Discovering Kinesin

00:00:07.14 Hello, I'm Ron Vale and today I'd like to tell you about
00:00:10.26 experiments that led to the discovery of a new type of
00:00:14.17 microtubule motor protein called kinesin. And in this
00:00:18.14 talk, I'd like to highlight this experiment shown in this
00:00:21.14 video, where kinesins are attached onto a glass coverslip
00:00:26.22 and are transporting these microtubules across the glass.
00:00:31.07 And I'll explain to you why this experiment came as a complete and
00:00:35.22 utter surprise to us. And also, how this experiment changed
00:00:39.17 the course of the research project, but also changed the course
00:00:43.13 of my life and career, as well. But before I go into these
00:00:48.16 experiments, I'd just like to share some general thoughts with you
00:00:51.15 about scientific discovery. We often teach that the way
00:00:56.14 scientific discoveries are made is through a process called
00:00:59.07 the scientific method. And you can see many different versions of
00:01:02.25 them here, but they mostly reduce to a similar theme
00:01:07.21 of first coming up with a question, then developing a
00:01:11.00 hypothesis, and then doing experiments to test that
00:01:14.11 hypothesis, analyzing the data, and then coming to a
00:01:17.24 conclusion. Now, many of the scientific projects I've been involved with
00:01:23.15 haven't followed such a linear path and been so predictable.
00:01:27.09 Oftentimes, you get started with an idea, but then the experiments
00:01:32.13 take you in all kinds of different directions. And there are
00:01:36.00 various twists and turns that happen in a project. And then maybe
00:01:40.02 if you're very lucky, you come across in one of your experiments,
00:01:44.07 some really unexpected result that tells you something very
00:01:50.04 important and very new about biological systems.
00:01:53.05 And I'd like to illustrate that with the story I'm going to
00:01:56.07 tell you. And this story, I think, is very much like a detective who
00:02:02.06 done it story. Where we started off following, in fact, the most obvious
00:02:07.08 suspect, but only found out later that that suspect was not
00:02:12.12 correct, and we were on the wrong track. So then we had to
00:02:15.12 go back and find new clues, develop new pieces of evidence
00:02:20.13 put things together in a new way that got us back on the right
00:02:24.17 track, and allowed us to come up with the correct answer.
00:02:28.11 And indeed, many scientific projects are really like detective
00:02:32.28 stories. So today, I'd like to share with you my detective story
00:02:38.18 that started off when I was an MD PhD student in the lab of Eric Shooter.
00:02:45.17 And I was interested in this problem of how material is transported
00:02:51.24 inside of nerve cells. In particular, I was studying this one hormone
00:02:55.28 called nerve growth factor, which interacts with receptors
00:03:00.16 at the nerve terminal and then some message must get
00:03:04.19 transmitted back through this axon, back to this cell body.
00:03:09.11 But this axon, in fact, can be fantastically long. Even in a mature
00:03:15.12 motor neuron in your body, the distal tip of that nerve terminal
00:03:20.28 may be at the tips of your toes, and that axon from that one nerve
00:03:25.18 travels all the way up your leg, and then the cell body would
00:03:29.15 be in your spinal cord. So to transport material over this long
00:03:34.03 distance, there had to be some machinery that was moving material
00:03:38.09 inside the axon. But in 1983, very little was known about what
00:03:43.22 that machinery was. But an opportunity to begin to understand this
00:03:49.24 problem came about actually one floor below me at Stanford, in the
00:03:56.02 laboratory of Jim Spudich, where a very exciting experiment was
00:04:00.05 going on between Jim and a sabbatical professor, Mike Sheetz.
00:04:04.16 And what Jim and Mike were trying to do was to develop
00:04:09.25 an in vitro assay to study muscle contraction. And what they did in their
00:04:14.16 experiment was first of all to purify muscle myosin, and then
00:04:18.15 add that purified muscle myosin onto plastic beads, and then they
00:04:24.04 added these myosin-coated beads onto actin cables, which were
00:04:29.00 derived from a plant cell, Nitella. And you can see these beads
00:04:32.16 or actually clumps of beads, being transported along these actin cables.
00:04:37.16 And from watching these movies that I saw one floor below me downstairs,
00:04:41.14 it was very tempting to think that maybe a similar process
00:04:46.04 was going on in the axons. Perhaps myosin was coating the
00:04:49.29 surface of membrane organelles and it was transporting these membrane
00:04:54.19 organelles along actin filaments inside the axon. So Mike and I
00:05:00.01 wanted to test this idea with a very simple experiment.
00:05:04.10 Which was to inject these myosin-coated beads inside of an
00:05:09.28 axon and see if they would move. See if they would be transported
00:05:12.23 along actin filaments. So to do this, we had to find a good
00:05:16.17 experimental organism. And we chose the granddaddy of
00:05:21.01 all axons, which is the giant axon from the squid, which has an axon
00:05:25.26 about 50 times bigger in diameter than a mammalian axon.
00:05:29.22 So the next step was to get some squid, so I called the Hopkins
00:05:35.24 Marine Station in Monterey and asked them if they would be able to
00:05:39.29 provide some squid for these experiments. And they said sure, we'd be very
00:05:43.05 happy to do that. We'll give you a call when we make a catch of
00:05:46.27 squid. Well, weeks went by, in fact, months went by. And we never
00:05:52.14 got a phone call from Hopkins Marine Station. So eventually, I called
00:05:56.09 them in June and said, well, where's our squid? And they said,
00:06:00.18 well, you know we've been trying to catch them but it's been a very unusual
00:06:04.22 year. We've been trying to fish for them and simply haven't been able
00:06:07.28 to catch any. And indeed, that was an unusual year, as shown in this
00:06:12.22 graph of the amount of squid that was caught in California
00:06:17.16 over various years. And that year was 1983, where there was a precipitous
00:06:24.00 decline in the amount of squid that were caught. And we now know why,
00:06:28.05 1983 there was an unusual meteorological phenomenon called El Nino
00:06:34.25 that warmed the coast of California. And when the water warmed,
00:06:38.17 the squid just disappeared. So this seemed like incredibly bad luck,
00:06:44.05 however, this bad luck turned into good luck in the end, as I'll
00:06:48.17 describe to you. And in fact, it's funny to think but perhaps, a lot of
00:06:54.07 the luck that I've had in my scientific career is due to this El Nino
00:06:58.27 phenomenon. Because what El Nino did was to force us to change our
00:07:03.06 plans and move and do the experimental work at this
00:07:07.21 beautiful location, which is the Marine Biological Laboratory
00:07:11.26 at Woods Hole. Now, not only was there a lot of squid at
00:07:17.16 the MBL in Massachusetts, but there was also incredibly
00:07:21.14 exciting research going on at the time. Indeed an entire new
00:07:25.16 revolution in microscopy that was developed independently
00:07:30.02 by Robert Allen and Shinya Inoue. And what these scientists did
00:07:34.28 was instead of looking through the microscope with your eyes,
00:07:39.10 they attached a video camera and also used computers to
00:07:43.06 enhance the contrast of these images and saw details
00:07:46.27 inside of cells that were never seen before by microscopy.
00:07:50.17 And Allen, Brady, and Lasek used this kind of microscopy technique
00:07:55.29 to investigate axonal transport in squid. And they made these
00:08:00.23 amazing movies, such as shown here, where you can see
00:08:04.03 now all of these fine details of small organelles being transported
00:08:09.07 up and down in this axon. So for the first time, axonal transport
00:08:14.03 could be directly visualized. So this was all very exciting, but again,
00:08:19.20 we had some bad news right when we arrived at Woods Hole.
00:08:24.00 And I opened up this issue of Nature, and found out to my great surprise
00:08:29.05 that someone else did the experiment that we wanted to do.
00:08:32.12 They took foreign particles, which were in fact beads, small plastic
00:08:37.17 beads, and microinjected them into an axon and found
00:08:42.00 that they were rapidly transported inside the axon. Except it
00:08:46.29 wasn't exactly the experiment that we wanted to do, because these were not
00:08:51.04 myosin-coated beads, these were inert beads that had nothing
00:08:53.23 on them. And in fact, this was a mystery. The investigators
00:08:58.03 themselves couldn't explain it. They thought, well maybe there
00:09:01.01 was a flow of fluid going on inside the axon and these beads got
00:09:05.21 caught up in this fluid flow. Or perhaps myosin got picked up by
00:09:10.05 these beads. Or maybe they were just piggybacking on membrane
00:09:13.25 organelles. So they didn't have a good idea of why this happened.
00:09:18.04 Undaunted, we went ahead and did our experiment with myosin-coated
00:09:23.01 beads. So we took our myosin-coated beads, added it to the squid axoplasm,
00:09:27.27 and the moment of truth came, and we saw nothing.
00:09:31.16 These myosin-coated beads did not move at all, so that was disappointing.
00:09:38.05 We then still had these control beads that didn't have any myosin
00:09:41.24 on them, and tried them. And surprisingly, these beads moved.
00:09:46.23 So we got the same strange result that these other investigators
00:09:50.15 did, which we couldn't explain either. But what we did know
00:09:55.12 was that our original idea of these myosin-coated beads
00:10:00.10 finding and moving along actin cables in the squid axon
00:10:04.12 was somehow incorrect. So we had to go back to the drawing board and
00:10:08.00 develop a new plan of investigation. And Mike and I did that by
00:10:13.00 joining up with Tom Reese and Bruce Schnapp. Tom had a
00:10:18.04 laboratory in Woods Hole, and both Tom and Bruce were
00:10:22.09 exceptional electron microscopists. Bruce also developed
00:10:25.12 a video microscope in Tom's lab. And the four of us worked as
00:10:30.09 a team to do all the experiments that I'm going to describe to you
00:10:33.14 next. So first of all, to probe this system deeper, we took
00:10:40.10 the inside of the squid axon out, the axoplasm, and kind of broke it
00:10:44.22 apart. And then found out in this dissociated axoplasm, transport
00:10:49.13 still continued. But now you could see that there were individual
00:10:52.19 tracks that were supporting this axonal transport.
00:10:57.11 However, with the lower resolution of this video microscope,
00:11:00.11 we didn't know what these tracks were. So we had to go to the electron
00:11:04.28 microscope to get that detail. And Tom and Bruce did this brilliant
00:11:10.00 experiment where they first looked at a field of view under the
00:11:13.18 video microscope and then found the same field of view under an
00:11:16.28 electron microscope, to find out what these filaments were. And then
00:11:20.28 indeed, when they went to the electron microscope, they found
00:11:23.17 organelles on single filaments that had the diameter
00:11:27.03 and the features of microtubules. So the main tracks of axonal
00:11:31.20 transport, the long distance tracks, were not actin. They were microtubules.
00:11:35.06 But the question then was, well the motor could be myosin, which
00:11:40.22 only worked on actin. So some other motor had to be at play here.
00:11:45.06 And we originally had some hypotheses for what that motor would
00:11:48.25 be. But those hypotheses also turned out to be wrong. So we had to take an
00:11:53.11 unbiased approach to understand the motor and the machinery that was
00:11:58.10 driving this transport. So one way to take this approach was
00:12:02.21 to reconstitute this system, essentially take it apart, put it back
00:12:06.01 together again, and find out what was the essential machinery
00:12:09.07 and perhaps the motor. So we did this by introducing tracks, which
00:12:14.13 were here made from purified tubulin. We then also took
00:12:18.23 the cargo, which were the membrane organelles, purified from
00:12:22.24 the squid giant axon, and added ATP as an energy source.
00:12:26.23 And we figured that the motors were going to be bound to
00:12:29.22 these membrane organelles, and when they had ATP they would move along
00:12:34.02 these microtubules. But again, disappointingly, the experiment
00:12:38.17 was negative. We didn't see any movement. Well then
00:12:42.29 we rationalized, okay maybe something is missing here.
00:12:46.07 Something else is needed. So we added to this mix a soluble
00:12:50.17 protein fraction containing hundreds of different kinds of proteins.
00:12:53.22 And to our joy, this worked, we now saw lots of membranes moving along these
00:12:59.13 microtubule tracks. And this happened late at night, but before
00:13:03.15 going home, we decided to do what was sort of a boring control
00:13:07.18 experiment, just to make sure that the things that we saw that were moving were
00:13:12.16 not particles coming from this soluble protein fraction, but in fact
00:13:15.27 were indeed the organelles. We mixed everything together
00:13:20.20 but now without the organelles. So we just added the microtubules,
00:13:24.16 ATP, and the soluble protein fraction, expecting nothing to
00:13:28.14 happen at all. Well, what happened blew our mind. What we saw
00:13:33.00 under the microscope was that these microtubules were attaching
00:13:37.11 themselves onto the glass coverslip and they were crawling
00:13:40.21 across the surface of the glass like little worms. And this was,
00:13:45.16 as I said, completely unexpected. We had no anticipation
00:13:49.09 that this was going to happen at all. But then in thinking
00:13:52.00 about this in more detail, what we thought was happening
00:13:55.26 here was that in this soluble protein fraction, there were motor
00:13:59.29 proteins that were just floating around in the cytosol. And
00:14:03.12 they attached onto the glass surface and they were grabbing hold
00:14:07.15 of these microtubules and then transporting these microtubules across
00:14:11.07 the glass surface. So suddenly, this old experiment that we did
00:14:16.06 with these control beads made sense. Maybe these control beads
00:14:19.08 were picking up this soluble motor protein and then moving along
00:14:24.07 these microtubule tracks. So we now tested this again
00:14:28.04 using our soluble protein fraction, and indeed, these
00:14:31.21 beads picked up a motor from the soluble protein mix and
00:14:35.27 were moving beautifully along these microtubules. So why was this
00:14:40.18 a surprise? Well, the reason is that most people in the field,
00:14:44.10 including us, thought that the motor for this transport would
00:14:47.21 be permanently bound either to the membrane organelles, which were
00:14:51.18 the cargo, or onto the filamentous track like the microtubule. But no one
00:14:56.03 expected there to be a lot of soluble motor floating around
00:15:00.15 in the cytoplasm. But the fact that there was a lot of soluble motor
00:15:04.21 gave us an opportunity to think about now purifying the motor
00:15:09.02 protein from this complex mixture of proteins. The problem was
00:15:13.25 this experiment happened at the very end of August, and I
00:15:17.21 was supposed to go back to medical school in two weeks
00:15:21.00 and start my clerkships. And it would probably be another three years
00:15:25.20 until I was going to get back into research again. So I called up Stanford
00:15:29.13 and I said, I had a very exciting experiment and I needed to keep
00:15:31.28 working on it, can I postpone my medical school clerkships?
00:15:35.24 And they said yes. So that fall and winter, I spent trying to purify
00:15:42.22 this motor protein. Now, Woods Hole is a very exciting place, lots of
00:15:47.25 people there in the summer. And in particular, in the '80s, there
00:15:51.26 was very few people in the winter. So I kind of had this interesting
00:15:57.17 monastic environment of just really trying to focus on this problem
00:16:02.19 of purifying the motor. And of course, it was really one of the most rewarding
00:16:06.10 and exciting times of my life. So the experiment really was to use this
00:16:11.17 motility assay in combination with protein purification techniques
00:16:15.20 to identify which protein of these hundreds of proteins that were in this
00:16:20.19 soluble fraction was the motor. So you could use this motility
00:16:25.04 assay for this purpose. So here are different fractions coming
00:16:29.07 off of a protein column, and here, each of these fractions now
00:16:33.10 is assayed for motor activity. And you can see this peak of
00:16:36.22 microtubule motor activity here corresponding to this polypeptide
00:16:43.00 which had never been described before. And since it was a new
00:16:46.17 type of protein, a new type of motor, we needed a name.
00:16:49.17 And a lot of people ask me where did the name kinesin
00:16:51.29 come from? Well, I could tell you that it came from a phone call with my mother.
00:16:56.29 And indeed, my mother from the Greek root "kine" came up with
00:17:02.14 the name kinesin. Since 1985, it's also been amazing to see
00:17:09.02 how the whole field of kinesin has developed. In 1985, we knew
00:17:12.17 almost nothing about how this motor moved objects along
00:17:16.23 microtubules. But since that time, single molecule assays
00:17:20.10 were developed. It was possible to get X-ray and EM structures
00:17:24.10 of kinesin. All kinds of protein engineering experiments. You know these
00:17:28.13 things were done in our lab and many laboratories around the world.
00:17:30.26 Now we actually have a pretty good idea of how this motor
00:17:34.07 is actually moving along a microtubule track. The same thing happened
00:17:39.11 in biology. At the beginning, in 1985, we had one kinesin motor
00:17:43.14 but since then, many kinesin motors have been discovered.
00:17:47.04 It's now known to be a large superfamily. They're known to be
00:17:51.13 involved in many different roles in cell biology, and several
00:17:56.00 neurodegenerative diseases have been linked now to mutations in
00:18:02.05 kinesin motors as well. So it's been a remarkable adventure,
00:18:06.06 a remarkable journey. This detective story that I told you
00:18:10.02 about had moments of frustration when we couldn't get squid,
00:18:14.21 we had times when we had results where we simply couldn't
00:18:18.04 explain, but there were also these fantastic eureka moments that
00:18:22.09 you really will remember for the rest of your life. And also
00:18:26.08 an opportunity to really dig into a problem and emerge with
00:18:29.27 the satisfying mechanism. Now what about my career in
00:18:35.02 medicine? I never did go back to medical school, I never did
00:18:39.04 complete my clerkships. And that was all for the best, I've
00:18:43.12 enjoyed being a scientific detective and I've enjoyed all the
00:18:49.25 problems I've worked on. And I hope you will enjoy your
00:18:53.05 scientific detective work as well. Thank you.

This Talk

Speaker: Ron Vale

Audience:

General Public
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Recorded: July 2016

Talk Overview

Vale explains how doing science often follows a winding path with unexpected, sometimes wonderful surprises, and uses his own story to illustrate his point. When Vale was a graduate student, he initially hoped to show that myosin was involved in axonal transport, but ended up discovering a new molecule which he called kinesin.

Speaker Bio

Ron Vale

Ron Vale is a professor at the University of California, San Francisco and an Investigator of the Howard Hughes Medical Institute. His lab combines cell biological and biophysical approaches to understand spatial organization, movement, and signaling within cells. Vale is a member of the National Academy of Sciences and a winner of the 2012 Lasker… Continue Reading

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