Discovery Talk: The Discovery of Tubulin

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00:00:16;01 ET: The mitotic spindle, the whole thing, the proteins and the chromosomes,
00:00:20;01 was isolated in 1952 by Mazia and Dan.
00:00:24;21 And this brought forward the hope that we would be able
00:00:28;00 to understand what this was made of and maybe how it works.
00:00:32;06 And there was a period of perhaps ten years in which many of us came,
00:00:37;12 isolated spindles, tried to isolate from them the proteins,
00:00:41;01 and we failed because the structure was too complex. There were too many proteins.
00:00:45;19 And we just didn't have an assay.
00:00:47;17 We knew that there was a fibrous protein, that is to say a filament,
00:00:53;02 because from the work of Shinye Inoue, he had shown that the spindle is birefringent.
00:00:58;05 And therefore it must be, to some extent, a parallel array of filamentous proteins.
00:01:04;20 And we wanted to find out what that filament was.
00:01:07;10 And Shinye Inoue will also give a talk on this subject,
00:01:11;15 which I suggest strongly that you listen to as well.
00:01:15;09 So we tried and failed both on sea urchin eggs and growing cells in culture
00:01:23;24 and isolating the proteins and nothing came out of it. There were too many things.
00:01:30;09 So we had the idea that maybe we could use a drug called colchicine.
00:01:35;23 A whole book had been written on colchicine, and it was known to be specific,
00:01:40;03 apparently specific, in blocking cell division,
00:01:44;07 although there were controversies as to what this really meant.
00:01:47;10 Maybe it didn't block cell division, maybe it stimulated cell division.
00:01:52;00 But everything suggested that this was a compound
00:01:55;03 that we might be able to use if a long shot suggestion
00:02:01;17 might be correct that this specific molecule
00:02:05;29 was binding in some way, or interfering in some way,
00:02:09;29 with the action of the formation of the spindle fibers.
00:02:13;12 And so we set out to synthesize tritium labeled colchicine
00:02:19;19 because this would allow us to see if it was binding to something in the cell.
00:02:26;01 This could be done by a method which was non specific, but easily done,
00:02:29;22 and we purified the tritium labeled colchicine.
00:02:32;15 And indeed showed that it was taken up by cells in tissue culture,
00:02:36;03 as well as into sea urchins eggs, which we had some sea urchins in the lab too,
00:02:40;26 and that it was concentrated by the cell,
00:02:44;16 which told us that it was binding to some component that was there
00:02:48;14 in relatively large amounts.
00:02:50;13 It couldn't be an enzyme. It had to be, possibly, a structural protein.
00:02:56;00 So it seemed like this was worth trying.
00:02:58;28 It seemed, on the other hand, that the chances of this project succeeding were very, very small
00:03:03;26 because it was a long shot idea, and I didn't like at first to give it to a graduate student,
00:03:09;19 to work on because he could have wasted two or three years and got nowhere.
00:03:13;09 But anyway, Gary Borisy joined the lab around that time
00:03:18;08 and well, what did you want to do, Gary, when you got to the lab?
00:03:23;00 GB: I mean before getting on the project I was an undergrad at the University of Chicago
00:03:29;00 and Frank Child was one of my professors
00:03:31;27 and he was teaching biology and showed me how cells divide,
00:03:37;05 actually we saw living cells divide and I asked him rather naively,
00:03:41;18 like all undergraduates are, I suppose, well how does it work?
00:03:45;29 What's the mechanism?
00:03:47;08 And it was clear that there was no molecular understanding
00:03:50;29 of the mechanism. And this is before I learned about your lab.
00:03:57;02 I thought this is a fantastic problem to be studying,
00:04:01;01 a problem that is worth spending one's research career on.
00:04:06;01 And then after graduating and looking at graduate schools
00:04:10;03 I learned about your work with colchicine,
00:04:14;08 and I thought, "Oh my gosh, this is the molecular entree to the problem of mitosis."
00:04:19;27 So that is why I wanted to join the lab.
00:04:22;06 ET: So then what we had to do was to get an assay,
00:04:25;28 and that was to get something that bound to the protein,
00:04:28;29 we could spin it out, separate it on a column,
00:04:30;17 later put it onto DEAE filter paper
00:04:35;06 and then what we could do would be to make an extract from a cell,
00:04:39;25 spin off the particles, look at the protein,
00:04:43;23 and say well how much colchicine is bound.
00:04:47;00 And then we could do what biochemists do,
00:04:49;16 we fractionated into different test tubes,
00:04:52;12 and tested each tube to see how much colchicine binding activity,
00:04:56;12 and then we could go, because that was whole issue.
00:05:02;01 We were trying to isolate the railroad tracks,
00:05:03;08 and there was no assay for railroad tracks.
00:05:06;02 It is not an enzyme, so the whole trick here
00:05:09;11 was to find an assay. And it was a long shot bet, but it turned out
00:05:13;20 as the work went on and Gary, and also other people, Mike Shelanski, Dick Weisenberg,
00:05:21;22 developed the approaches
00:05:23;26 and then once we had figured out that it wasn't just in mitosis, but it was present all over,
00:05:30;14 and particularly in brain, and that was just, as we said,
00:05:35;03 we were testing to see if it was a distribution of binding related to mitosis.
00:05:41;11 And the brain was the control, and that changed our thinking
00:05:45;13 because clearly this was a widespread protein,
00:05:47;26 which was in mitosis, but was all over the place.
00:05:50;17 GB: Well now we know that it's a widespread protein,
00:05:55;20 but at that time when we got the brain result it was very puzzling.
00:05:59;01 ET: It was.
00:05:59;23 GB: Very puzzling. So the idea was to assay different tissues varying in mitotic index,
00:06:07;24 and the expectation was those tissues high in mitotic index
00:06:10;29 would have high colchicine binding,
00:06:13;04 brain very low in the mitotic index should be very low,
00:06:16;25 and then we got this astounding result that brain was very high.
00:06:19;16 And now the students at that time would meet around Botany Pond and talk about results,
00:06:25;08 and I remember very distinctly all, you know, my fellow students
00:06:30;03 were saying it's clearly an artifact
00:06:32;01 of this drug partitioning into the membranes of nerve cells
00:06:37;20 because they are so rich in membrane structure for all the neuronal processes.
00:06:41;25 So it's an artifact.
00:06:43;15 So we didn't immediately conclude from the brain result
00:06:47;12 that this was telling us something about the
00:06:49;07 widespread nature of the colchicine binding protein, tubulin.
00:06:54;05 We had to do further tests.
00:06:55;24 One further test was to take advantage of a previous laboratory association
00:07:02;15 that you had, as you recall, when you had your adventure in Chile
00:07:07;17 and getting squid axoplasm from the marine station in Chile,
00:07:12;00 and so you arranged to get some of that shipped up to us, and we tested it.
00:07:16;16 And we thought that if there was something important in brain,
00:07:20;20 it might be even more abundant in the pure axoplasm,
00:07:25;06 and in fact it was.
00:07:26;09 And this would also provide a test for whether or not the drug was partitioning into the membranes
00:07:32;13 because the membrane wouldn't be present in the axoplasm.
00:07:35;19 And here this positive result gave us reassurance that
00:07:41;09 maybe it wasn't an artifact, this binding in brain, but it had a deeper significance.
00:07:46;16 And it was around that time as I recall,
00:07:49;10 that I came out to Woods Hole, or around that time.
00:07:53;12 Maybe we don't have the sequence right.
00:07:55;05 But I came out to Woods Hole to learn how to isolate spindles
00:07:59;28 and extract protein from spindles,
00:08:01;28 and we also learned how to isolate sperm tails and cilia and flagella
00:08:07;03 because those systems also have microtubules,
00:08:10;23 and the word microtubules had only recently been coined
00:08:13;25 because of the introduction of a new fixative that preserved them.
00:08:17;02 Previously they hadn't been preserved.
00:08:18;18 So I learned how to isolate mitotic spindles here
00:08:24;21 and extract the protein in a more gentle way
00:08:27;09 than had been done previously
00:08:28;20 and found that we could identify the colchicine binding protein
00:08:33;03 with the same molecular properties.
00:08:34;25 We used sedimentation at that time to identify how rapidly it moved in a centrifugal field,
00:08:41;15 so it had the same properties as the colchicine binding protein from cells and from brain.
00:08:47;11 And so then we felt that there was something real here.
00:08:51;22 We had this same molecule from these different sources,
00:08:53;16 and we asked what's the structural basis? Is there a structural basis for this?
00:08:59;13 And the structure that we saw that was in common among all of these sources was the microtubule.
00:09:04;29 And that's when we made that suggestion.
00:09:08;20 ET: Yeah, because we hadn't been able to do the crucial experiment.
00:09:11;29 The crucial experiment was to take this purified protein
00:09:15;02 and turn it into microtubules. And we struggled.
00:09:18;02 GB: And we struggled with that.
00:09:20;03 ET: And I think we sometimes got some polymerization,
00:09:22;16 but we never really were able to quantitate it.
00:09:24;27 I am not sure, but I think you made-we had some microscopy,electron microscopy.
00:09:30;04 We made rings, I think.
00:09:32;02 GB: We made rings. There was also this confounding protein that we extracted from the spindles.
00:09:40;03 This very large protein that had a cylindrical shape
00:09:44;05 that at one time we thought could be the subunit of microtubules,
00:09:48;08 but it turned out to be yolk protein.
00:09:50;07 ET: Yeah, oh yes.
00:09:52;29 GB: That was another red herring and blind alley in this study.
00:09:55;11 Do you recall the discussion of the naming of this protein?
00:10:01;03 ET: Yes, we got scooped on that. We should have named the protein.
00:10:04;12 GB: We should have named the protein, but do you recall that,
00:10:06;04 I think it was in your office, and we were talking about what we should name the protein.
00:10:10;19 I recall a discussion where we said, "Well, it's a microtubule
00:10:15;26 so the subunit could be called tubulin." With -in being the common suffix for a protein,
00:10:23;01 but it didn't sound very good to our ears,
00:10:26;04 and we reverted to the name colchicine binding protein.
00:10:31;04 Morhi, Hideo Morhi later felt that clearly the protein needed a name
00:10:37;19 and so gave the protein the obvious name.
00:10:40;02 But for several years we in the literature
00:10:43;25 referred to it only as the colchicine binding protein.
00:10:46;29 What do we take as messages that we might give to the students watching this?
00:10:54;12 ET: Well, you gave and I gave the same message: choose an important problem,
00:10:58;05 and when you are just starting out choose what you think is the most important problem.
00:11:02;20 I thought that this was the most important, and so did Gary when he joined the lab,
00:11:06;25 and that is the first important thing.
00:11:09;28 Don't work on a trivial problem.
00:11:11;19 Do something where if you succeed, you have done something really good.
00:11:16;11 And don't be confined in your thinking because we thought we were doing one thing,
00:11:22;17 isolating something specific to mitosis, because of our thinking, which was, well colchicine
00:11:29;14 is specifically blocking mitosis and therefore it is probably
00:11:33;06 some protein that's specific. And what we found as we went on,
00:11:37;15 we were completely wrong. It turned out to be not only general,
00:11:41;16 but one of the most important fibrous proteins in the cell.
00:11:45;19 GB: So I would add to that the observation that sometimes when you are doing work
00:11:53;03 you encounter apparently contradictory results.
00:11:57;15 You encounter a paradox. Don't sweep it under the carpet.
00:12:00;11 Look at it more deeply because
00:12:02;29 sometimes the deepest understanding comes from resolving those paradoxes.
00:12:07;23 Here we have Borisy/Taylor, Take 1.
00:12:16;11 ET: Drop everything we are going to work on the brain.
00:12:18;29 That's how I remember it, and then you, and well, Mike...
00:12:26;23 GB: I think with, ok, see, so this is interesting.
00:12:28;16 We may need to compare some notes and check some things for accuracy.
00:12:33;18 My recollection is that the first hints of binding in brain
00:12:40;29 actually came before I came to Woods Hole.
00:12:44;07 And part of the motivation for coming to Woods Hole was to look at isolation of the spindle again to
00:12:51;18 try to resolve this paradoxical result with the brain tissue.
00:12:55;04 GB: And then... ET: OK. GB: So we can check that, of course,
00:13:00;08 if we just look at our old papers and notes.
00:13:05;06 ET: We no longer have the notebooks.
00:13:07;17 GB: So I thought that actually came first, but maybe it doesn't matter.
00:13:14;15 Borisy/Taylor, Take 3.
00:13:16;14 GB: I recall having to beg you to get onto the project
00:13:20;20 because you said that it was not fit for a graduate student. It was too risky.
00:13:27;04 ET: Yeah, well that is worth bringing up.
00:13:28;17 GB: Do you remember that?
00:13:29;18 ET: I may have said that, yes...
00:13:32;25 GB: Also, I had to beg to go to Woods Hole,
00:13:35;09 which I think you discouraged strongly because it would be, you know...
00:13:40;20 ET: Yeah, letting your graduate student go away is..
00:13:42;07 GB: You don't want to let the graduate student leave the lab. It is sort of a fundamental principle.
00:13:47;15 Borisy/Taylor, Take 42.
00:13:49;16 GB: You decided at some point, and I guess I never asked you this.
00:13:53;15 You decided at some point that it was worth
00:13:58;14 investigating the mechanism of action of that drug.
00:14:01;04 What made you... what led you to that conclusion?
00:14:05;29 ET: Yeah, well, gee, you should have taped this. This is better than what you are going to get.
00:14:10;22 I have been.
00:14:11;09 GB: You have been?
00:14:12;26 Absolutely. I tape everything.
00:14:15;16 ET: Right well....

This Talk

Speakers: Edwin Taylor, Gary Borisy

Audience:

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

Recorded: November 2011

Educator Resources for this Talk

Talk Overview

More than 40 years after Gary Borisy was a graduate student in Ed Taylor’s lab, they reminisce about the events that led to the discovery of tubulin, the protein building block of microtubules. Entertainingly, their recollections don’t always match.