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Imaging Synaptic Vesicle Transmission

Part 1: Imaging synaptic vesicle transmission

00:00:06.25 So, hello I'm Erik Jorgensen from the University of Utah.
00:00:11.04 And today we have Drs. Tom Reese and John Heuser,
00:00:14.02 and we're going to be interviewing them and discussing
00:00:17.20 some of their seminal work that they did in the 1970s.
00:00:20.18 They were using electron microscopes, which you see in the background,
00:00:24.02 this is a device that uses electrons to image cells at a resolution
00:00:30.18 close to 1 nanometer. So this is the highest resolution microscope
00:00:34.17 possible. In the 1970s they performed experiments that allowed us
00:00:39.16 to understand neurotransmission, the fundamental behavior of a
00:00:43.17 cell during communication, and they're going to tell us about some of
00:00:48.07 their experiments that they performed then. So, what was the
00:00:52.12 experiment, what was known, what was not known,
00:00:54.11 and what was the experiment and what did it demonstrate?
00:00:56.14 We were interested in a little structure in the brain called a
00:01:00.26 synapse. And a synapse to me is about the most important
00:01:05.16 structure in the universe, because it's the way the nerve cells
00:01:09.12 communicate with each other. and it's these networks of
00:01:12.27 communication that allow us to think and remember. And
00:01:17.17 be conscious, and be who we are. So the synapse is sort of
00:01:23.29 the rockstar of biology because of its tremendous interest
00:01:29.08 and importance. I'd been interested in the synapse
00:01:34.26 when there was a great deal already known about it,
00:01:37.05 about how it worked electrically, it was known that it
00:01:40.08 released drugs, but nobody really had an intimate understanding
00:01:47.14 of how an actual individual synapse works. And that's what we wanted.
00:01:54.13 Yeah, and I was equally interested. And graduating from medical
00:02:00.23 school. And made a beeline for the laboratory where the original
00:02:06.22 work had been done that set the stage, where electrophysiologists
00:02:11.05 had demonstrated that the communication event at the synapse
00:02:14.29 involved release of little packages of chemical transmitters.
00:02:19.26 In little electrical blips that they could see. Now the man that did
00:02:24.15 that in his lab I was joining, Sir Bernard Katz, went on to get the Nobel
00:02:28.24 Prize after that. But his insight was profound and on top of that,
00:02:33.09 he'd written unbelievably beautiful papers that terribly impressed
00:02:36.20 me as a student. So made a beeline for that lab. He had
00:02:41.28 already looked at electron micrographs and seen that these
00:02:45.28 synapses on the inside were filled with tiny membranous
00:02:50.26 membrane vesicles, little spheres of membrane. And
00:02:55.07 he made the brilliant, but logical, interpretation that those
00:02:59.24 little spheres contained the packages of transmitters that he was
00:03:03.20 measuring electrically as little blips. And so he had the insight to
00:03:07.19 propose that the little electrical blips were being generated
00:03:11.13 by chemicals coming from those tiny packages. That was established,
00:03:16.17 I mean it was such a clear cut idea that it almost had to be
00:03:19.24 that way. And so my project when I entered that lab as a postdoc
00:03:24.25 in those days, you're basically assigned a project, was
00:03:28.18 to quote "prove it." Get an image of these little vesicles
00:03:32.06 popping on the surface of the nerve when I stimulate the
00:03:35.18 nerve. And during that postdoc, I didn't get it. But I had the
00:03:40.04 opportunity to join Tom Reese at the NIH immediately after that.
00:03:44.07 We reformulated the problem and came up with a whole new
00:03:47.29 approach to either proving or disproving that idea that
00:03:52.24 Katz had had.
00:03:53.18 So Tom, maybe you could tell us how it came to be that you
00:03:57.24 met John and how that led to a collaboration.
00:04:02.04 When John came to the lab, we decided to try a whole different
00:04:06.13 approach. As John pointed out, you usually went to a lab
00:04:09.16 as a postdoc and were assigned a project, but in his case,
00:04:12.22 he came to the lab and assigned me a project.
00:04:17.15 We were originally thinking about looking at the aftermath and trying
00:04:22.22 to interpret back to what might've happened. And that
00:04:27.04 was a good approach.
00:04:29.12 As it turned, yes. And the idea is if these little packages
00:04:34.02 are opening to discharge transmitter, and that's the mechanism
00:04:36.25 of communication, then during that moment of opening, they should be
00:04:41.25 able to capture material from outside the cell and carry it back in.
00:04:44.22 And it would be indirect proof that at least they had interacted with
00:04:47.29 the surface.
00:04:48.18 So what you demonstrated was that the membrane, the synaptic
00:04:51.26 vesicles were normally found in the synapse, in the
00:04:55.09 bouton, and that they had visited the surface, that's all you
00:04:59.05 could show and the problem was a question of time scale or
00:05:02.22 was there something missing?
00:05:04.12 There was the missing fact that we had nothing but
00:05:09.14 embalming fluid, and we were dropping these things into
00:05:12.06 embalming fluid every time. And life did not continue
00:05:15.28 in any kind of appropriate manner in that solution.
00:05:19.19 So we didn't capture it.
00:05:21.09 There's a missing piece here, and the next step of that,
00:05:24.07 was when we decided, well why don't we just stimulate
00:05:28.05 the nerve for various periods of time and then fix it
00:05:34.10 immediately or let it sit for a while and have a look.
00:05:37.10 So we did a series of different times and different amounts of
00:05:42.04 stimulation, and different time intervals for recovery. And
00:05:45.02 that turned out to be very informative, because when you
00:05:50.06 stimulated the nerve, the nerve ending where all the vesicles were,
00:05:55.18 it got quite a bit bigger as if the vesicles inside were being
00:05:59.23 added to the surface. And so that was a second piece of
00:06:04.24 evidence. And then if you waited a while after you stimulated
00:06:09.25 before you fixed, you went back to a perfectly normal looking
00:06:13.13 terminal. As if there was a cycle of activity and recovery.
00:06:21.23 And that cycle, now we know that that cycle involved
00:06:26.17 some recycling in the vesicle compartment. And I have to say, John,
00:06:31.19 it's always been very clear to me that the idea of vesicle recycling
00:06:36.05 was totally yours, okay?
00:06:39.29 I wish. -- No, I mean really!
00:06:41.28 At the time, I was astonished by this idea because it had never
00:06:48.14 occurred to me. And I'll take some credit later for some other stuff.
00:06:53.17 Well, it is true that the very first paper that we wrote on the
00:06:58.09 attempts in London to get it working, we didn't. But we got
00:07:00.28 this aftermath effect. We said maybe the vesicles are
00:07:06.18 dissolving and being reformed or --
00:07:08.26 You sort of had that in your head.
00:07:11.01 Or maybe it's merely recycling. Merely recycling.
00:07:16.09 Is the final sentence of my paper in London.
00:07:19.22 Merely. It became our career to pinning that down and
00:07:24.22 work it out.
00:07:25.08 So, back in 1973, one, when you published this, was it accepted at
00:07:33.20 that point? Was there anybody else who was doing these experiments
00:07:37.04 at the time? Tell me a little bit about what was going on
00:07:40.09 in 1973 when this paper came out.
00:07:44.04 73, there was a whole cadre of people doing this kind of work
00:07:50.24 all fascinated by the nervous system, all having electron
00:07:54.25 microscopy as their tool, they'd become experts at it. That was really
00:07:59.23 the heyday of electron microscopy. It was the opportunity
00:08:04.29 to see things for the first time at that resolution.
00:08:08.10 So everything was a gold mine and everything was fascinating and exciting.
00:08:11.23 People were going crazy. So there were a number of people
00:08:14.24 that were neurobiologists that also focused on the nerve terminal.
00:08:18.16 And the remarkable thing was that 2-3 groups, including groups
00:08:23.10 here at the Marine Biology Labs and in Europe, particularly in
00:08:27.24 Milan, were focusing on the exact same preparations as
00:08:31.10 us. And doing the exact same experiments. And we were
00:08:34.19 publishing in the exact same journals pictures that could've
00:08:38.29 been swapped from one paper to the other.
00:08:41.27 And just because of human nature, they were drawing completely
00:08:45.17 different conclusions about how to interpret those
00:08:48.24 micrographs. So there has never been peace in this field,
00:08:52.21 there's never been consensus. And we are still, to this day,
00:08:58.02 battling out the interpretation of what you see in an electron
00:09:01.16 micrograph. But the way we were thinking was indicative of
00:09:05.13 the way it's always been. We were -- why we left everybody
00:09:09.20 in the dust right then was because we were saying
00:09:12.19 how do you see it? How are we going to see that?
00:09:15.04 And so we do this, we do this, we put it on that
00:09:17.17 substrate, we look at it on this instrument, this and that, etc ...
00:09:20.14 We'd move instantly into the technology of it.
00:09:23.29 And getting the right image in the period. But you can't forget the rationale
00:09:30.07 for doing it at all, for working and developing this machine
00:09:33.11 was basically to capture this event of neurotransmission.
00:09:38.07 And the physiologists had taught us the event occurs on
00:09:42.25 1/1000 of a second. From when activity spreads into the nerve and
00:09:48.11 the nerve communicates with the next one by secreting these
00:09:51.09 little chemicals from synaptic vesicles. It takes only 1/1000
00:09:55.21 of a second for it to happen. And we needed to capture that event
00:10:00.17 on that same time scale. We needed to capture it on
00:10:04.03 its own, so that's why we went through on the effort to
00:10:08.17 develop the freezing machine further.
00:10:10.00 So maybe John, you could describe the instrument for
00:10:13.11 our viewers and all its glory and complications?
00:10:16.22 Well, I wish it were glorious and complicated.
00:10:21.09 But it couldn't be more trivial. It's a copper block, copper
00:10:27.08 chosen because it's very highly conductive metal, the most
00:10:31.16 conductive, particularly at those temperatures. So you have a heat
00:10:34.25 sink, the best heat sink you can think of or imagine, and
00:10:38.27 you want to apply the thing you want to study in the electron microscope,
00:10:42.15 in our case these little nerve terminals doing their secretory
00:10:45.04 event. Want to apply that prep to the copper block as abruptly
00:10:51.22 and precisely timed as possible. So we just plain plunked it down
00:10:57.03 on top of it. Just popped it into it. Touched it to the surface.
00:11:01.11 To the copper surface at 4 degrees Kelvin. And hook up all
00:11:05.01 the stimulating electrodes and develop methods of synchronizing them.
00:11:08.21 And how was it done? How did you time when you would
00:11:13.00 stimulate before it hit the copper block?
00:11:14.26 Simply had a magnet mounted right next to the sample,
00:11:20.09 which could be used by a little sensor that sensing magnets.
00:11:24.22 As the magnet passed the sensor, it said, "You're about to hit the
00:11:29.22 copper block!" And that stimulated the electrode that
00:11:33.19 fired the action potential in the nerve. So we could synchronize
00:11:36.21 that action potential arrived, the vesicle's discharged,
00:11:40.04 and the sample hit the copper block in 1/1000 of a second.
00:11:43.08 But you also had a problem with the sample freezing before
00:11:47.04 it ever hit the surface.
00:11:48.17 It got so close to the copper that it pre-froze before it would
00:11:53.06 get stimulated. But the real trick was and still is, and you need to use it,
00:11:58.02 the drug. The drug that augmented release, our physiology
00:12:04.27 friends who helped us work on it said, either between 1000
00:12:08.04 or 10,000 times, one nerve impulse would release 10,000 times
00:12:12.23 more vesicles than normal. If you make a nerve stimulate
00:12:17.11 -- release 10,000 times more than normal, it isn't going to want to do that
00:12:21.17 very often. It does it once. And it's shot. So we have to make sure
00:12:25.25 we don't pre-stimulate it as we're getting the whole experiment
00:12:28.26 ready.
00:12:30.07 So, you do the experiment, what do you see? Was there a eureka
00:12:34.22 moment where you were on the scope? And you said,
00:12:38.22 oh my god, this is it! We've seen the light!
00:12:42.12 This is the image we were looking for. I mean, can you
00:12:45.00 describe a little bit about that? Were you together, where did it happen?
00:12:48.02 As it happened, Tom wasn't there at the particular time that it happened.
00:12:51.11 He was back at NIH, and I was in San Francisco. But at that time,
00:12:54.29 I had Mike Denitz was a very fine physiologist who was doing
00:12:59.04 all this treatment with this magical drug that increased
00:13:02.12 things so much. And we had two young, even pre-docs,
00:13:08.20 maybe they had just gotten their postdoc, and they
00:13:10.28 wanted to learn some EM. So they were wandering around
00:13:13.27 my lab getting in the way underfoot, but one of them,
00:13:18.25 Lily Jan and her husband Yuh-Nung Jan, was the other
00:13:22.29 person wandering around. Both have gotten on to great careers.
00:13:25.29 But they, so they're sitting on the microscope, and they called me
00:13:30.08 into the microscope, and they said, John, I think we've got it.
00:13:34.03 I mean, look at that. And I came in and looked over their
00:13:36.16 shoulder and I said, no, no, no. That's a capillary.
00:13:39.09 You don't recognize there are whole bunch of little vesicles
00:13:42.23 attached to the surface of the capillary all the time.
00:13:45.06 That's part of transport of blood. Nothing to do with
00:13:48.19 nerves. You know, forget it. Looked a little harder, my god,
00:13:52.24 it is a nerve terminal! Look at all those openings. That really was a
00:13:56.28 eureka moment.
00:13:58.05 What was it you saw? What was it that you were actually
00:14:00.19 looking for that was the eureka?
00:14:03.08 Tom had explained how at that point were doing it by freeze
00:14:06.05 fracture. It's a method of looking at the surface of the
00:14:08.25 nerve by cracking open this frozen tissue. Particularly
00:14:12.21 nice, because after all, we'd started by freezing it.
00:14:14.22 So why not take advantage of that new technology.
00:14:16.19 So we're cracking all these nerves open. It's this tube,
00:14:20.07 so here's a long cylinder. And on that cylinder, dotting
00:14:24.20 all the way down are these small craters. Like craters on the moon.
00:14:29.14 And it's a very small jump to --
00:14:32.18 Remember the vesicles were originally lined up on this membrane.
00:14:37.04 Inside, and you couldn't see it from the outside.
00:14:40.13 You wouldn't have seen them at all. And so the fact that
00:14:43.01 you could now see openings, it meant that they had actually
00:14:47.08 exploded open under the surface of the -- just right before
00:14:53.22 the muscle hit the copper block and froze. So we'd captured
00:14:59.26 them in millisecond timescale. I don't think anybody had ever done
00:15:07.09 any anatomy structures of things at that time scale.
00:15:11.23 There was another story that emerged right after that, and that is
00:15:13.27 after the vesicle opened, we noticed that in the wall
00:15:19.06 -- in the membrane of the vesicle, there were various kinds of
00:15:22.10 structures and that as time -- you see it with this freezing,
00:15:26.02 you do a time experiment. You can stimulate at 10ms, 20ms,
00:15:30.17 30ms, so we noticed that as time passed, that the vesicle
00:15:34.27 was getting wider and wider open, and that some of these
00:15:38.17 particles inside, these structures inside, were coming out
00:15:41.22 and fusing into the surface membrane. And so that was now
00:15:45.07 the story where we measured all that. And that story,
00:15:48.02 we made a story out of that. So that was a third leg.
00:15:53.25 That was actually -- I enjoyed, actually, that part of it.
00:15:56.22 Well, it's a critical piece of the puzzle. Because if you have
00:16:00.09 these little packages of transmitter and they're capable of
00:16:02.25 fusing with the surface in 1/1000 of a second, activated
00:16:06.28 by calcium, what happens to them after that? And why
00:16:10.27 were we seeing all these strange changes at much longer
00:16:13.26 times? Well, that's when we really had fun with the freezing machine.
00:16:19.07 We were no longer just trying to capture the immediate moment
00:16:22.26 but the aftermath of the event. As Tom described it, we
00:16:26.12 could now do aftermath in really close coordination. We could do
00:16:29.28 looking just milliseconds later, and it's our conviction that
00:16:35.00 it's in the nature of the tiny membrane vesicles, something about
00:16:40.04 biophysics of membranes, that when that joins a larger membrane,
00:16:44.27 like the surface of the nerve terminal, it has no alternative
00:16:49.23 but it has to collapse. It has to flatten out, join that
00:16:53.23 membrane completely. Therefore, deposit all of its
00:16:57.19 contents and its whole nature.
00:17:00.20 Did this end the debate? So you showed the vesicle touches,
00:17:03.16 flattens out, and you basically, it's irreversible.
00:17:07.09 Absolutely. It ended the debate. Erik, you're baiting us because
00:17:11.04 you know perfectly well that our colleagues in Italy,
00:17:17.19 our colleagues at the MBL and such, did not accept
00:17:20.19 that this was happening. Yeah, they had to accept
00:17:25.09 that Katz was fundamentally right, that's how neurotransmission
00:17:30.27 occurs, by the fusion of these vesicles. But then is when interpretation
00:17:35.16 of electron micrographs end and how you did the experiment,
00:17:39.11 because they looked at the same micrographs of a vesicle
00:17:45.22 fused with a cell surface and concluded that that was it.
00:17:50.26 That that vesicle did that transiently, a little kiss. Kissed the
00:17:55.06 surface, let out transmitter, and simply reversed the process.
00:17:58.25 Now I was earlier describing to you that we thought
00:18:01.22 it's in the biophysics in membranes that they can't do that.
00:18:05.06 That if they've joined another bigger guy, they're going to merge
00:18:08.11 with it completely. And so there's a controversy that
00:18:12.16 is embedded in the literature, in scientific thinking
00:18:16.22 to date. And everybody asks about this still, are you
00:18:20.27 really correct that all of your aftermath stuff is really
00:18:25.25 true? Or should we believe the Italian group that it's just
00:18:30.24 a kiss? The vesicles are kissing and coming right back in
00:18:33.10 again and again, in exact momentary exposure and
00:18:38.02 enclosure. And that's the true biophysics.
00:18:41.15 And so you then did experiments that were over longer
00:18:45.28 and longer periods of time. And then you saw something
00:18:48.28 that said, well there is evidence that in fact these
00:18:52.26 vesicles are collapsing fully into the membrane.
00:18:55.21 Yes, the aftermath experiments of milliseconds.
00:18:59.05 Particles inside the membrane, the fusing into the membrane.
00:19:03.26 Particles being our marker, and membrane.
00:19:05.19 Particles are places where the membrane is impinged by
00:19:09.27 a transmembrane protein, and freeze fraction will show up as bumps.
00:19:14.15 And the bumps were diffusing out of the vesicle membrane
00:19:20.01 into the whole other membrane. Now it's not going to do that
00:19:22.26 in a kiss and run.
00:19:24.23 What is it that you know, you saw in your images that
00:19:27.27 convinced you that vesicles had to be recycled?
00:19:31.12 You know, the reason that I would've had a preliminary,
00:19:33.07 that I had a preliminary glimpse of that and said that maybe
00:19:37.11 merely recycling happened was because of the magic
00:19:40.26 clathrin coated vesicles that appeared in the nerve
00:19:44.18 terminal as a consequence of stimulation. And I had just
00:19:48.27 come from my undergraduate work, where I was exposed
00:19:53.06 to the father of electron microscopy, Keith Porter, and
00:19:57.19 his -- one of his, he had trained so many outstanding
00:20:01.07 people, but the person who was still present when I was in
00:20:04.10 college, was Tom Roth. And he had just discovered that
00:20:08.29 the mechanism for endocytosis, the mechanism of uptake of material
00:20:12.15 from the outside world in all cells is mediated by a vesicle
00:20:17.08 that has a coat, a beautiful cytoplasmic coat that actually is
00:20:21.09 polygonal. Like a geodesic dome. And you can see that
00:20:25.12 dome in the electron microscope. And so here I'm the young guy
00:20:29.01 in London, and I see these domes in nerve terminals as a
00:20:34.23 consequence of stimulation.
00:20:36.00 You'd seen those in London?
00:20:37.04 Yeah.
00:20:38.03 Oh, I didn't know that.
00:20:39.00 And I said, oh my god, it's endocytosis!
00:20:41.16 And then it's also interesting that we managed to end a
00:20:46.16 total contentious situation with our Italian competitors
00:20:50.19 over that. Because they couldn't see these coated vesicles.
00:20:55.19 And why couldn't they see it and we did? Well, it was simply
00:21:00.09 that technique of embalming that we used. Our was a little bit
00:21:03.13 different than theirs. Ours had a little bit more heavy metal
00:21:07.00 than theirs. So, the coats looked a little more dramatic in our pictures
00:21:11.02 than in theirs. But that's the way that electron microscopists
00:21:14.17 butt horns and can enter total disagreement. Total contentions
00:21:20.18 over this kind of issue.
00:21:21.20 In the end, what I think solved the debate, are those freeze
00:21:27.16 etched images, where you see the clathrin coats.
00:21:30.16 This is absolutely clear that geodesic structures, absolutely
00:21:34.12 clear. Maybe you could tell us a little bit about the invention
00:21:37.06 of the freeze etch technique and how you used that to show
00:21:40.02 that there were clathrin coated vesicles?
00:21:43.04 That was a late chapter that arose from being able to
00:21:46.15 directly freeze things. Because if you freeze things out
00:21:52.06 of their natural juice, when you try to draw the water away
00:21:56.23 the contents of the juice collapse down on top of everything,
00:22:00.16 you can't see anything. So something we were able to
00:22:02.03 do was freeze directly and not using a fixative, which also
00:22:06.22 destroys things. We had a whole world of looking at the inside
00:22:14.06 of membranes. And that was invented in our summer neurobiology
00:22:17.24 course at Woods Hole by one of John's students.
00:22:22.02 They made a mistake on one of the machines and
00:22:24.13 John decided to have a look at it anyway, and that was one
00:22:29.12 of those eureka moments. All of a sudden, for the first time
00:22:33.13 we could actually look inside the cytoplasm. Not just look
00:22:37.04 at the membranes like we do with freeze fraction.
00:22:40.08 We could look inside the cytoplasm and you could see all
00:22:44.18 these wonderful coats. And John went on back to San Francisco
00:22:51.09 and began a decade of marvelous work looking at the
00:22:58.28 coated vesicle and coated vesicle endocytosis and cycling.
00:23:03.02 I think that's a good place to end it, but before we do,
00:23:06.21 I would like to ask if whether you guys have anything
00:23:10.12 like reflections, or memories or anything else you would like to add?
00:23:13.15 Thank you for being my friend all these years. In spite of
00:23:19.03 a few differences we have every now and then.
00:23:20.22 I can say exactly the same thing. Thank you for being my
00:23:25.00 friend. And muse, and a colleague, and it's just like.
00:23:32.08 I don't know. Hahahaha.

Part 2: The Future of Electron Microscopy

00:00:00.23 So, that decade really starts in the late 60s to 1980, was a
00:00:07.25 marvelous time for cell biology. I mean, this work came out of
00:00:12.25 that time was a revolution for understanding how synaptic
00:00:15.12 transmission works. There was just an era of great cell
00:00:21.10 biology. So why do you think that happened?
00:00:24.15 And why did it end?
00:00:26.02 Well I don't think it did end.
00:00:29.03 Are you kidding me? It has just started up again.
00:00:32.05 The focus, you know, is molecular biology sort of swept away.
00:00:35.28 Oh yeah, I mean there was a period of time in the summer, of course,
00:00:38.06 you get these guys at Caltech. In fact, we had a student and
00:00:45.01 she got up and she said, well I want everybody to know that
00:00:49.28 electron microscopy is a dying field. And so we wanted
00:00:54.21 -- we made a plot of what a dying field looked like over our door.
00:00:58.21 A dying electromagnetic field. Remember that?
00:01:01.20 No!
00:01:02.09 It just showed this thing where all of a sudden the curve went
00:01:05.00 down. And it said "Dying Field." She was in Keith Porter's lab, too.
00:01:09.10 Really?
00:01:10.23 The founder of modern electron microscopy had become disillusioned.
00:01:15.04 And so there was a period of time when people thought that
00:01:18.00 we were going to understand everything by understanding genes.
00:01:21.16 And we've understood a lot from genes, but you don't understand everything
00:01:25.04 from genes. Now, finally, the technical side of electron microscopes
00:01:33.12 is having a drastic -- and new kinds of electron microscopes
00:01:37.25 are appearing. And the challenge is on people like John and me,
00:01:43.02 people who actually prepare stuff for electron microscopes
00:01:47.29 to take advantage of these new kinds of optical things that these
00:01:52.18 microscopes can use. And that's going to happen big time.
00:01:56.20 We're going to have a whole new understanding of cells,
00:02:01.21 not at the level of membranes running around, but of the individual
00:02:05.16 protein molecules running around each other.
00:02:09.00 Back up to what Tom was talking about, the death of electron
00:02:13.01 microscopy. That period when its imminent death seemed
00:02:17.28 unavoidable. There are a couple of other factors that I'd like
00:02:21.19 to bring up. One is that that was a period when light
00:02:25.04 microscopy took off, when it first started. And light
00:02:28.15 microscopy was providing two incredibly fundamental things that EM
00:02:32.23 did not provide. Number one, the dynamics of cells.
00:02:36.17 The actual movements of all of these things. Mind you,
00:02:41.03 light microscopy is 1/1000 the resolution, that far away.
00:02:47.07 But it was a whole process displayed for you in a living cell.
00:02:52.04 Second, the light microscopy was allowing you to begin to identify
00:02:59.11 which protein was doing it, because new technology of fluorescence,
00:03:03.29 fluorescent proteins and so on, were coming in to be examined by
00:03:07.09 light microscopy. So, it was not that electron microscopy died, it was
00:03:12.13 eclipsed by light microscopy and the huge potential of it.
00:03:16.11 You went through a light microscopy phase.
00:03:17.29 I fell in love with it myself, and lost myself in it.
00:03:21.03 But, the critical thing we've always been up against, and we're still
00:03:29.16 up against, no matter how fancy the electron microscopes get,
00:03:33.06 or how perfect our means of viewing is, we'll never get around
00:03:36.15 the fact that the electron microscope gives you a static
00:03:39.21 image. A still, a snapshot of something. One instant in time.
00:03:45.00 For each picture that you take, each preparation that you make,
00:03:49.13 is one instant in time. And you have to back extrapolate
00:03:54.11 to what was happening in life. You have to say, well I timed it
00:03:58.22 perfectly, I had 5 milliseconds, I had 10 milliseconds,
00:04:01.11 20 milliseconds, so I can say it did this, that, and that.
00:04:05.05 But that's an interpretation in and of itself.
00:04:08.05 Open to questions if you got competitors in your field.
00:04:11.27 It's always that extrapolation event that's an interpretation.
00:04:17.13 And that made people shy off and led to the demise,
00:04:22.27 of which we're now trying to recover. Always will be trying to
00:04:27.03 recover.
00:04:27.25 I mean, if you were a young student in the course and you came in
00:04:31.25 and you had a choice of spending a week doing meticulous
00:04:37.12 preparation to get something ready for the electron microscope
00:04:39.20 to get one picture of it, or you could take some genes and create some
00:04:44.15 beautiful colors that would run up and down inside your cell,
00:04:48.05 you could sit there all afternoon looking at them, which would you do?
00:04:50.28 Got me. I'd sit here and look at this. That's one picture.
00:04:55.23 So what is the future of electron microscopy then? Why
00:04:59.08 -- what recommends it to students?
00:05:03.07 Well, you asked two questions there. The future and
00:05:08.15 what recommends it to students. It might be that there's little to
00:05:12.11 recommend to the students. That's what we're really facing.
00:05:15.05 How many people are going into this field? It's heartbreaking to see
00:05:18.16 how few people are being trained, even in these labs that
00:05:21.29 are totally cutting edge. And the whole culture of electron microscopy
00:05:27.07 is in dire danger.
00:05:31.17 So still, you haven't answered why they should be trained,
00:05:35.26 what are they going to see that you can't see in a light
00:05:38.00 electron microscope?
00:05:39.05 Everything. I mean you're very limited. I mean the idea people
00:05:46.25 got all excited about high resolution microscopy and super
00:05:50.29 resolution. Super resolution, that's about a 100% improvement.
00:05:57.08 You go from seeing something that's 200 nm to something
00:06:01.20 that's 50nm. But we see stuff all the time that's 1nm!
00:06:06.06 2nm. I mean, the gap between resolution in electron
00:06:13.00 microscope and a light microscope is huge. And I don't
00:06:17.02 -- my goal in my current work is that I want to understand
00:06:21.10 how individual proteins fit together to make protein motors.
00:06:25.15 Now you can examine that with light microscopy, but
00:06:28.21 with some fluorescent dyes and watch what happens,
00:06:33.14 make some inferences, but you don't actually know until you
00:06:36.23 get the images and reconstruct the proteins.
00:06:42.15 This is -- I've just listened to Tom give his lectures
00:06:47.09 to this year's students at the Marine Biological Laboratory
00:06:51.04 and was impressed again by what an appearance he has.
00:06:57.10 He, as he's saying, and it's apparent in every word he says
00:07:03.04 to students, he believes, and I totally agree with him, that
00:07:07.25 it's at the EM resolution, a level of electron microscopy,
00:07:12.08 that we're going to understand cell function, organelle function,
00:07:17.19 and everything about the cell. Because it's at that level
00:07:20.15 that you can actually see molecules. And the whole name of the game
00:07:25.05 is seeing molecules interacting with each other.
00:07:27.19 That's -- to understand this machine called life, we have
00:07:33.02 to see the individual molecules interacting with each other.
00:07:36.09 Light microscopy gives you these wonderful clues that
00:07:39.23 they're dancing together, or going over here, but to see
00:07:43.13 how that machine works, you have to have the resolution of
00:07:49.22 this instrument. And it's a simple fact. It's amazing that we have
00:07:57.01 the instrument. It's great that we've had it for 50 years,
00:08:00.28 60 years almost, but it's got a niche in cell biology
00:08:07.01 and in medical science that is irreplaceable.
00:08:10.08 Never will be the same.

Part 3: Why Collaborate?

00:00:00.14 You guys strike me as a Mutt and Jeff team.
00:00:02.29 And so that must have been --
00:00:05.08 Elvis and Costello.
00:00:06.10 An Elvis and Costello team maybe?
00:00:09.19 So you know, what did you guys see in each other?
00:00:13.07 How did it work? Were there times when you guys couldn't
00:00:16.00 see eye to eye? You know, it's a little bit like a marriage, right?
00:00:19.22 We fought in the lab like cats and dogs. People said they didn't want to
00:00:23.13 be around us. Like it was being around a married couple
00:00:25.09 where we were getting ready to get a divorce.
00:00:27.03 No, they literally, people would leave the lab when the two of us were working together.
00:00:31.06 But the fact is, when you have a collaboration that's as close as John
00:00:38.06 and as effective as John, part of the reason for that is because
00:00:43.27 there's a complimentary compensation of each other's weaknesses.
00:00:49.15 Oh really?
00:00:52.04 Do either of us have any?
00:00:54.04 That's the point!
00:00:56.05 I mean, I'm the messiest person on Earth. I mean, I could take a picture
00:00:59.28 and lose it the next day. And you know, you have every picture.
00:01:03.13 You told me the other night, you have 90,000 pictures, right?
00:01:07.29 CDs, we were talking about. CDs of pictures, yes.
00:01:11.21 CDs of pictures, you know. And I can mention, I can have some crazy
00:01:15.24 idea. I'm sort of a crazy idea guy. And I can have some crazy
00:01:20.02 idea, and 10 minutes later, John shows up with a picture.
00:01:23.17 You know, I don't know anybody else like that.
00:01:27.28 I think really the bottom line here is that we are both
00:01:35.16 crazy about structure. I mean, we just have it in our blood to understand
00:01:40.09 the world through understanding and interpreting structure.
00:01:44.24 And we shared that immediately. And I had a lot of people
00:01:49.04 come to my lab, and some of this have this love for structure
00:01:56.01 and understanding of structure, and some don't.
00:01:58.20 And so our field attracts those people. And this was the basis,
00:02:07.02 really the basic bottom line for John and I working together.
00:02:11.04 Yeah, but I want to add one thing, because there's another
00:02:14.15 fundamental way that we're similar, and that is that we're problem
00:02:17.18 solvers. And I mean, if I was going to get stranded on a boat
00:02:21.03 with no motor, somewhere off at sea, I wanted to be with
00:02:25.12 Tom. Because he's the only person that I feel we could solve
00:02:30.11 the problem of our situation appropriately together. That's why
00:02:35.13 it's worked between us. Because we are some complementary in that.
00:02:38.19 And how are you different?
00:02:40.21 Avogadro's number.
00:02:43.03 No, no, I mean the point, remember I love to do theoretical calculations.
00:02:52.02 Even if they're wrong.
00:02:53.08 It's in my blood, though I'm not really precise. And I just want to see how
00:02:57.26 the math goes. And you know, I'll throw in any old number
00:03:01.17 and run the calculation. And I did. We were trying to figure out
00:03:07.22 how many molecules you could put in a vesicle, so I ran the
00:03:10.26 calculation. And in order to do that, you need a constant
00:03:14.21 called Avogadro's number. And so I threw in it in the calculation, later
00:03:19.19 on I think you went and talked about or we published it. But
00:03:23.06 the point was that I had the number wrong by an order of magnitude.
00:03:26.20 Four orders of magnitude.
00:03:27.24 No, it was a critical calculation. You and I were calculating
00:03:32.04 how much tracer we would need to get in a nerve terminal
00:03:35.19 in the medium to hope to get a bit of that tracer into these
00:03:39.02 little synaptic vesicles. And it was going to be our first evidence
00:03:42.00 that they actually merged with the cell surface. So we're calculating
00:03:45.21 on the blackboard what was the concentration of these little tracers
00:03:49.14 that we would need to add. Tom figured out the volume of
00:03:52.15 the vesicle, had Avogadro's number wrong by four orders of
00:03:55.25 magnitude, so we calculated it would have to be 10 Molar
00:04:01.16 HRP or something.
00:04:04.12 And luckily you got it wrong.
00:04:07.15 So that worked out.
00:04:10.29 The experiment worked.
00:04:14.27 Despite us.
00:04:16.12 So, I'd like to ask you. Why bother collaborating, right?
00:04:21.10 You're both smart, you could've done these experiments on your own.
00:04:24.04 What was the -- why deal with each other?
00:04:27.06 That's something that seems like a silly question.
00:04:30.05 Like asking why get married? You know, we're not businessmen
00:04:32.28 calculating a profit. We feel these things in our heart and we
00:04:39.08 -- I felt an attraction to John's way of thinking about stuff and
00:04:43.28 it happens. This happens.
00:04:45.27 Also the issue of as you advance, as you make little observations
00:04:52.05 that are even big ones and get excited about your work,
00:04:55.10 it's just a whole lot more fun to share it with somebody
00:04:58.14 who's totally with you and got the understanding of it.
00:05:01.18 And is on the same path and is struggling to get it with you.
00:05:05.27 As opposed to doing it all by yourself.
00:05:07.20 We were together late one night and we sort of asked a
00:05:12.07 -- I think you asked you a very great question, you know, why are we
00:05:18.05 doing this together? Why are we here doing all this stuff?
00:05:20.16 And you said to me, you said the most important thing
00:05:23.29 in my life is I want to be -- I want to share discovering something,
00:05:30.01 having the truth unfold with somebody. I want to be there
00:05:35.10 -- I want to be there when it happens. And you know, we
00:05:39.14 were sort of committed to each other at the beginning
00:05:42.06 when it happened.
00:05:43.03 All scientists want to share their results with a broader community.
00:05:48.13 They want to make an impact, make a contribution, and so on.
00:05:52.21 But this is a more intimate thing. It's just your day to day
00:05:55.26 operation, day to day thinking about science, somehow it's
00:05:59.23 just a whole lot more fun to share it with a friend if you can.
00:06:02.22 It's just great.
00:06:03.22 So the first thing is that it's fun to work with somebody,
00:06:06.23 but the second is that there's a potential story that lies beyond just
00:06:11.25 having fun with each other. Some insight that you wouldn't have
00:06:14.19 if you were working alone.
00:06:16.13 Well there's a whole other end. Let's say I might come out to Colorado
00:06:20.24 because I want to bring some specimens and work with somebody who's
00:06:24.25 really good in a mass spec lab. That doesn't even touch the kind of thing we're
00:06:30.17 talking about here. Scientists work together sometimes because
00:06:33.07 science has become so complex and so specialized that you can
00:06:37.20 get things done by working together. And you have to give up
00:06:46.08 the kind of, I want to do everything myself, I want the
00:06:48.23 the glory. You need to know how to share and the ethics
00:06:54.17 of sharing, and the ethics of writing papers together,
00:06:57.26 and all that. But that's a different issue.
00:07:01.27 Yeah, that's the issue of symbiosis in science. And yes, that's
00:07:06.11 a critical ingredient. But this is a more personal thing, it's just a
00:07:10.15 lot more fun to do it with a friend, and particularly when
00:07:15.26 you've been working together for a long time and had a common
00:07:21.15 struggle. A common goal. And it just feels that much sweeter.
00:07:26.19

Videos in this Talk
  • Part 1: Imaging synaptic vesicle transmission
    Part 1: Imaging synaptic vesicle transmission
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: The Future of Electron Microscopy
    Part 2: The Future of Electron Microscopy
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 3: Why Collaborate?
    Part 3: Why Collaborate?
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
Speakers: Thomas Reese, John Heuser
Total Duration: 0:40:06
Recorded: July 2015
All Talks in Neuroscience

Talk Overview

Two pioneering electron microscopists, John Heuser and Tom Reese, reminisce about their early experiments to understand synaptic vesicle transmission.  They describe the development of freeze fracture electron microscopy, a technique that allowed them, for the first time, to image events at the nerve terminal just milliseconds after stimulation.

In their second talk, Heuser and Reese explain why they are still enthusiastic about the future of electron microscopy even in the age of “super-resolution” light microscopy.

And, in their third talk, Reese and Heuser explain why they have remained close friends and collaborators for years, despite occasionally fighting like cats and dogs.

Speaker Bio

Thomas Reese

Thomas Reese

Tom Reese is a Senior Investigator at the National Institute of Neurological Diseases and Stroke at the National Institutes of Health and a summer researcher at the Marine Biological Laboratory. He continues to study synaptic structure and function using advanced light and electron microscopy techniques. Reese was elected to the National Academy of Sciences in… Continue Reading

John Heuser

John Heuser

John Heuser is a Professor Emeritus in Cell Biology and Physiology at Washington University in St. Louis.  Heuser developed and spent his career using quick-freeze, deep-etch electron microscopy to study all aspects of cell biology.  In recognition of his work, Heuser was elected to the National Academy of Sciences in 2011. Learn more about Dr…. Continue Reading

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