Discovering the Cell Cycle Regulators

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00:00:06.27 Hi. I'm Sue Biggins,
00:00:08.23 and it's a privilege to talk to Lee Hartwell today
00:00:11.13 about his pioneering work in understanding the regulation of the cell cycle.
00:00:16.07 This of course led to the Nobel Prize in Physiology or Medicine in 2001.
00:00:21.06 Just for background, I want to remind everyone
00:00:23.16 that the cell cycle is an ordered process
00:00:26.05 where the genome and organelles are duplicated and accurately partitioned into daughter cells.
00:00:30.26 And it's really one of the most fundamental things for all organisms.
00:00:34.13 By studying the morphology of temperature-sensitive mutants in yeast,
00:00:38.09 Lee Hartwell identified many of the key regulators of the cell cycle.
00:00:42.01 His seminal discoveries included identifying the cell division cycle, or CDC, genes
00:00:46.22 that are now known to be universal regulators of the cell cycle
00:00:50.01 in organisms from yeast to frogs to humans.
00:00:53.20 Perhaps the best known of these genes is cdc28,
00:00:57.02 which is the master regulatory kinase
00:00:59.14 that controls the progression of cells through the start of the cell cycle.
00:01:03.02 Hartwell is also credited for discovering cell cycle checkpoints,
00:01:06.00 such as the DNA damage checkpoint.
00:01:08.07 Checkpoints are signals that let the cell know if a step in the cell cycle is not completed,
00:01:12.13 and then generate a cell cycle arrest.
00:01:15.03 And Lee, it's really fun to be talking here in Seattle,
00:01:17.06 where you did your groundbreaking work that led to the Nobel Prize.
00:01:20.29 Thanks for doing this.
00:01:22.21 HARTWELL: My pleasure.
00:01:24.06 BIGGINS: So, to get started, Lee, can you tell us what got you started or interested in science?
00:01:28.24 HARTWELL: Oh, going back a long ways?
00:01:32.17 I think the turning point for me was in high school.
00:01:38.00 I hadn't been a very serious student,
00:01:41.29 but I had a great physics teacher,
00:01:47.18 who challenged me with hard problems to take home and work on.
00:01:52.12 And I just look back on that as a turning point
00:01:55.10 where I got interested in science.
00:01:58.04 BIGGINS: And what about biology? When did you get interested in biology?
00:02:02.02 HARTWELL: Not until I was in Caltech.
00:02:05.08 As a sophomore, I took a... I was... I was planning to be a physics major,
00:02:09.14 and I took a course from James Bonner.
00:02:14.05 And it was supposed to be sort of a biology... general biology course.
00:02:21.18 But as I remember it,
00:02:23.25 he spent the entire semester ranting and raving about how great DNA was.
00:02:29.11 So, this was about 1957 or so.
00:02:31.19 So, it was only a few years after the structure of DNA had been discovered.
00:02:35.11 And he was so exciting,
00:02:38.27 I just changed my major on the spot, and became a biologist.
00:02:42.27 BIGGINS: Great. What inspired you to work on the cell cycle?
00:02:46.06 HARTWELL: Well, I... looking back,
00:02:49.18 I think that somewhere I gained the perspective that
00:02:55.14 scientists ask big questions.
00:02:57.29 I suppose that I got that idea at Caltech.
00:03:01.25 But after I'd gotten my graduate PhD,
00:03:06.01 and was deciding where to go for a postdoc,
00:03:12.08 and thinking about what I wanted to work on,
00:03:14.22 I chose cell division.
00:03:16.26 Because, as you say, it's very fundamental to biology.
00:03:19.28 And it seemed like almost nothing was known about it.
00:03:23.17 So, that seemed like an interesting direction to go in.
00:03:28.15 BIGGINS: And do you want to tell us a little bit about the CDC screen?
00:03:33.17 HARTWELL: Well, let me first just mention how we got into yeast.
00:03:38.01 BIGGINS: Yeah, I was gonna ask that question.
00:03:40.03 Everyone asks that question.
00:03:41.14 Why yeast?
00:03:43.06 HARTWELL: Yeah. I wrote a grant to do cell division studies in mammalian cells,
00:03:48.17 as I had been doing.
00:03:50.10 But I knew I didn't wanna do that.
00:03:52.24 And I was really frustrated about what to do.
00:03:57.00 And I had about... a period of about three months
00:04:00.21 where the equipment I'd ordered was on...
00:04:04.10 coming but not arrived yet, when I just had time to think.
00:04:09.13 And I remember talking to one of my colleagues about this, Dan Wolf.
00:04:14.10 He was from Delbrück's group at Caltech.
00:04:18.04 I had worked in Delbrück's group as an undergraduate.
00:04:20.20 And he said, well,
00:04:22.29 why don't you take... look for a simpler organism?
00:04:25.20 And that was the key for me.
00:04:29.14 And I immediately went to the library
00:04:32.03 and started scouring for eukaryotic cells
00:04:36.09 where you could do genetics and cell physiology.
00:04:40.13 And so that's how I came to yeast.
00:04:43.07 And it was really because of a frustration with what I couldn't do with animal cells.
00:04:49.11 BIGGINS: And so that leads me to wonder about looking for temperature-sensitive mutants.
00:04:53.17 Why did you make that decision?
00:04:55.26 HARTWELL: Well, I wanted the essential genes.
00:04:58.25 And I had, as an undergraduate, as I said,
00:05:03.02 worked in Delbrück's group with Bob Edgar.
00:05:05.23 And Bob Edgar did a beautiful piece of work on how
00:05:13.01 a T4 virus is put together -- 150 proteins or so --
00:05:18.29 getting mutations in genes
00:05:22.19 that were part of the structure of the virus.
00:05:26.06 Now, the problem is a mutant
00:05:30.29 that's defective in an essential function for the organism is dead.
00:05:36.08 And so you have to make them conditional,
00:05:38.21 so they'll grow under one condition but not under another.
00:05:42.00 And Bob had used temperature-sensitive mutants.
00:05:45.06 And Horowitz, at Caltech,
00:05:50.26 very early in genetics,
00:05:52.28 had gotten temperature-sensitive mutants in Neurospora.
00:05:55.14 So, I knew about that conditional phenomenon,
00:05:58.18 and so that was the obvious way to go.
00:06:01.08 BIGGINS: Now, why don't you tell us the story
00:06:04.00 about the morphologies and how you...
00:06:06.17 what you were looking for, and how you went about that?
00:06:09.22 HARTWELL: When I moved to the University of Washington,
00:06:13.03 the genetics department,
00:06:15.24 there was an undergraduate in the laboratory there,
00:06:19.01 Brian Reid, who is now at the Fred Hutchinson Cancer Center
00:06:22.16 studying esophageal cancer.
00:06:24.26 And I suggested to Brian that
00:06:29.22 he look at some mutants that formed odd morphologies.
00:06:35.01 And the question was, once you...
00:06:40.07 there was some really interesting work in Paramecium
00:06:43.05 that showed that if you surgically changed the surface of the cell
00:06:46.27 that was inherited.
00:06:51.17 And so that was very interesting.
00:06:54.04 Some kind of structural inheritance on the surface.
00:06:57.22 So, we had mutants that at a high temperature
00:07:00.28 became morphologically abnormal.
00:07:03.09 And I suggested to Brian that he
00:07:05.26 look to see if that was inherited.
00:07:09.12 So, he would shift them to the high temperature,
00:07:11.19 and then shift them back down to the low temperature.
00:07:13.08 And it turned out it wasn't inherited.
00:07:15.13 But then we...
00:07:18.00 he got interested in the question, well,
00:07:19.24 how do they go from abnormal to normal?
00:07:22.17 And so he started taking pictures of them in the microscope.
00:07:26.11 And as I remember it,
00:07:28.25 immediately we realized how much information there was
00:07:32.23 in these photographs about the cell cycle.
00:07:35.22 And started, then, screening all of our mutants
00:07:39.07 for their morphological changes
00:07:42.26 when we shifted the temperature.
00:07:45.06 BIGGINS: So, another question I had is,
00:07:47.15 how did you order the cell cycle using your mutants?
00:07:51.13 HARTWELL: Well,
00:07:55.07 the mutants provided some order
00:07:57.18 because, for example, there were mutants that didn't bud;
00:08:01.20 there were mutants that didn't make DNA,
00:08:05.05 and then everything else stopped;
00:08:06.27 there were mutants that didn't divide the nu...
00:08:09.21 that made DNA, but didn't divide the nucleus;
00:08:12.07 and different steps around the cell cycle.
00:08:14.21 So, in principle, what the mutants told us
00:08:19.15 was that there's basically two cycles.
00:08:22.05 When the cell passes the commitment to division
00:08:28.00 that we called Start,
00:08:30.04 the nuclear cycle is one series of events --
00:08:34.09 the DNA synthesis and the division of the nucleus.
00:08:37.03 And another series of events has to do with the cell surface,
00:08:39.24 which is the formation of the bud,
00:08:42.11 and then eventually the division of the cell wall at division time.
00:08:49.00 And then these two things, these two pathways,
00:08:51.19 have to hook up at the end.
00:08:53.22 So, the the pathways and events...
00:08:57.06 order of events...
00:08:59.11 was largely determined by the phenotypes of the mutants.
00:09:03.17 BIGGINS: Can you explain sort of the two big models
00:09:07.02 you were trying to distinguish between in terms of the regulation of the cell cycle,
00:09:10.07 i.e. the substrate model versus a timing model?
00:09:13.21 HARTWELL: So, when the cells stop in the cell cycle
00:09:18.19 because something hasn't been completed,
00:09:23.26 the question becomes why they stop.
00:09:26.01 So, let's say a cell can't make its DNA.
00:09:32.24 Why does the cell stop
00:09:36.15 with the chromosomes not separated?
00:09:39.14 Why doesn't it just go ahead and try to separate the chromosomes,
00:09:43.12 and get incomplete chromosomes and then die?
00:09:48.01 Why does it stop?
00:09:49.18 How does it know?
00:09:50.23 How does the mitotic spindle know that the DNA is incomplete?
00:09:56.22 So, that was the question.
00:09:58.24 And... and there's sort of two ways to think about this.
00:10:03.20 One is that the DNA is part of the mitotic spindle,
00:10:09.27 and if it's not complete then the spindle just can't function.
00:10:12.28 So, that's sort of the substrate model.
00:10:15.05 The other model is that there are signals in the cell.
00:10:20.14 The cell has a communication system that tells it what's happening,
00:10:24.21 and what hasn't happened.
00:10:26.12 And that when the DNA isn't replicated,
00:10:29.02 a signal is sent to the mitotic spindle
00:10:31.09 that says wait, okay?
00:10:33.22 So, that's the other model.
00:10:36.04 BIGGINS: So, which one did you discover was correct?
00:10:39.06 HARTWELL: So... yeah.
00:10:41.21 So, that was a story
00:10:45.08 that begins with a postdoctoral fellow named Ted Weinert.
00:10:50.01 And Ted wanted to study regulation.
00:10:56.21 And as we were discussing what regulation might mean,
00:11:04.07 I had collected a bunch of radiation-sensitive mutants in the freezer.
00:11:08.04 And so I got 'em out, and Ted looked at 'em.
00:11:10.25 And it turned out that...
00:11:13.14 I think it was just a few days...
00:11:16.13 that he discovered that some of the radiation-sensitive mutants,
00:11:20.23 when you X-ray them,
00:11:22.28 arrest cell cycle.
00:11:25.08 So... and they just stay arrested,
00:11:28.17 whereas the normal cell would arrest, repair, and then go on.
00:11:33.21 So, these mutants were defective in the repair process.
00:11:37.19 But there were other mutants that just didn't stop dividing.
00:11:41.13 They just kept dividing and produced dead colonies.
00:11:45.00 And so we realized that these were defective in the signaling.
00:11:49.19 And that there was a signal that told the cell when there were breaks in DNA.
00:11:53.29 BIGGINS: Right?
00:11:55.23 And you defined this as a checkpoint? H
00:11:57.14 ARTWELL: Yeah.
00:11:58.12 BIGGINS: And do you want to say a little bit more about what you view
00:12:00.14 the definition of a checkpoint as?
00:12:02.28 HARTWELL: Well, we tried to define it relatively clearly,
00:12:08.29 as... as something which is...
00:12:17.15 is needed to arrest division when something goes wrong.
00:12:23.01 BIGGINS: At the time, when you discovered this checkpoint,
00:12:25.18 did you think that there might be other checkpoints for other cellular processes?
00:12:31.23 HARTWELL: We certainly wondered that.
00:12:35.00 And I think over the years,
00:12:41.24 a lot more have been discovered.
00:12:44.12 And so... you know,
00:12:49.07 one of the, I think, really fundamental principles about biology,
00:12:53.05 that fascinates me,
00:12:55.21 is the tremendous accuracy of biological processes.
00:12:59.12 So, yeast cells lose a chromosome
00:13:02.14 about once in 100,000 divisions.
00:13:04.27 That's remarkably precise and reproducible,
00:13:10.13 especially when you watch mitosis
00:13:13.19 and see the chromosomes jiggling around and everything.
00:13:15.24 You wonder how they keep track of them.
00:13:19.09 And I suspect that's true for all kinds of cellular processes,
00:13:23.21 that normally we can't measure the accuracy
00:13:27.11 because it is so accurate.
00:13:29.11 And... and so the extent to which evolution
00:13:36.15 has driven the accuracy of biological processes
00:13:41.03 is really enormous.
00:13:42.26 And it... I think it must mean that,
00:13:45.07 for most things that go on in biology,
00:13:49.04 there's the basic machinery,
00:13:52.22 there are things which repair the basic machinery when it gets in trouble,
00:13:56.29 and there are things that coordinate that repair
00:14:00.20 with everything else that's going on.
00:14:02.28 And we probably only know the tip of the iceberg yet
00:14:07.10 in terms of cell biology.
00:14:09.24 BIGGINS: cdc28 is the most famous, conserved
00:14:14.27 cell cycle molecule, probably,
00:14:17.02 since that was in large part...
00:14:20.18 winning the Nobel Prize with Tim Hunt and Paul Nurse's contributions.
00:14:26.13 What did you think... before we knew what that...
00:14:29.15 what that protein did, did you have a feeling,
00:14:32.18 what you thought that molecule would end up being?
00:14:36.17 HARTWELL: No, I really didn't think in terms of biochemistry.
00:14:40.08 I wasn't interested in biochemistry.
00:14:42.23 It's funny.
00:14:45.02 But I was just much more interested
00:14:48.17 in the order and and regulation of events in the cell cycle.
00:14:54.05 But it's really interesting that before that story broke,
00:15:02.26 Fischer and Krebs, who had gotten the Nobel Prize for protein phosphorylation,
00:15:10.05 came to me and said... and we had a meeting,
00:15:13.21 and they wanted to know whether I thought kinases
00:15:16.08 could play any role in the cell cycle.
00:15:19.03 And I said, well, you know,
00:15:20.27 I just don't know, you know?
00:15:22.27 But it was interesting, because that was actually the right guess.
00:15:26.09 And cdc28 turned out to be a kinase,
00:15:29.04 and their suspicions were correct.
00:15:33.27 BIGGINS: We now know that many of the CDC genes are conserved?
00:15:37.18 Was it a surprise to you how many were conserved,
00:15:41.16 now that we know what they all are?
00:15:43.27 Were more conserved than you thought? Less?
00:15:46.08 HARTWELL: I think, in general,
00:15:50.26 biology has surprised us all in the degree of conservation
00:15:54.16 at the molecular level.
00:15:57.20 And we're even seeing...
00:16:02.19 so, the... so, the answer is yes, for me.
00:16:03.15 It surprised me.
00:16:05.08 I was just hoping that cell division in yeast
00:16:08.07 would be enough like mammalian cells to be a useful model.
00:16:11.21 And only after genes were cloned and sequenced
00:16:16.07 and various things
00:16:19.03 did we find out how conserved they are.
00:16:22.02 I think it's something like 50% of yeast genes
00:16:27.20 can be complemented by human proteins.
00:16:32.07 BIGGINS: I think it's amazing what you are able to learn
00:16:35.02 given how little you could really measure.
00:16:38.27 Now we have so many tools to
00:16:42.09 know what's happening to different structures in the cell.
00:16:45.01 And you were really limited to just looking at budding
00:16:47.11 and the nuclear position.
00:16:51.04 I'm curious if there was, at the time,
00:16:54.20 one extra thing you could have seen
00:16:57.13 or one tool you could have had,
00:16:59.13 what would you have chosen?
00:17:01.01 What would have given you the most information
00:17:03.13 that you couldn't get at that time?
00:17:05.03 HARTWELL: Well, we actually had that experience,
00:17:07.02 in that when we first learned to do photomicroscopy,
00:17:12.04 and could look at the morphology of the cells
00:17:14.11 and pick out those that looked like they might be cell cycle mutants,
00:17:18.02 we didn't know what was going on inside the cell.
00:17:21.26 So, we had to be able to see the nucleus.
00:17:24.25 And at that time, that was very difficult,
00:17:26.29 to stain the cells to see the nucleus.
00:17:29.20 And there was only one guy who could really do it.
00:17:32.06 His name was Robineaux.
00:17:33.23 And it turned out there was a... some international meeting,
00:17:38.05 and he was coming to Seattle.
00:17:40.07 And I got in touch with him and asked him if he'd come to the lab a day ahead
00:17:43.24 and show us how to see nuclei and stain them.
00:17:46.20 And he did.
00:17:48.12 And so, then we were able to verify
00:17:50.29 that the mutants that looked like cell division mutants
00:17:52.23 were actually defective in nuclear division as well.
00:17:58.28 BIGGINS: What was so hard about getting the nuclear staining to work?
00:18:02.09 HARTWELL: It was just tricky
00:18:06.26 because the amount of DNA is so small in the yeast.
00:18:12.00 And you're trying to stain the DNA.
00:18:15.07 And there's 100 times as much RNA as there is DNA.
00:18:19.05 And so it's hard to get the staining conditions just right
00:18:24.29 so you can see that DNA.
00:18:27.01 BIGGINS: So, if there could have been one more structure you could see,
00:18:29.13 what would you have chosen at the time?
00:18:31.20 HARTWELL: Oh, well that's an interesting story too,
00:18:33.18 now that you bring it up.
00:18:36.06 My colleague, Breck Byers,
00:18:40.21 was in the genetics department.
00:18:44.13 And I went to him at one time
00:18:46.07 and said, well, we've got all these mutants.
00:18:48.09 We should, you know, look at their detailed three-dimensional structure.
00:18:52.25 Do thin sections and reconstruct the cell and everything.
00:18:55.22 And he looked at me, and he said,
00:18:57.17 there's only one thing of interest.
00:18:59.05 And that's the spindle.
00:19:01.25 And I said, okay.
00:19:04.00 So, he started looking at the spindle on electron microscopy,
00:19:07.02 and found that some mutants were defective in making the pole duplicate,
00:19:10.27 and some others were defective in forming the spindle.
00:19:13.23 BIGGINS: You mentioned earlier, and in your Nobel lecture as well,
00:19:16.27 that when you started your lab
00:19:18.24 you didn't really have a plan of what you were gonna work on.
00:19:20.26 And this is so different than how things work today,
00:19:23.12 where people have to have
00:19:26.11 brilliant, well-developed research proposals to get a job,
00:19:30.10 and they know exactly what they're gonna work on.
00:19:33.04 And I'm just curious if you think this change
00:19:35.13 actually hinders creativity,
00:19:37.28 or if this is a good change.
00:19:41.03 HARTWELL: I think the present system does hinder creativity.
00:19:44.06 I think that opening up new fields
00:19:53.17 requires a long incubation period.
00:19:59.24 I just recently experienced it myself,
00:20:01.27 because the last decade I've been interested in education.
00:20:08.01 And it's taken me ten years
00:20:10.28 to sort of figure out what I wanna do there.
00:20:13.24 And the current system
00:20:16.23 just doesn't allow students
00:20:20.05 to go beyond their immediate experience.
00:20:22.24 So, you can keep extending
00:20:25.24 what's already going on and what's interesting and productive,
00:20:28.11 but to really start something new
00:20:31.06 is very hard to do in the current environment.
00:20:35.04 Established labs can do it
00:20:37.19 if they have a little bit of leeway to try something new
00:20:40.25 for a year or something.
00:20:42.11 But... that and the whole system of,
00:20:45.21 you know, counting publications, and impact factors, and grants,
00:20:51.18 and all the things.
00:20:53.02 I think the best system for sponsoring science
00:20:55.04 was the old system at the MRC, in England,
00:20:58.19 which doesn't exist anymore.
00:21:00.03 The MRC exists but the system doesn't exist.
00:21:02.21 They've adopted the American system unfortunately.
00:21:05.22 But their scientists used to be given
00:21:09.11 a budget, yearly,
00:21:12.29 that would support themselves and a technician
00:21:14.24 and maybe a graduate student or two.
00:21:17.05 And that was guaranteed,
00:21:19.15 regardless of whether they produced anything or not.
00:21:23.19 And then, they could give grants on top of that.
00:21:26.23 But I think that basic level of stability
00:21:30.07 allowed some people to do very creative things
00:21:32.22 and take five years to do it before they published.

This Talk

Speakers: Leland Hartwell, Sue Biggins

Audience:

Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: June 2018

Talk Overview

Dr. Leland Hartwell started his scientific career studying a fundamental question in biology: how do cells know that they have everything they need in order to divide. By studying the morphology of temperature sensitive mutants in yeast, Hartwell identified many of the key regulators of the cell cycle. In this conversation, Hartwell talks to Dr. Sue Biggins about his Nobel Prize winning discoveries and the experiments that led to his seminal findings.

Speaker Bio

Leland Hartwell

Dr. Leland Hartwell is a former president and director of the Fred Hutchinson Cancer Research Center in Seattle, Washington. He earned his bachelor’s of Science from the California Institute of Technology in 1961. Hartwell continued his graduate education at Massachusetts Institute of Technology in Boston under the mentorship of Dr. Boris Magasanik. After his graduation… Continue Reading

Sue Biggins

Dr. Sue Biggins studied biology as an undergraduate at Stanford University and initially thought she would apply to medical school after receiving her degree. However, after a summer working in a research lab, she changed her mind and decided to apply to graduate school. Biggins received her PhD in molecular biology from Princeton and was… Continue Reading