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.
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…
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.
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.
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?
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…
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.
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.
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.
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.
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.
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.”
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.
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…
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…”
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…
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.
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…
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.
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.
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.
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.
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?
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.
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?
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.