So, he wrote a letter back to Markert.

A year or so later….the science part of the Japanese/American mutual treaties had a sort of a Japanese and American scientists’ exchange program….Markert and Holtfreter and Hamburger and, I don’t know if Dr. Gross was included or not….Anyway, Fred Wilt, young or established people all together, came to Japan.

And I had a chance to see Markert in Japan, so I talked with him. George Nace was also there, and we three agreed that I could have a chance to go to his, Markert’s, lab. But at that time Markert was at Johns Hopkins. So, he sent me a letter saying that he was going to move to Yale, and as soon as he was settled in New Haven, I could come over to his lab. That was 1966. So, I went to his lab.

Ruderman: So was that a big change for you?

Masui: Well, yes. I requested one…I thought at first, you see, two years, usually, people went to the States one year, and they extended one more year. So that they got to stay in the United States or any other country for two years…So I just requested two years, and they said, “Okay, first one year, and then you can extend.”

I thought, “Well, this is a good chance to see the science in the States.”

At the time I was coming, I thought I was going back to Japan. I then accepted Markert’s offer to see what’s going on.

Ruderman: Okay, so now I want to ask you about the penguin embryo project.

Masui: Yes, yes.

Ruderman: What’s the deal with penguins?

Masui: Yes, penguins. In the 1960’s, in the early times, I was looking for any way to look into the differentiation problem through biochemical methods. Of course, Markert was working very actively at that time on isoenzymes. I knew that, and I was working [with] electrophoresis myself, using frog embryos and so forth.

I was not a trained biochemist, and I wanted to learn isoenzymes from him. So I went out there and told him I wanted to learn such things. He said, “Fine. Why don’t you practice working with this material?” He brought out a penguin, a frozen penguin.

Ruderman: He just happened to have a frozen penguin in his freezer?

Masui: Yes, yes. I learned that he and his friend in Johns Hopkins…I think a Dr. Slotting or Slatten or something, Slodden, S-L-A-D-E-N?…that is an ecologist…

Ruderman: Yes.

Masui: He went to the Antarctic at that time and collected a lot of materials that he sent back to Johns Hopkins in a frozen state. One of his materials was penguin embryos collected in the shell, as well as a few adults. So he was keeping penguin tissues and embryos in the freezer.

He published the year before I came to Yale in “Nature,” the isozyme patterns of penguin tissues, adult tissues. That shows a very abnormal pattern. Usually isozymes, that is LDH, lactic dehydrogenase isozymes, show five patterns in a mouse or rat. But penguin embryos show a very unusual pattern. And that was published in “Nature.”

Ruderman: Unusual in what way? Many more or fewer?

Masui: The number of the bands is not five. He said nine or eleven…very fuzzy separations. He couldn’t make it very clear what it meant, so he wanted to continue this work.

That’s where I came in. If you analyze penguin LDH isozymes, perhaps you can learn isozyme enzymology as well as other techniques. So I thought this might be a good sort of start. Then I devised new buffer solutions to make the resolution better, and I came up with fifteen bands. Fifteen bands. And then, as you know, the isozymes consist of four subunits, two different, an “A” and a “B,” or that sort of thing.

So usually four tetramers. Two subunits. Random distribution gives rise to five different combinations. And this is the case of the mammalian tissue.

Ruderman: What’s the explanation for penguins?

Masui: You see, penguins, if you assume that three subunits…that all combinations give rise to fifteen different combinations. So I just used three different subunits and tetramers, so that you can have fifteen bands.

Using this hypothesis, I explained whole patterns.

Ruderman: So what’s the function of the third subunit isotypes? Is that ever discovered?

Masui: “A”, “B”, “C”…so you can have a, say, “AABB” or “AACB”—all combinations you can make with fifteen. At that time, you see, the…there was no way to know the different combinations of…why, say, heart has BBBB, why leg muscles have AAAA. People were trying to figure out the physiological meaning of that.

Anyway, pyruvate and lactate and equilibrium and that sort of a way, and they try to find out optimum concentration, relative concentration—aerobic or anaerobic, and so forth. So anyway, I just invented some explanation that is the penguin is a diving animal. And the way they live, all tissues have to have a sort of resistance to oxygen deficiency.

Ruderman: Yes.

Masui: Most land animals have a better oxygen supply than diving animals. Diving animals have to live under oxygen deficiency for a long time. So I thought…you see…this combination [is] kind of [an] adaptation to oxygen deficiencies…

Markert liked that sort of imagination. So, anyway, he found it satisfactory, and so he said, “That’s fine. Everything’s fine. Why don’t you write a paper?”

Then he said, “That’s it. Okay. Forget about isoenzymes. You have enough learned. Why don’t you try something else?”

Ruderman: Yes. I suppose it’s possible now for someone to go back and by doing transgenic analysis in penguins or using much more sensitive markers to antibodies or gene probes to actually figure it out now. Maybe you should think about returning to this.

[both laughing]

Part 3: Focusing his Research
While searching for a research topic easily relocated back to Japan, Dr. Masui decided to try replicating the work of a Russian embryologist. The project failed, but Masui was onto something that turned out to be not only the key to the mystery of oocyte maturation, but the mechanism responsible for driving all cell divisions.

Masui: No, no. It’s not..Yes, anyway, then my mind came back to George Nace, you know?

Ruderman: Yes.

Masui: I was fascinated by his demonstrations of ovulation, oocyte maturation. So I was interested in this problem before coming to the Markert lab.

I proposed two different projects. One was oocyte maturation. And the other one was metamorphosis and tail resorption during metamorphosis. So, that sort of process and then maybe a more biochemically defined process rather than induction, that sort of thing. So I brought this proposal to him on the tail resorption experiment.

Induction is…embryonic induction is…so fuzzy. Nobody knows what can induce tissues. Then neural induction, whatever, you cannot define the specific agent.

But tail resorption is very well defined and induced, that is, it has an inducer very well defined. So I proposed this tail resorption…using isotopes and biochemical agents.

Ruderman: Yes, yes.

Masui: He looked at it and said, “Hey, Masui, can you continue this work after going back to Japan?”

I said, “Well, I’m sorry. That’s a little difficult. Economically, my university is not rich enough.”

He said, “Then, forget it. You have to continue after going back to Japan.”

At that time I still hadn’t decided.

Ruderman: Right. So, there’s very good practical advice.

Masui: Yes, yes. And then, so, I think…and then on the other hand, this work…I was looking at Dettlaff’s paper. That was a very important paper for me. Anyway, he advised that I had to start something that I could continue in Japan. So I just looked at this oocyte maturation and things that don’t cost much.

Ruderman: Yes, yes.

Masui: So, after his advice, I took it. Also, Markert, himself, liked that oocyte maturation project…because in his mind…he always talked to everybody that if you suppress the meiosis, the first meiosis, and then you can get a diploid by parthenogenesis, which should have all gene combinations the same as the mother…so you can have pure strains…that sort of thing. It’s very beneficial economically…animal husbandry. He was also interested in oocyte meiosis. His purpose was to suppress. But I said, “Well, before suppressing, we should know something about how meiosis works, the mechanism.”

Ruderman: Right, the natural state.

Masui: Yes, yes. First of all we have to know the mechanism, or you cannot suppress successfully. As a starter, I just repeated the Dettlaff group’s experiments.

Ruderman: That work by the Russians had been done, am I right, with sturgeon eggs? Or some kind of fish eggs?

Masui: No, no. They used Bufo, toad eggs. Toad eggs. But, you see, the Russians also learned from the Chinese.

Because in 1939, Heilbrunn published a paper that showed in vitro ovulation and maturation using pituitary extract. Then, nobody had followed that work—only a couple of people. I think one of them was a Vermont University professor, Paul Wright. He’s a guy who used this technique to see the conditions of ovulation in vitro. And that is the only person. [At that time] nobody in Europe and nobody in North America was working on this project.

But in China, in the 1950’s Tchou-Su was working completely independent from Western science. He was working on in vitro ovulation and maturation.

Ruderman: Oh, how interesting.

Masui: Yes. Tchou-Su, who was disciple of Bataillon, a French scientist long ago. At that time, in the Cold War, there was no good relation and exchange between [the United States and] the Soviets, so we never knew what was going on there. They never knew what was going on here. But, you know, China and Russia were very close. So, Dettlaff heard of Tchou-Su’s work, so she went to Beijing and learned from Tchou-Su about oocyte maturation in vitro.

She brought back to Moscow that knowledge, and there she started working with oocyte maturation. She published in 1964 in JEEM a paper. When I started oocyte maturation two years later, that was the latest paper about oocyte maturation.

I repeated this experiment because my mentor, Dr. Takaya at Konan University, told me that anything it is you are interested in, you have to find out the best paper about that subject, and then you should repeat it, exactly what they did. I followed his advice—I repeated Dettlaff’s work. Then I found that they were wrong. They thought that pituitary and gonadotropin act directly on the oocyte nucleus.

Ruderman: But, it’s not that way at all.

Part 4: Discovering Maturation Promoting Factor (MPF)
Discovering MPF was “interesting,” says Dr. Masui, but discovering CSF (Cyto Static Factor) was “really unexpected.” Dr. Masui describes how in research puzzles and clues, refinements and repetitions eventually lead to discoveries. While searching for a research topic easily relocated back to Japan, Dr. Masui decided to try replicating the work of a Russian embryologist. The project failed, but Masui was onto something that turned out to be not only the key to the mystery of oocyte maturation, but the mechanism responsible for driving all cell divisions.

Masui: Right, right. So that is the starting point of my oocyte maturation work. Yes.

Ruderman: Was that a surprise to you initially to find out that it wasn’t nuclear? Because at that time there was so much interest focused on gene activation as the primary driver of changes. You know the time course of frog oocyte maturation is several hours, depending on the species. You have plenty of time for new genes to be turned on.

Masui: Right. But you see the…Jacob’s mRNA hypothesis came out in 1961 or so in “The Journal of Molecular Biology,” and everybody was, as you say, interested in the sort of gene activation. Then, also in 1963 or so, Jensen’s group was working with the uterus and progesterone…

Ruderman: Yes.

Masui: …and also they thought that progesterone directly acts on the nucleus.

Ruderman: Well, yes.

Masui: Yes, yes. And that is true in his case, you see. So everybody was saying, you know, “The hormone action’s directed toward the nucleus.” So Dettlaff thought that this hormone, pituitary hormone, also acted on the nucleus to induce a new mRNA. And her papers…

Ruderman: Well you know, even, what is it…thirty-five years later, people are still surprised when I tell them that in the oocyte, progesterone works through an unknown surface mediated mechanism.

Masui: Right, right. So anyway, you see, the…it was not a revelation, just a frustration, in a sense, because I couldn’t repeat her work.

Ruderman: Yes.

Masui: And then so…it took a little while to come to the indirect actions. But, finally, something on the surface must react with progesterone. That’s an idea that I came upon in 1967.

Ruderman: So in all of this work, you know, showing how progesterone works through a novel mechanism in the injections of cytoplasm from the egg back into the oocyte, which led to the discovery of MPF and later to cytostatic factor, it seems to me that there were two things that were key: One is the microinjection assay itself.

Masui: Right, right.

Ruderman: Which is conceptually very simple, and once you learn it, technically very simple, and the second is that there is an assay, that there is a starting point and an end point, which can be quantified.

Masui: Right, right.

Ruderman: And that the amount of time you have for this assay is actually quite small, so you can come in one day, you can do the experiments, you can see the results, you can think about it, come back the next day and set it up different.

Masui: Yes, yes.

Ruderman: It’s just a very powerful combination of a simple technology and a fabulous assay.

Masui: Well…

Ruderman: I mean how…at what point did you learn or develop the microinjection assay for use with the frogs?

Masui: Well, I think of course, this came from Briggs and King’s nuclear transplantation.

Ruderman: Yes, yes.

Masui: Also I think that Dettlaff used microinjection technique to inject a nucleus into the maturing oocytes to see if enucleated oocytes have an ability to cleave after injections and that sort of thing she described in her paper. And, also, John Gurdon was doing nuclear injections and that sort of thing in the early sixties. So I think the injection was not the strangest thing at that time, and frog embryologists knew that injection into frog oocyte, blastomeres, were not so difficult things.

So, the only way that I had to improve this technique was quantitatively….You have to inject in something. Until that time, nobody knew how much they injected. So I made a kind of micropipette…a graduated micropipette. I used a microforge and a Leitz micromanipulator.

Ruderman: Yes.

Masui: They happened to be in Markert’s lab because there were people doing microinjection in his lab. First Heinrich Ursprung was injecting proteins and so forth into frog eggs. And also Markert’s former student at Johns Hopkins, Charles King, and that sort of people…they did a lot of microinjections. So that the microinjection equipment was in Markert’s lab.

Ruderman: Yes.

Masui: That was very fortunate for me…everything was there, and nobody was using it. So I used this equipment to make a graduated micropipette. So, quantification of cytoplasm I can inject is the first step to the assay, to the MPF or CFS, that sort of thing. That helped me to develop a more accurate or exact idea of what was going on.

Ruderman: Do you remember the first day you injected egg cytoplasm into the oocyte and saw maturation?

Masui: I don’t exactly remember. But, anyway, you know…yes, I was little bit excited. [I thought] “Yes, [this] really is something I should pursue. It’s worthwhile to go further.” That was my feeling.

Ruderman: Yes.

Masui: Then I thought that if this is the case, I should quantify how much we need, and that sort of thing. You know? So that was why first, you know, just a trial, and it worked. The reason was …actually Dettlaff was injecting nucleus and germinal vesicle into oocytes to induce oocyte maturation.

Then that was sort of…Okay,..so then she said that the nucleus contains something that induces oocyte maturation. So…that is true in a sense…when she took out a nucleus from an oocyte just before germinal vesicle breakdown…and she injected it. Then all the data was very very fuzzy—she didn’t mention how much of anything. Also, she mentioned that this is a very, very stable substance in the nucleus. Because one of the needles that she or her associate used…that dirty needle which was not washed. They used this again. It worked without anything, just injecting and using this dirty needle. They injected Ringer or something, and it worked.

So, they suspected that something remaining inside the glass needles was still active or something. Very strange things they mentioned.

Ruderman: Oh how bizarre!

Masui: Yes. So…

Ruderman: I wasn’t aware of all of that.

Masui: Yes, yes. I was impressed that, first of all, it was a very sloppy way of handling things. But you know that was not the case, [there were] some mistakes.

But anyway, I thought, just before germinal vesicle break down you took out a nucleus, put in, and it works and that’s fine. I saw a few cases. So it was not totally false. I thought, “This has to be more cleaned up.”

Ruderman: Yes.

Masui: Yes. So, first of all, I had to quantify the material I had to inject and that sort of thing. So, I started from scratch to carefully reexamine their work. Then it turned out to be MPF. But anyway, I just removed the nucleus—if Dettlaff was right and if you remove the nucleus nothing happens, nothing should happen.

Ruderman: Yes, yes.

Masui: So, I enucleated, and then it still worked, you see. So, I thought that first of all, her site of hormone action was totally wrong. And the second thing, this is totally wrong, so I lost all confidence in their work, you see.

But, anyway, [I thought], “Okay, now I have to go [on] myself.”

Ruderman: So what was the initial reaction of your colleagues to this experiment?

Masui: Well I think…I had people like Rick Elinson as a grad student. He was watching me. I don’t remember, [but] he said that that first time he heard me, I said “That’s very interesting.” That was my expression.

Yes, yes. “Masui said, ‘That’s very interesting.'”

I don’t remember. But he remembers. He mentioned this sort of thing a few times to other people. But, anyway, that was one. I was not as much surprised as [when] I found CSF, you know. That was in a really sort of…I was more excited.

Ruderman: Yes, yes.

Masui: Because that was really unexpected. When I was looking at [the] level of MPF in oocytes and also blastomeres, I found some residual activities in the cleaving blastomeres. So, I thought initially the MPF was doing something during cleavage, and so I thought MPF might accelerate the cleavage, you see.

If you take out MPF from a maturing oocyte and inject it into fertilized eggs, it should accelerate mitosis as well. And, therefore, cleavage should occur a little earlier.

It takes about three hours in Rana pipiens, and in Xenopus one and a half hours. Rana pipiens takes a little longer, just twice longer. So I injected MPF into fertilized eggs and waited, [thinking] perhaps it occurs [in] less than three hours. But they didn’t cleave, and I thought I had failed. First of all, frog eggs are fertilized….You don’t know for sure where the frog eggs are normally fertilized until they cleave.

Ruderman: Yes.

Masui: So I thought, “Okay, uncleaved eggs [are] not good test systems. So I should inject [at], say, [the] two-cell stage.” The two-to-four cell stage takes about one hour…

Ruderman: Yes.

Masui: So I thought that it should cleave again [in] less than one hour, and I injected, and then they didn’t cleave. I found only the other side cleaved. I was very perplexed.

I repeated and repeated this experiment many times. I was convinced that injections stopped cell divisions. That was more, sort of, surprising [it] seems…

Ruderman: Yes, yes. And also a very dramatic result.

Masui: Yes, yes. And then Rick Elinson was also impressed by this experiment, so he repeated my experiment. And he got half a blastula. So he proposed this as his PhD thesis and project.

He brought it to the committee meeting. The committee meeting said, “That’s not a sure thing, yet. Just a…”

Ruderman: Wow!

Masui: “…found just yesterday or something.” So as far as I remember, he said, “The committee feels kind of unsafe going on that sort of results as a PhD thesis.” That he should go with a more solid thing. This is just still a…

Ruderman: Phenomenon.

Masui: I would like to know, whether it’s true or not, you know? But he took a half-arrested blastomere picture, and he gave [it to] me. So, he repeated my work, and it turned out to be right… in his hands too. I was very happy about that.

Ruderman: Did Rick and you move independently to Toronto?

Masui: Yes, yes. Independently. I went to Toronto, and there were assistant professors there, embryologists, but I was a little bit senior, so they hired me as an associate professor. Then, two assistant professors who were there didn’t do well, so the chairman didn’t give them tenure. So, vacancies were suddenly created.

They asked me if I knew anyone I could contact, so, I contacted Markert and asked him to recommend someone he knew. He recommended Rick and other people. So they went through the selection. I was not a member of the committee, but anyway I submitted Markert’s letter to the committee. And the committee searched using this letter. Finally, a few people were short-listed and came, and Rick was included in the short list. So, he got the job there.

Ruderman: Good choice.

Masui: Yes, yes. I was happy, too, because at least I had my colleague who I knew very well.

Ruderman: Yes. It’s very important to have colleagues that share your point of view and your experience.

Masui: Yes, yes. I met him in 1967, when he came as a student and from Johns Hopkins, so [I’ve known him] more than 30 years.

Ruderman: I want to ask you more about MPF.

Masui: Yes.

Ruderman: When did you begin to realize that MPF was actually something that had much broader importance in cell division, rather than just driving oocyte maturation. And we now know it’s, in fact, responsible for driving all cell divisions in all plant and animal cells on the face of the Earth.

Masui: Yes, yes. As I said, the experiment that happened to lead me to the discovery of what CSF was, based on the idea that MPF was doing something with cleavage or mitosis. That’s why I injected maturing oocyte cytoplasm into cleaving blastomeres…

Ruderman: Yes. So, it had an effect on the somatic or the embryonic cell divisions, which was the first clue that it was more general.

Masui: Yes. That residual MPF was still there in the blastomere, and that started my idea that something—maybe MPF— causes something, some effect on mitosis, and that should be tested.

So I did that. But it turned out to be the other way around.

Ruderman: The opposite effect. Yes.

Masui: Yes, yes. Because CSF was there. But you see, this thing was hanging in my mind. So, when Frank Ruddle, you know, at Yale…

Ruderman: Yes.

Masui: He came to give a seminar, and we were talking. I said, “I think MPF is doing something with mitosis, but I have not had any proof of that.” Then I was talking with my students and that sort of thing, but I didn’t have a chance to test it.

I never thought of the other way around experiment. That is you take out the cytoplasm from the two-cell or four-cell stage and then inject it back into the oocytes to induce maturation.

I didn’t do this experiment myself. One of my students, William Wasserman went to Dennis Smith’s lab, and then shortly after he went to Dennis’s lab, he published a paper with him, in the “Journal of Cell Biology” that, you know…

Ruderman: Oh, yes. I remember this paper well.

Masui: Yes, yes. That was, perhaps, 1970 sometime. That was the first demonstration that MPF existed in mitosis and mitotic cells. Then after that, Sunkara and Rao’s group published.

Ruderman: Yes.

Masui: But when I wrote about oocyte maturations for a review. International…

Ruderman: Yes, I think the “International Review of Cytology.”

Masui: Yes. Yes. I was comparing the oocyte maturation and mitotic cell cycles, and I speculated that MPF might work. That was a hypothesis that I wrote in that review.

Ruderman: Well, you were right.

Masui: Yes, well anyway, and then it was…and then, I think somebody, Vogelstein, also his group did…

Ruderman: Oh that’s right. Wasn’t it they tried to…

Masui: Yes, 1979-80

Ruderman: I had forgotten about that.

Masui: Yes, yes. Vogelstein and, Sunkara and Rao’s group, and Vogelstein’s group and Nelkin, Nelkin is the name…first author, I suppose…

But, anyway, they published that mitotic cell cultures and mitotic mammalian cells have MPF.

Ruderman: Yes.

Masui: Then after that, then suddenly, many people [were] working on this problem. French people did work with sea urchin eggs, and then Weintraub, you know Harold Weintraub, he was working with a French Group, in…Baulieu’s group in Paris. They published about the existence of MPF in yeast.

Ruderman: Right. They induced the frog oocyte to mature.

Masui: Right, right.

Ruderman: So it made it seem to be very universal.

Masui: Yes, yes. So that was around the 1980’s.

Part 5: The Hallmarks of Dr. Masui’s Lab in Canada
Asked to mark defining events in his lab at the University of Toronto, Dr. Masui recalls discovering that MPF is a protein and developing the in vitro cell system that allows scientists to analyze cell cycle processes biochemically.

Ruderman: Well, you know most labs goes through a series of evolutionary changes that I think can be split into eras of evolution. They’re often defined by an exciting discovery, or a particularly engaging student, or a post-doc who tries something new and gets it to work. Once you started your own lab in Toronto, how would you look back on the evolution of your own lab? Sort of the key important things and people that happened?

Masui: Well, I think you know in my lab the first important point was we succeeded for the first time in extracting MPF. It was not stable enough to go on [with] further molecular characterizations, but at least we could separate [it] using sucrose density gradient and centrifugation. So there was something in [the] molecules there, and we determined the sort of sedimentation constant type of thing.

Then we also tested for protease sensitivities and RNAse sensitivities. To find [if] it perhaps [was a] protein kind of molecule. So that was the first step in 1976 or so. We published in “Science.”

The second step was [the] in vitro cell cycle system. We first developed [it] in 1983…

Ruderman: So this was the work of you and Fred Lohka.

Masui: Yes, Fred Lohka and Bill Wasserman and myself. I think these two points are kind of landmarks in my lab.

Ruderman: And certainly the development of the cell-free system had a tremendous impact on the field. It made it possible to study the cell division cycle in the test tube.

Masui: Yes.

Ruderman: It opened the way for everybody, including molecular biologists who couldn’t get a needle in a frog oocyte to save themselves, like me, to really inquire into an enormous number of questions that have an impact on development, on normal cell division, and cancer. I mean it just really broke the field wide open.

Masui: Yes, thank you. Well anyway, this technique we wrote [about] in the “Science” paper makes biochemistry of the cell cycle amenable to everybody. That was what we hoped for. But, actually, it took another several years to make it widespread. Andrew Murray’s improvements [made that possible], and as soon as Fred Lohka moved to Jim Maller’s lab, he adapted this technique. We were using Rana pipiens but he adapted this work to Xenopus oocytes.

Ruderman: Yes.

Masui: It works much better than the Rana pipiens system, because the Rana pipiens system takes a longer time to make one cycle. Usually, normal development of Rana pipiens takes twice longer than Xenopus. Fred Lohka’s system is better than we did in Toronto.

But then Andrew Murray improved Fred’s system, further, more. So now his system works…three cycles and that sort of thing.

Ruderman: Yes, yes.

Masui: And so, I think these gradual developments from Rana to Xenopus systems made it very useful, this system.

Ruderman: You know when we saw your report of the cell-free system from Rana and when we saw the Murray report from Xenopus, we thought, “Well, we should be able to do this in sea urchins or clams. Basically, they’re rapidly dividing embryonic cells, and the only difference with the sea urchin and the clams is that they’re smaller, they’re marine.” But for some reason they just don’t work as well.

Masui: Right, right.

Ruderman: In the sea urchin system, the sea urchin lysates get stuck in mitosis for hours. Very bizarre.

Masui: Yes. You know I happened to have sea urchins for a lab, a student lab when I was teaching an embryology lab in Toronto… We bought sea urchins from California…[and the] leftover sea urchins we used for this kind of work. I was trying to extract MPF from sea urchins, and it’s very difficult. The reason was, I thought, those animals are used to kind of a hypertonic solution compared with frogs, right?

Ruderman: Yes.

Masui: And sea water is very, very hypertonic, compared with frog eggs. I think that makes things more difficult…marine animals are difficult to work with in this way. However, I think Kishimoto was doing something at that time, and I don’t know how successful he was. They reported something…an in vitro system…using starfish eggs?

Ruderman: Yes, yes. It’s certainly possible to take the eggs in the early part of the cell cycle and drive them in to M phase by adding MPF or a cyclin. But they don’t go on to the next part, which is to complete mitosis and inactivate the MPF.

Masui: Yes, I see.

Ruderman: So, presumably, there’s some interesting phenomenon going on there that either has to do with the cell being intact or some sort of check point control system operating in vitro. Perhaps that will explain it.

Masui: Yes. How about cultured mammalian cells? They include extract MPF from M phase cells, right? So in theory they also can make sort of in vitro extract system.

Ruderman: It’s certainly possible to make extracts that destroy the cyclins, but I don’t know of anyone who’s tried to do the biphasic cycle and have it succeed.

Masui: I see. It is still difficult.

Ruderman: I think so. Because so much depends on new transcription, and the cell cycle is so much more complicated.

Masui: I see. So far, still frogs are the only animals that we can use routinely?

Ruderman: I think, as far as I’m aware..

Part 6: Attitude is the Key to Success
Research, like all human activity, does not obey plans, says Dr. Masui. The challenge to research is not planning, but how you think through anything new or unexpected.

Ruderman: I want to ask you one more question.

Masui: Yes.

Ruderman: You know most winners of the Lasker Awards are heads of big labs that are doing big team science.

Masui: Yes, yes.

Ruderman: And your work is to many of us, one of the most wonderful examples of how basic scientific research that’s driven simply by curiosity, by trying to understand how nature accomplishes a specific interesting task, how that actually led to a major breakthrough that has profound impacts on all aspects of human biology and medicine. It’s really an inspiration to all of us.

And I wonder if you have any words of advice to young scientists who are facing the next part of their scientific career?

Masui: Well I cannot say that sort of “big things.” You know any human event [is] not very well predictable. We cannot predict which way we [will] go. The research [is that way] too, you see? If anything we can plan comes true, life is so easy, you know?

Human life is a kind of thing that we can try, of course, but more often it doesn’t come as we planned. I think research is a part of human activities. And we never expect what things we planned go and come true.

Reflecting on my sort of way, you know, when I write, say, a research proposal, usually things turn out in unexpected ways. The only [thing] I think important is that when we face something, we have to know how to handle it. So that it seems to me what challenge my research goes through…it is not planning, but the attitude when I face something, anything new, or unexpected or whatever, and how seriously I think it over and to know what it means, you see? So that attitude has helped to me, rather than my research and planning. I’m very bad at planning. I never planned to go through this way.

Ruderman: Well I think those are very good words indeed.

Masui: Is that right?

Ruderman: Very philosophical.

Masui: Well anyway, so that you know, no matter how well written the proposal is, [there is] no guarantee. It doesn’t guarantee how it will work, you know. In my case at least. So if you write a really sure plan…you cannot get something unexpected or something very new, you know, novel? If you plan something, and things [are] coming out as the plan says, then that’s not novel, right?

Ruderman: Yes.

Masui: So I think that novelty and planning are always contradictory things.

Ruderman: Well, I think these are very good words. Dr. Masui, I feel very privileged to have had this opportunity to talk to you for such a long time.

Paul Nurse interviewed by Gerard Evan

Nature Medicine Essay

Dr. Gerard Evan interviews Lasker Award winner Paul Nurse, September 1998. Dr. Evans is currently the Service Chief for the Infectious Diseases Service of the Southeastern Ontario Health Sciences Centre and has an interest in medical education, both at the undergraduate and postgraduate level, antimicrobial use, clinical trials in new anti-infective agents, and home parenteral antibiotic therapy. Active interests in the sphere of clinical infectious diseases include infective endocarditis, sexually transmitted diseases, fungal infections, and CAPD peritonitis.

Part 1: Interest in the Cell Cycle Sprang from Desire to Do Something ‘Important’
Paul Nurse describes his genetic approach to cell reproduction and tells interviewer Gerard Evan how a grueling night in the laboratory as a graduate student prompted him to search for something “interesting and important” to work on. Nurse then details why he chose to study the cell cycle in yeast.

Evan: Hi, my name is Dr. Gerard Evan, and I’m interviewing Dr. Paul Nurse, who is a joint recipient of the 1998 Lasker Award for his work on the cell cycle. Paul, can you describe to me what you work on?

Nurse: Yes, Gerard. I have been interested, in fact for many years, more years than I would like to remember, in the cell cycle—that’s the process by which cells reproduce themselves—from one to two. And I’ve taken a genetic approach to this problem, working with a very simple organism, yeast—that’s a single-celled fungus. And I’ve isolated mutants, which are altered in the cell cycle and its control, and then studied these mutants to find out what the basic process is that is underlying the cell cycle. And then used these mutants to clone the genes and then establish what the molecular basis of how they work might be. By using these various approaches, you can then work out exactly what molecules are controlling cell cycle progression and how they are regulated.

Evan: As you say, you’ve been working for a very long time on the cell cycle. So why is this? Is it because it is innately so interesting? Or does it tell us a lot about other aspects of biology, or is it relevant to particular diseases, or what?

Nurse: Well, I have to say, my interests in the cell cycle, they started when I was a graduate student. And I was working all night with a machine that was actually in the development stage, so it kept breaking down. I remember I had to keep putting rubber bungs in all the safety switches to keep the thing working. And this was extraordinarily tedious, and my mind drifted off into other things about what sort of better world might there be somewhere else outside of this room with this little machine. And I thought it’s very important, if you are going to suffer like this doing science, to actually work on something that’s interesting and important. And the cell cycle came to mind then for several reasons.

The first is that one of the basic properties of life is its ability to reproduce. And that is seen in its simplest and most basic form with the reproduction of a cell, during the cell cycle, the division from one to two.

In fact, the reproduction of all living things, including complicated organisms, such as human beings, can be understood in terms of the division of a cell from one to two. So, it was a process that was central to understanding life, of defining one of the major distinguishing characteristics of life. So that was one reason.

A second reason was that the cell cycle is an example of a simple developmental pathway. Now, lots of biology has got to do with development, but it’s usually quite complex. Whereas, the growth and division of a cell, although actually quite complicated, is relatively simple compared with most development that we’re trying to understand. I mean, working out how a frog is made is certainly much more difficult than working out how a cell is reproduced.

Evan: So this is the advantage of yeast as a single-celled organism, when doing this sort of thing?

Nurse: It is, indeed, because a yeast is so simple that it barely does very much more than actually reproduce itself…and make beer and bread, of course. Many of the genes that it has are really devoted towards this process of cell reproduction. And the point about this is this—that I felt this was an example of a simple developmental sequence which, in principle, we could perhaps one day completely understand. Whereas, I think it will be a very long time before we understand how, for example, a tadpole is made. So that was another reason—that we could understand a developmental sequence in detail.

And the final reason was that because cells are in all living organisms, because they all have to undergo cell division, there was the possibility that we were looking at a process that was likely to be universal across all of life. And that had its attractions because it meant that it was relevant to all different sorts of living things and situations. And also, that perhaps we could use a whole variety of different tools, exploiting the different advantages of different organisms, to actually work this problem out. So I think that was what originally attracted me to this. And I suppose it was mainly thinking well into the night whilst looking after this ghastly machine that prompted most of this.

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Part 2: On the Need to Identify Crucial Elements Amid Biological Complexity
Nurse discusses the growing complexity of the cell cycle field and cautions that it is necessary to identify crucial elements. On the other hand, any biological problem is, indeed, complex, Nurse says, and figuring out how to analyze this complexity will be “one of the greatest challenges of the first half of the 21st century.”

Evan: So, I mean, these processes that underlie all biological organisms, I mean, some would say that they are all very well, but the thing that distinguishes different organisms is the complexity of the processes. And certainly as an outsider to the cell cycle field, it looked very simple when it first started, and it has gotten immensely complicated. Every gene, every protein involved seems to have multiple copies, and the more complex the organism the more complicated the system. I do have this tendency when I read a review, that it seems to have gotten very bogged down in the detail. I mean how do you feel about this? Because the work that you did seemed to show that the underlying simplicity was the thing that was most important about it.

Nurse: Yes, I know what you mean. You know, when you read these reviews, it’s a bit like looking at the underground map in London or the metro in New York. And you’ve got all these different things connected by these different lines. And it’s difficult to sort out the wood from the trees. Well, I think this is a very interesting question you’re asking here, because I think the answer is also complex. Some of this complexity is very real, and we have to face it, and some of it is, in a sense, generated by the work that’s being done on it.

Now what do I mean by the latter? When one is faced with a biological problem, biology being very complex, I think the key question is to try and focus on those parts of the problem which are, in fact, illuminating and are most important. And the key is to try and approach that—there’s different ways of doing that and that depends on the system to system that you use—but it’s very important to hold that in your mind, because not everything is of equal importance in the system.

I think that at the beginning, the people who were working on the cell cycle thought a lot about this and tried to focus on those parts of it and those aspects which were, indeed, most important and which were particularly relevant for control. Now when the field became established, many more people became attracted to work on it. And they identified new aspects of it, because they were trying to perhaps make a new contribution that they naturally were excited about. And it may be that not all of this was actually of equal interest and importance. But when you now are trying to describe it all, what you have is a whole set of bits out there, and unless you really think about it very carefully, they may look as if they are all of equal importance, when, in fact, they are not.

And I think what is absolutely crucial here when we are thinking about problems like the cell cycle—in fact, any biological problem—is to try and get to grips as to what is, in fact, the crucial part of the problem. And that does get lost as more and more people get into a field and, perhaps, try and promote their little bit of it. And unless you are ruthless in your assessment of what’s important or not, that can get lost. So that’s what I meant by saying that maybe it may not be as complicated, in that sense, as perhaps as it was in the beginning.

In another real sense, of course, it is complex. There are many different steps that have to be carried out, and there are many different steps that we have to understand. There’s no doubt that biology is about to move into a new dimension, where it has to cope with this complexity. And I would suggest that one of the greatest challenges of the first half of the 21st century will be managing this…how to analyze this complexity. We already see it with sequencing the genomes and identifying so many of the genes, and so on, that are there—that we are being overwhelmed with information, and we’ve got to make sense of that information.

And this is going to require new ways of thinking about it; analyzing complex patterns of data, the computer analysis that is required there; conceptual ways of thinking about how you cope with complex networks, mathematics there, the conceptual basis of dealing with that; actually putting together all these functions of molecules so that we know how they operate in real time and space. And I think that’s another important issue there that often gets lost in most analysis—that we don’t actually know what’s happening in the single cell, in different parts of that single cell.

Part 3: The Importance of Interactions Over Space and Time
Biochemists “mash everything up” and loose sight of interactions over space and time beyond the local molecular interactions, Nurse says. He also notes that he is interested in turning his attention to larger-scale structures and to how cells organize themselves in space.

Evan: Most technologies give a static picture, I guess.

Nurse: We do! We lose the dynamics. We lose the information in space and time. What we do—we are biochemists—we mash everything up, and we lose this organization. And we have to put that organization back. Now, at first sight, that may seem as if it makes it more complex, but I have a feeling that when we put that extra information back (which is only the information that is present in life itself) that in fact there’s a chance that it may make some of this complexity more straightforward. Because, once again, I suspect it will make us focus on that which is more important, rather than that which is simply necessary for something to work.

Evan: There also seems to be a problem—I don’t know what you think of this—when you’re teaching, which I sometimes call the hardware/software problem, which is when people come into science at the undergraduate level, they often think there’s a basic machine and then the system that regulates it. And, of course, in biology it’s all mixed up.

Nurse: Yes, that’s quite right. In some ways, the analogy of the software program driving the computer is not such a useful one, but because we are now so computer-minded, we’re starting to think like that. As you absolutely are right to say, the information flow—which is really what you mean by the software—the information flow is built into the hardware. There are some interesting issues here. The fact is that we tend to think about biology and molecular biologies as interactions of molecules at a very local level—that is, one molecule touches another, and it’s limited over space and time in a very limited way. Whereas, in a living system, although that’s extremely important, there is also communication between different regions of the cell, at different times in the life of that cell, as well as, of course, between cells. And this requires transduction mechanisms, means of detecting information, means of transferring information. And all that adds an extra dimension and expands the interactions over space and time beyond the local molecular interactions. And there’s a lot of exciting stuff in there that I think we have really barely got to grips with yet.

Evan: So, I mean, obviously there is a lot of complexity, and I guess for younger scientists, they’re all trying to carve out their particular gene or process. And that’s why, in a sense, more and more information comes out, and it’s quite difficult to consolidate it. From your point of view though—you’ve made some of the very important basic discoveries in the basic mechanisms of the cell cycle—do you now ever get the feeling that there may be other things that you would become interested in or have become interested in?

Nurse: Yes, I do, because I also have to recognize what my own limitations are and what hopefully some of my strengths might be. And I’m very much a biologist. In some ways, quite a lot of the cell cycle—this isn’t yet quite true, but may be in a relatively few number of years—that we understand the basic outline of how the cell cycle works. And perhaps much work now has got to work at a rather more detailed level, which is perhaps not my particular forte. Increasingly, I’m thinking about other issues which I think are important and central to life where I can take a more biological approach, where I think my own skills perhaps are best found.

And another problem which very much interest me, it’s certainly related to the cell cycle, is how cells organize themselves in space. You know, we’re not going into Star Wars or anything here. What I mean is that…the point about biology is that it is extended in time and space. What we have is a cell, which although the basis of it is molecular interaction, is, in fact, extended over a much larger scale.

Evan: Large-scale structures.

Nurse: Exactly. And so how is that organized? How does a cell know what a front end is from a back end? How does a cell know where its middle is? How does it grow in particular directions? There’s a whole series of problems there, which are to do with organizing itself in space, which are relevant, of course, also to larger organisms. I mean, I’m looking at a fish in an aquarium over here, which has got extraordinary shapes. How are these actually made? I think this is one of the wonders of life, yet another characteristic of life. Just like reproduction is seen in the dividing cell, the generation of form—which is a central characteristic of life—is also seen in the single cell and perhaps has not received as much attention as, for example, developmental biologists have given to the acquisition of form at a bigger scale in the formation of embryos and adult organisms.

Evan: So, is your thought then that perhaps large-scale structures are sort of just iterations of the processes that allow a cell to know where its back and front is, and where its middle is?

Nurse: No, I don’t. I think that the problems are analogous, rather than homologous, by which I mean that I don’t think the same molecular mechanisms will be involved, but I think the ways of thinking about the problem…

Evan: So maybe the same sorts of general rules…

Nurse: There may be general rules that can be derived. And I, as before, think that understanding this in single cells will be simpler. And because all living things are made of cells, there may be, as with the cell cycle, universal answers to the questions of how a cell organizes itself in space. Whereas, I suspect that the way in which this fish is organizing the structure of its fins will have differences compared with the way in which we organize our feet. I’m, of course, very much aware that there are conserved systems, so that it is interesting to think about the conservations. But I suspect that it will be much more conserved at the level of the cell than at the level of the organism. I could be wrong there, but that’s my view.

Part 4: Nurse to Young Science Students: Be ‘Totally Motivated,’ Curious and Bold
Nurse tells Evan that curiosity, above all, keeps him motivated. Asked to offer advice for young people going into science, Nurse says they need to the “totally motivated” because this is a difficult profession. Curiosity and boldness are pluses, he adds. And finally, he notes, “completely ignore the advice of your elders.”

Evan: Okay, so I mean, anyone looking in from outside would say, “Hey, this guy has spent the last 20, 25 years working on one particular thing. He’s at last thinking about perhaps getting involved in some other things.” To most people this sounds either crazy, bizarre or as though we are mentally ill (and, of course, a lot of people would think scientists are). What keeps you interested in your work and keeps you motivated?

Nurse: I think really I can answer that with one word, and that’s curiosity. Scientists can be motivated by different things, actually. That’s one of riches of science. We shouldn’t all try and be put in the same mold. Different types of scientists are good at different stages of the problem. Some scientists are motivated straightforwardly by competition, being the best in that field. There’s nothing wrong with that. It means that they generally like being in the hurly-burly of competition and being the best in that situation. I’m not really quite of that sort. I think that I’m…

Evan: But they’re all legitimate ways of deriving motivation, is what you’re saying?

Nurse: All legitimate ways of driving motivation. I mean even, I suppose, it’s perfectly legitimate to want to earn your salary. The problem is that in the UK we don’t get good enough salaries for that to be such a motivation. At least the US is much better in that respect. But what I was going to say is that, for me personally, I think it has to be curiosity. That is, I’m very curious about living things, and they fascinate me. I like thinking about the detail of a problem. I like concentrating really hard on particular problems which are very simply stated. I mean, I’ve stated them already. Like, you know, with respect to space, how a cell knows where its middle is. It sounds so ridiculously simple, but is, in fact, very fundamental. So I’m very naturally curious about that. I think that type of scientist, you can make a case that they’ve suffered from retarded development, because curiosity is something that is seen very much in young children.

Evan: And puppies.

Nurse: And puppies. And it usually gets knocked out of them by about 14, 15. I think often scientists, who in my scientific management role seem like a lot of adolescents most of the time, I think a common trait is retarded development, which maintains curiosity to late in life.

Evan: So, obviously younger people coming into science, I mean, they’re going to look at you and they’re going to say, “What do you advise for me?” Did you have any advice for younger people coming in?

Nurse: Well, given what I have just said, clearly you have to be totally motivated, because it is a difficult profession. And often it can be depressing, because if you’re working on something important, and you’re at the cutting edge, then frequently you will fail, perhaps even 90 percent of the time, in what you’re trying to do. So you have to be highly motivated. So I think that’s absolutely certain. I am clearly going to also promote, from what I have just said, curiosity…is extremely important. To remain curious, not only is it wonderful—nature and what’s around us—but curiosity is a very good motivator.

Secondly, I would argue strongly for boldness. Be bold, aim high, try and address something that’s very important. Not only is that satisfying, but why waste your time on something that isn’t bold. You know, at least aim high, and you never know, you might get to it. And I suppose the last thing I have to say is really to repeat what Max Perutz said when I think he was asked this question: That is, completely ignore the advice of your elders.

Evan: Paul Nurse, thanks very much, indeed. It’s been a pleasure talking to you.

Nurse: Thank you, Gerard.

Key regulators of the cell division cycle

1998 Albert Lasker Basic Medical Research Award

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Key Publications of Lee Hartwell

Key Publications of Yoshio Masui

Key Publications of Paul Nurse

Award presentation by Ira Herskowitz

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Interview with Lee Hartwell and Rochelle (Shelly) Esposito

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Yoshio Masui Interviewed by Joan Ruderman

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Paul Nurse interviewed by Gerard Evan

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