The Discovery of Tubulin
Transcript of Part 1: The Discovery of Tubulin
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....