Discovery Talk: Developing GFP as a Biological Marker

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00:00:17.15 Hello, I am Martin Chalfie. I am in the department of
00:00:19.14 Biological Sciences at Columbia University.
00:00:22.08 Green fluorescent protein and other fluorescent proteins
00:00:24.27 are now used quite commonly in biology,
00:00:29.11 particularly cell biology. What I would like to do
00:00:31.29 in the next few minutes is tell you a little bit about
00:00:34.27 my lab's involvement in the origin of using GFP as a biological marker.
00:00:40.11 I did not start out being interested in developing a new biological marker.
00:00:45.28 Actually, my lab has been interested for many years in mechanosensation,
00:00:50.13 particularly trying to understand the genes
00:00:52.16 that are needed for touch sensitivity.
00:00:54.28 But in the late 1980s, my lab had cloned several genes
00:01:01.10 that were needed for touch sensitivity in the nematode, Caenorhabditis elegans
00:01:05.13 and we wanted to know where those genes were being expressed.
00:01:09.17 And particularly, we wanted to know
00:01:11.13 which of our cells were, in the end, touch sensing cells.
00:01:16.05 At this time, there were several methods that could
00:01:18.25 and did help us to answer these questions.
00:01:21.14 These included antibody staining,
00:01:25.17 markers such as beta-galactosidase,
00:01:28.10 in situ hybridization. All of these could answer the question:
00:01:33.22 what cells activated what particular gene?
00:01:37.01 But there were problems with these methods.
00:01:39.27 First, they actually required a fair amount of work. We had to prepare
00:01:44.17 the tissues, and that meant killing them, fixing them, permeabilizing them so that
00:01:49.13 we could get the antibody or the substrate for beta-galactosidase,
00:01:53.22 or the probe for in situ hybridization into the tissue.
00:01:56.24 But the main problem I think was the fact that although we could tell
00:02:02.02 at the moment we prepared the sample that genes were being expressed there,
00:02:07.02 we couldn't look at more than just this static picture.
00:02:11.15 If we wanted to look over time, we'd have to do preparations
00:02:14.24 for each time point. Then on April 25th, 1989,
00:02:19.07 I heard a seminar that really changed my life.
00:02:21.14 It was a talk being given by Paul Brehm, who at the time,
00:02:25.06 was a neurobiologist at Tufts University.
00:02:28.06 And in the introduction of his talk, he started talking
00:02:32.02 about the work of this man, Osamu Shimomura,
00:02:33.16 and the work he had done in isolating proteins
00:02:37.29 from the jellyfish, Aequorea victoria.
00:02:40.14 In the early 1960s, Shimomura had discovered
00:02:46.00 the protein aequorin, which was a bioluminescent protein.
00:02:49.06 It produced light.
00:02:50.23 What he found was that when aequorin and calcium were together,
00:02:56.03 they would produce light, and this was the problem he was trying to address.
00:02:59.27 But he had one slight difficulty, and that was that the light
00:03:05.04 that was produced by this reaction
00:03:06.21 was blue. The jellyfish, however, gave off a light that was green.
00:03:13.05 And he knew that there had to be something else.
00:03:15.18 So he went back to his protein fractions to look at
00:03:16.23 what was there, and he found that there was another protein,
00:03:21.11 that when added to aequorin and calcium, now gave green light.
00:03:26.26 And this protein, he called the green protein.
00:03:29.10 We now call it GFP or green fluorescent protein.
00:03:31.24 And because I was working on a transparent animal,
00:03:37.00 Caenorhabditis elegans, I was looking at gene expression,
00:03:41.20 I suddenly realized that this protein, GFP,
00:03:45.10 would make a terrific marker for our experiments.
00:03:49.00 And I spent the whole rest of the seminar fantasizing about
00:03:52.11 this work. I actually don't remember what the rest of the seminar was about.
00:03:56.14 The next day, I got in touch with this man, Douglas Prasher,
00:04:00.08 and found out that he was in the process
00:04:03.17 of cloning the cDNA for GFP from the jellyfish.
00:04:07.20 We had a wonderful conversation, culminating in a decision
00:04:11.26 to collaborate to see whether GFP could be used in other organisms.
00:04:16.03 A few years later, I was lucky enough to get a very talented graduate student
00:04:21.03 into my lab, Ghia Euskirchen,
00:04:24.18 who came to the lab having just finished a Master's degree in chemical engineering
00:04:29.04 working on fluorescence. She came into the lab,
00:04:31.21 to do a rotation, and this was the project that she was given.
00:04:34.23 We got back in touch with Douglas who had cloned the gene by this time;
00:04:39.07 he sent it to us, and Ghia proceeded to see whether this would work in, at first, E. coli.
00:04:46.13 When she did these experiments, there was hanging over us a problem.
00:04:51.11 And that problem was that we weren't really sure that it was going to work
00:04:55.22 because of what was known about the GFP molecule.
00:04:58.12 By this time investigators had found that GFP had a rather unusual
00:05:03.19 post-translational modification. The peptide backbone in GFP
00:05:10.04 cyclized, and the making of this five-membered ring was real mystery.
00:05:16.03 People speculated that it might take one, two, or maybe even more
00:05:19.29 converting enzymes to make the mature protein from the translated product.
00:05:26.00 So it wasn't a sure thing that this was going to work.
00:05:29.07 Nonetheless, Ghia did try the experiment,
00:05:32.14 and to our great joy and excitement, it worked very nicely.
00:05:36.10 This is a page from her lab notebook that day,
00:05:39.19 approximately one month after she entered graduate school
00:05:43.05 where she found strongly fluorescent E. coli.
00:05:46.10 and took this picture. She was also lucky in another sense, and that is how
00:05:50.22 we did the particular experiment.
00:05:51.25 Douglas's clone had a cDNA from the jellyfish that was really the coding sequence
00:06:03.01 which I have diagrammed here in green,
00:06:04.24 and non-coding sequence, which is in red.
00:06:08.12 He had gotten this as an EcoRI restriction fragment.
00:06:13.13 Now we could have simply taken that restriction fragment.
00:06:17.18 and used that in our experiments, but I decided
00:06:20.09 that I didn't want to get extra stuff,
00:06:22.12 and that it would be better to use PCR to amplify
00:06:25.15 just the coding region. I wasn't sure what the rest was going to be,
00:06:31.01 but this has turned out to be a very lucky choice,
00:06:33.25 because it turns out that at least three other labs that I know of
00:06:37.10 tried the experiment, but used the EcoRI fragment.
00:06:41.01 And when they did that, they never got any fluorescence,
00:06:44.08 so there's something in these extra sequences
00:06:47.00 that seemed to interfere with the production of a fluorescent protein.
00:06:49.28 We didn't do that. We didn't have that problem.
00:06:52.29 Within one month we knew that this was going to work.
00:06:55.09 We were very excited, and it now meant that we had to do...
00:06:58.18 we were going to go off and do many other things.
00:07:01.13 We put this into worms, and we decided that this was time now
00:07:05.06 to publish the material, and we had a little bit of difficulty with publishing the paper.
00:07:09.25 So we sent the paper in to be reviewed
00:07:13.18 in Science, and the first thing we found was that the editors
00:07:17.11 were not going to send it out to reviewers
00:07:20.20 for consideration because they did not like the title.
00:07:24.14 The original title was "Green Fluorescent Protein: A New Marker for Gene Expression"
00:07:28.13 and the editors told me that all the papers in the journal were new
00:07:32.12 and novel and so we couldn't use new in the title and would I change the title.
00:07:36.25 I was a little bit miffed at this, so in retaliation,
00:07:42.05 the title I gave them was considerably longer,
00:07:45.02 "The Aequorea victoria Green Fluorescent Protein
00:07:47.23 Needs No Exogenously-Added Component to Produce
00:07:50.15 A Fluorescent Product in Prokaryotic and Eukaryotic Cells".
00:07:54.08 This is essentially the entire paper.
00:07:57.11 The paper got reviewed; the reviewers liked it.
00:08:00.15 And then the copy editor got in touch with me and said,
00:08:02.25 "You know your title is a little long,
00:08:04.21 could you possibly shorten it?"
00:08:07.08 And I said, "I think I can do that,"
00:08:08.20 and I changed it so the final title is
00:08:11.03 "Green Fluorescent Protein as a Marker for Gene Expression".
00:08:14.10 This was not the end of our troubles though,
00:08:16.16 we had sent in this picture that you see here
00:08:18.18 that eventually made it to the cover of Science
00:08:20.17 and I was very proud of this picture because what it shows
00:08:25.10 is a growing nerve cell in a living larva of the animal.
00:08:29.28 And I wanted to point out the fact that GFP could be used in living tissue.
00:08:35.11 The art editor, the cover editor called me up and said,
00:08:39.14 that they really liked the picture, that they wanted to use it on the cover
00:08:42.15 but there was one problem. And that problem was that they
00:08:46.03 never like to use the color green on the cover,
00:08:48.17 and would I consider changing the color
00:08:51.10 of the picture. I said, "no, I really wanted it to be green,"
00:08:55.00 and fortunately they kept it that way.
00:08:57.16 The final problem that we had in publication
00:08:59.18 was by this time we had already given out samples
00:09:02.26 of the plasmid to people to try for themselves.
00:09:04.24 and we were getting wonderful reports back from people saying that
00:09:07.18 it had worked. So I wanted to put that as personal communication into our paper,
00:09:13.27 and the people that we asked were all uniformly very generous,
00:09:19.22 and one person, however, asked for some additional
00:09:24.06 considerations, specifically, and you probably can't read it in this letter,
00:09:30.27 but the letter asked that I prepare coffee every Saturday morning
00:09:36.03 for two months, prepare a special French dinner,
00:09:39.21 and take out the garbage nightly for the next month,
00:09:42.16 these were requirements set out by my wife, Tulle Hazelrigg.
00:09:45.01 But I really wanted to use her work, and although we still debate about whether
00:09:49.23 I have actually paid up on this.
00:09:51.23 The work she did was really wonderful,
00:09:54.17 and it was published a few months later.
00:09:56.19 But she's the person that made the first protein fusion with GFP,
00:10:01.22 so she was able to show that GFP could be linked to other proteins,
00:10:05.25 and that one could follow those other proteins, check their localization,
00:10:08.24 but also see their movements within cells and tissues.
00:10:13.21 A beautiful example of the use of GFP protein fusions can be seen
00:10:18.06 in this movie by Rosalind Silverman-Gavrila
00:10:21.04 of the nuclear divisions in an embryo
00:10:24.16 of Drosophila. I have taken this from the cell image library
00:10:27.28 of the American Society for Cell Biology.
00:10:30.05 It's on their website. It's a really beautiful picture
00:10:34.06 showing, in this time-lapse film, cell division in which you can see the spindle labeled
00:10:39.21 and you can see the spindle forming and then dissolving and forming once again.
00:10:44.26 GFP, other fluorescent proteins, and their derivatives,
00:10:49.18 have been used for thousands upon thousands of experiments
00:10:53.06 throughout biology. Their small size and inheritability
00:10:57.03 provide a dynamic and essentially a non-invasive
00:11:00.12 means of following biological processes in living cells.
00:11:05.17 We've already learned much, and we will continue
00:11:07.16 to learn more by using these molecules in the future.
00:11:10.29 I want to close with some more general
00:11:13.11 lessons that I take from the story of the development of GFP.
00:11:17.02 First, many discoveries are accidental.
00:11:22.29 Certainly the discovery of GFP is one of those cases.
00:11:26.29 Shimomura was not looking for a fluorescent protein;
00:11:31.04 he was looking for a bioluminescent protein.
00:11:32.28 But his experiments led him to this rather wonderful molecule.
00:11:36.25 Second, scientific progress is cumulative.
00:11:42.00 It is not the product of just Shimomura or myself or Douglas Prasher,
00:11:48.05 or Tulle Hazelrigg, or many other people-
00:11:51.05 Roger Tsien, who developed the first molecules with different emission colors,
00:11:58.17 who also developed the first FRET based
00:12:00.26 molecular monitors using the fluorescent proteins,
00:12:04.29 or the Lukyanovs or Mikhail Matz who discovered the first red fluorescent protein
00:12:10.03 by looking at corals. And really the thousands of people
00:12:14.20 who have added to the usefulness of GFP.
00:12:17.09 Third, I think this is a good example of saying that all life should be discovered
00:12:23.01 We should not simply be working with model organisms.
00:12:26.05 I think we can now all be very grateful that Shimomura
00:12:29.21 was interested in a fundamental biological question,
00:12:32.17 that had nothing to do with human biology or human health,
00:12:35.27 but how it is that some organisms can produce light.
00:12:40.04 And it was that curiosity that led him to work on the jellyfish,
00:12:44.05 and then l ed him to discover GFP.
00:12:47.19 And finally, the GFP story is a good example of how essential basic research is.
00:12:57.05 When Shimomura discovered GFP,
00:13:01.02 I don't think anyone would have imagined the usefulness
00:13:05.26 that it would have for other discoveries and basic research
00:13:08.05 throughout the biological sciences, or for investigations into human health.
00:13:13.24 It's been used to study HIV, AIDS, inherited diseases.
00:13:17.25 Or that it would be useful in biotechnology
00:13:21.03 The usefulness comes from other people and what they've done,
00:13:24.09 but it all starts with that basic research that was so necessary.
00:13:28.14 So what I want to do is close with one of my favorite quotes about basic research.
00:13:33.09 It comes from Robert R. Wilson who is the physicist
00:13:37.05 that was the first head of the Fermilab,
00:13:39.18 the particle accelerator in Batavia, Illinois.
00:13:41.22 He was asked in 1969 to go before a congressional committee to
00:13:47.17 justify why they should pay for this rather enormous science experiment.
00:13:53.03 He was asked many times to try to describe the security benefits,
00:14:01.15 the benefits for national defense that would come from this accelerator,
00:14:07.11 from what was going to be learned there.
00:14:08.27 And every time he was asked he said no, and then he finally
00:14:11.28 said that the accelerator had "only to do
00:14:15.15 with the respect with which we regard one another.
00:14:18.10 The dignity of men, our love of culture.
00:14:20.21 It has to do with whether we are good painters,
00:14:23.14 good sculptors, great poets.
00:14:25.22 I mean all the things we really venerate in our country
00:14:28.24 and are patriotic about. It has nothing to do directly with defending
00:14:33.03 the country except to make it worth defending." Thank you.

This Talk

Speaker: Martin Chalfie

Audience:

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

Recorded: June 2011

Educator Resources for this Talk

Talk Overview

In this talk about developing GFP as a biological marker, Martin Chalfie describes the events, both serendipitous and insightful, that led to the discovery that the green fluorescent protein (GFP) could be used to track the expression and localization of proteins, thus revolutionizing modern cell biology. He also presents a strong argument for the importance of basic research in moving science forward.

Speaker Bio

Martin Chalfie

Martin Chalfie is a Professor of Biological Sciences at Columbia University where his lab investigates the development and function of touch sensitive nerve cells in C. elegans. In 1988, Chalfie realized that GFP had great potential as a marker of gene expression in living cells and, with his colleagues, he went on to develop this… Continue Reading