Gary Borisy & Edwin Taylor
Questions
- Did Gary Borisy and Edwin Taylor discover the mitotic spindle?
In the video, Dr. Taylor and Dr. Borisy tell the story of the discovery of tubulin. In the paper, however, Dr. Taylor and Dr. Borisy presented their research goals slightly differently. - Describe the difference between the description of the discovery in the talk and in the research papers.
- In the talk, Dr. Taylor says that the first step to finding tubulin was developing an assay.
Describe the assay his team was able to develop. - After developing the assay, Dr. Borisy said they assayed “different tissues varying in mitotic index” and were surprised to find that brain tissues displayed high colchicine binding.
- Describe the evidence supporting this statement in the paper. Why were the authors surprised to find that brain tissues displayed high colchicine binding?
- Dr. Borisy explains that the first response to these results from his peers was that “it was an artifact.” Explain what Dr. Borisy’s colleagues meant by that, and what was Dr. Borisy and Dr. Taylor’s own interpretation of the data.
- How did the authors show that the presence of colchicine-binding protein was NOT, in fact, an artifact?
- Why didn’t the authors name the colchicine-binding protein ‘tubulin’?
- In the talk, Dr. Borisy says: “sometimes when you are doing work, you encounter apparently contradictory results. You encounter a paradox. Don’t sweep it under the carpet. Look at it more deeply because sometimes the deepest understanding comes from resolving those paradoxes.” (11:45).
Describe a situation where Dr. Borisy and Dr. Taylor decided not to “sweep results under the carpet”
Answers
- No. Video (0:16) “ET: The mitotic spindle, the whole thing, the proteins and the chromosomes, was isolated in 1952 by Mazia and Dan.”
- In the talk, Dr. Taylor explains that he and his team decided to use colchicine as a tool to isolate microtubule proteins after a decade of failed experiments:Video (0:32) “And there was a period of perhaps ten years in which many of us came, isolated spindles, tried to isolate from them the proteins, and we failed because the structure was too complex. There were too many proteins. And we just didn’t have an assay. We knew that there was a fibrous protein, that is to say a filament, because from the work of Shinya Inoue, he had shown that the spindle is birefringent. And therefore it must be, to some extent, a parallel array of filamentous proteins. And we wanted to find out what that filament was.”
Video (1:15) “So we tried and failed both on sea urchin eggs and growing cells in culture and isolating the proteins and nothing came out of it. There were too many things. So we had the idea that maybe we could use a drug called colchicine. A whole book had been written on colchicine, and it was known to be specific, apparently specific, in blocking cell division, although there were controversies as to what this really meant. Maybe it didn’t block cell division, maybe it stimulated cell division. But everything suggested that this was a compound that we might be able to use if a long shot suggestion might be correct that this specific molecule was binding in some way, or interfering in some way, with the action of the formation of the spindle fibers.”
In the paper, Drs. Borisy and Taylor write that their research goals were to elucidate the mechanism of action of colchicine, an inhibitor of cell division:
p.525: “The present report’ is concerned with studies on the nature of colchicine binding. The primary objectives were to (a) demonstrate a colchicine- macromolecule complex in colchicine-treated cells, (b) study conditions for the colchicine-binding reaction in vitro, (c) determine whether the bound colchicine is chemically unchanged, and (d) survey colchicine-binding activity in various cell types and model systems. The results are consistent with the hypothesis that the binding site is the subunit protein of microtubules.“
In the Shelanski research paper (https://www.ncbi.nlm.nih.gov/pubmed/6035644), which was published in the same issue of the Journal of Biological Chemistry, Dr. Shelanski and Dr. Taylor write that their research goals were to isolate the subunit of the microtubules, and to demonstrate that it was the colchicine-binding protein.
Shelanski et al., p. 549: “It was shown previously (4, 5) that colchicine forms a complex with a protein present in the isolated mitotic apparatus (MA), cilia, and brain tissue, and it was suggested that the binding protein is the subunit of microtubules. The object of the present investigation was to obtain a protein from sea-urchin sperm tails which could be identified as the subunit of microtubules and to demonstrate that this protein is responsible for the colchicine-binding activity. Once this point has been established, the isolation of the protein in large amounts for physical and chemical studies can be carried out with a more easily available source of material.”
- The assay is described in the paper as follows:
- Incubation of cells in colchicine
- Removing colchicine and harvesting cells
- Homogenization and fractionation of cell extracts
- Gel filtration
p. 527: “Intact Mammalian Cells Assay – 1. Suspension cultures of KB cells were incubated for 12-18 hr with colchicine-3 H at concentrations of 10-6-10 – 7 M. (…); 2. Cells were harvested and washed three times in phosphate-buffered saline by centrifugation to remove free colchicine. (…); 3. The cells were homogenized in 0.24 M sucrose (SMT solution) and the homogenate was fractionated by differential centrifugation. (…); 4. In order that the amount of colchicine could be determined quantitatively, aliquots of the supernatant were subjected to gel filtration on G-100 Sephadex columns.”
p. 528 – “Mammalian Cell Extracts Assay – 1. Cell homogenates prepared in 0.24 M sucrose (SMT solution) were incubated with 2.5 X 10-6 M colchicine for 1 hr at 37°C. (…); 2. Material was centrifuged at low speed to remove nuclei, and at 100,000 g for 2 hr to sediment mitochondria and microsomes. The supernatant showed high binding activity as assayed by gel filtration (30,000-60,000 cpm per milligram protein) which was of the same magnitude as that obtained with supernatants prepared from cells labeled in vivo. (…); 3. Zone Sedimentation: Applying an aliquot of the bound radioactivity fractions from a Sephadex column to a 5-20% sucrose density-gradient.”
- p. 530: “Colchicine Binding by a Variety of Cells and Tissues – A survey of colchicine binding activities of various cells, tissues, and model systems was made to determine the distribution of the binding sites and to test for a correlation with mitotic activity or other parameters. (…) Among six tissues with the exception of brain, those with a higher mitotic rate, e.g. spleen and bone marrow, showed higher binding activity but considerably less than cultures of a mammalian carcinoma (KB or HeLa) or sea urchin eggs. However, the high activity of brain established that binding is not confined to a protein found only in dividing cells. Although some division may occur in glial cells, mitotic processes can be ruled out since the binding was equally high in extracts of white and gray matter. In addition a sample of lyophilized axoplasm of the giant nerve of the squid showed an even higher binding activity than whole mammalian brain.“p. 532: “Colchicine-binding activity was found to correlate strictly with neither mitotic activity nor motility, but rather with those sources abundant in microtubules. Microtubules form the framework of the mitotic spindle (21, 22); they make up the 9 + 2 array of filaments in cilia and spermtails (23); and they are present in large numbers in neuronal processes (24).“
- The division rate of neurons is very low, and therefore its “mitotic index” is expected to be very low. However, the authors found that neurons displayed high colchicine binding, which hinted a role for microtubules that would go beyond cell division.Video (05:35) – Dr. Taylor: “We were testing to see if it was a distribution of binding related to mitosis. And the brain was the control, and that changed our thinking because clearly this was a widespread protein, which was in mitosis, but was all over the place.”
Video (05:59) – Dr. Borisy: “So the idea was to assay different tissues varying in mitotic index, and the expectation was those tissues high in mitotic index would have high colchicine binding, brain very low in the mitotic index should be very low, and then we got this astounding result that brain was very high.”
- An artifact refers to an “undesired alteration in data, introduced by a technique and/or technology” (Source: Wkipedia.org, http://en.wikipedia.org/wiki/Artifact_(error)) By this, Dr. Borisy’s colleagues suggested that colchicine was not binding to the protein they had identified in this study but instead, that it was binding to the cellular membranes.Video (06:19): “And now the students at that time would meet around Botany Pond and talk about results, and I remember very distinctly all, you know, my fellow students were saying it’s clearly an artifact of this drug partitioning into the membranes of nerve cells because they are so rich in membrane structure for all the neuronal processes. So it’s an artifact.”
- To rule out the possibility that colchicine was partitioning to the nerve cell membranes, the authors decided to use a neuronal model that did not have cell membranes – the squid axoplasm.Video (06:55) – Dr. Borisy: “One further test was to take advantage of a previous laboratory association that you had, as you recall, when you had your adventure in Chile and getting squid axoplasm from the marine station in Chile, and so you arranged to get some of that shipped up to us, and we tested it. And we thought that if there was something important in brain, it might be even more abundant in the pure axoplasm, and in fact it was. And this would also provide a test for whether or not the drug was partitioning into the membranes because the membrane wouldn’t be present in the axoplasm. And here this positive result gave us reassurance that maybe it wasn’t an artifact, this binding in brain, but it had a deeper significance.”
- They didn’t think it sounded right. The name tubulin was later attributed to the protein by Hideo Morhi.Video (09:55): “GB: Do you recall the discussion of the naming of this protein?
ET: Yes, we got scooped on that. We should have named the protein. GB: We should have named the protein, but do you recall that, I think it was in your office, and we were talking about what we should name the protein. I recall a discussion where we said, “Well, it’s a microtubule so the subunit could be called tubulin.” With -in being the common suffix for a protein, but it didn’t sound very good to our ears, and we reverted to the name colchicine binding protein. Hideo Morhi later felt that clearly the protein needed a name and so gave the protein the obvious name. But for several years we, in the literature, referred to it only as the colchicine binding protein.”
- When Borisy et al. found the surprising high colchicine-binding results in brain tissues, which they were using as a control for low mitotic index, they didn’t discount it as artifact. Instead, they decided to look closely at colchicine-binding in squid axoplasm, which corresponds to the content of axone cytoplasm without its membranes, to demonstrate that colchicine was in fact binding to a structure contained in the cytoplasm of neuronal cells.
Video (06:55) – Dr. Borisy: “One further test was to take advantage of a previous laboratory association that you had, as you recall, when you had your adventure in Chile and getting squid axoplasm from the marine station in Chile, and so you arranged to get some of that shipped up to us, and we tested it. And we thought that if there was something important in brain, it might be even more abundant in the pure axoplasm, and in fact it was. And this would also provide a test for whether or not the drug was partitioning into the membranes because the membrane wouldn’t be present in the axoplasm. And here this positive result gave us reassurance that maybe it wasn’t an artifact, this binding in brain, but it had a deeper significance.” [00:07:46]
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