Discovering Kinesin

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

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

Professor of the Department of Cellular and Molecular Pharmacology; Investigator in the Howard Hughes Medical Institute
University of California, San Francisco Continue Reading