Scientist Stories: Ron Vale, Molecular Motor Proteins & the Cytoskeleton

Good evening. My name is Ellen Lumpkin. I'm a co-director of the MBL Neurobiology Advanced Course. It is a great pleasure to welcome you here tonight to our Monday night seminar series. This is a long tradition in the MBL Neurobiology Course. And it's a particular pleasure because tonight is our third annual Thomas Sargent Reese Lecture. And to introduce Tom and the lecture, I invite Joanne Buchanan to come to the podium. Hi everyone, thank you, Ellen. I'm here to introduce the Thomas Sargent Reese Endowed Lectureship. So Tom was a member of the neurobiology course for 40 years. He taught hundreds of students over that time, and his specialty is electron microscopy. So his favorite thing was to have students sitting at the console of the electron microscope, looking at the green screen, and teaching them about the beautiful ultrastructure of neurons and synapses. And he was so generous, he opened up his lab used to be in the basement of the Lerb building, and allowed the entire course to take over, including all the students and faculty. And many people over the years have fond memories of working with Tom. So you're going to learn more about Tom tonight from Ron Dale and their wonderful collaboration together. I also want to thank the people who gave generous donations to fund this lecture. Membership, so we can honor Tom year after year. And with that, I'm going to have Dave Finkel come up.

Thanks, Joanne. It is my distinct pleasure to introduce tonight's speaker, our speaker for the 3rd Thomas Sargent Bees endowed lecture, Dr. Ronald David Vale. Dr. Vale has been a professor at the University of California in San Francisco since 1986 and an investigator at the Howard Hughes Medical Institute since 1995. Dr. Vale has recently been named executive director of the Janelia Research Campus and the vice president of the Howard Hughes Medical Institute. Dr. Vale earned his bachelor's degree in chemistry and biology at the University of California, Santa Barbara. He then entered an MD-PhD program at Stanford University, where he did his dissertation on nerve growth factor receptors and axonal transport. Research at the Vale lab employs biophysical, cell biological, and microscopy techniques to study the microtubule-binding motor proteins kinesin and dynein. Together with James Budich and Michael Sheets, he was awarded the 2012 Albert Lasker Award for Basic Medical Research for their discoveries of cytoskeletal motor proteins that convert chemical energy to mechanical work, which allow our muscles to contract, our cells to divide and migrate, and in the case of Bale's kinesin, delivery of molecular cargo to the tip of axons. Dr. Bale is not only a scientist but also an educator. Having founded iBiology, a nonprofit organization that produces and disseminates free online videos by leading biologists speaking about biological principles and their research, as well as scientific training and professional development of practicing scientists. Ever an educator, Dr. Vale co-directed the physiology course from 2004 to 2008 here at the Marine Biological Laboratory, transforming that into an interdisciplinary training environment that brings together biologists, physicists, and computational scientists. Vale's recent project, The Explorer's Guide to Biology, or xBio, is a free online undergraduate textbook that provides a storytelling approach to learning biology. His lecture tonight is titled An Eye for Biological Structure: Lessons from Tom Reese. So without further ado, please join me in welcoming Dr. Vale to the podium.

Well, thank you for that very kind introduction. At least I remembered what a podium is. I haven't been at a podium for about a year and a half, but I guess I can take this off because other people did. But I couldn't think of a better time to restart my— oh, this is like all complicated. Complicated, isn't it? Um, than by giving this third annual Tom Reese Lectureship. It's a great honor for me to do this. As you'll hear, Tom is a hero of mine, a great mentor, and a great friend. It's great to talk to you here honoring him tonight. Also great to be introduced by Joanne. We have a very long friendship at the MBL going back to the '80s. We and a bunch of people shared a house together, including her now husband, Stephen Smith. And I think this is something very special about the MBL that many of you will experience, that you do make lifelong friendships here that start at the MBL. And that's part of the MBL magic. So yes, today I'd like— title of this lecture is An Eye for Biological Structure: Lessons from Tom Reese. My lecture will not be so much neurobiological. I'll talk about some of the early work that was done with Tom, but I'll then transition into some recent research that involves electron microscopy. And I think I probably wouldn't be doing that electron microscopy if it wasn't for that experience with Tom many years ago. Perhaps many of you didn't read the abstract, but this is how I started this abstract for this particular lectureship with this quote from Richard Feynman. And of course, Richard Feynman, a Nobel Prize-winning physicist and, you know, quite flamboyant scientist in general. Said it's very easy to answer many of these fundamental biological questions. You just have to look at the thing. And I do believe there's actually a lot of truth to this. You know, we've now kind of catapulted in this age of genomics, but I think there are many believers that still view the power of seeing what biological structures look like and the incredible amount of information embedded in those images. Now, I then continue the abstract, again for those of you that read it, that, you know, I do believe what Feynman said is correct, but what's missing from this quote is that it's not just so— it's not so easy just to look at the thing. I mean, it makes it sound like Well, maybe if you're, you know, studying subatomic particles, it may sound really easy. But, you know, looking at things in biology is, in fact, a real skill. And in Tom's case, I would even frame it at the boundary of science and art, even. And what the abstract then continues is the looking involves technological innovation. It involves deep experience in sample preparation and knowing what organism and method is best for a particular question. It involves patience of achieving a clear and not a confusing result. And all of those things, I think, summarize Tom's achievement in science and as a microscopist. And that's how he really stood out, that not everyone can just look at the thing. It takes all of these skills to come together embodied in, in a great scientist like Tom, who could actually look at things in the way that basically all other scientists, or very few scientists, could. So, you know, this was, as Joanne said, LERB looks very different right now, but the basement was almost like Tom's Temple. That's the only way I could describe it. It was a little bit chaotic. It doesn't look like LERB now. There were— it was chaotic, you know, rabbit warren of rooms down there and microscopes and chemicals and all kinds of stuff. But that's the environment that was Tom's lab. He was an NIH scientist, but he had a full-time lab at— in the Lur Building. And that's where every summer he opened up his lab totally and completely to the neurobiology course students that were roaming around there doing experiments. Um, uh, and he just viewed them as a continuation of his laboratory. So your share is stuck on the first slide. You want to try restarting the share? Yeah, you know, this is the Zoom, uh, hybrid world that may be slightly problematic. And I know what the problem is, and I've never tried a hybrid talk, but okay, let me, let me do this, and then we may have to figure this out, but I think it'll work. In Keynote, you have— yeah, it won't go full screen, but It will— yeah, I can't go full screen and do this on Keynote. So anyway, I just learned about this. Okay. Yeah, okay, this will work for both audiences. I should go back to my Zoom audience and I I do hope that Tom is here listening, and if you are, hello, Tom. And yeah, so this was just the quote that I put up already that I guess the remote audience couldn't see, but I'll just leave it up here just for one second so people in the Zoom world and maybe Tom can just see this quote and statement. Okay, hi Tom. I miss you. I'm glad you're joining. And I'd love to give you a call later. Okay, so this was also the slide, Tom, that I showed that you couldn't see, but Tom in two of his native environments. One with, um, uh, in the lab and one with, uh, uh, one of his prize poodles. I don't know if this is Wicked or, uh, we're, we're speculating that this is Wicked, but, uh, also one thing that you have to know about Tom, especially when we go to, uh, his science, um, when Tom decides to do something, he doesn't go halfway and with science, as you'll see, he was all in. I mean, more than all in. But he was the same way with any hobby that he took up. So, you know, like I have a pet, I have a pet dog, but, you know, Tom has, you know, prize-winning retriever poodles, you know, that won like competitions around the United States. You know, when Tom picked up tennis, you know, it wasn't for like light recreation, you know, he got a coach and he trained and trained and trained and trained. And, you know, he went from not knowing how to play tennis at all to, you know, really being one of the best tennis players at MBL. So, you know, that's Tom, he just has He's full in when he wants to do something, he's full in and commits to it with utter passion. So, and this is just to say a little bit before I get on to our work, I just want to say a few words about, I mean, Tom accomplished many things throughout his career, but certainly one of the most important scientific pieces of work that Tom did is this collaboration with John Heuser. And I don't know if you're learning about this in the course. I hope you are. What I, what I would say about this is, first of all, in my opinion, this is really one of the great experiments in science overall. I'm not talking about neurobiology, I'm talking about biology. And in my view, too, is also one of the incredible collaborations between, you know, Heuser and Reese. I mean, you think of other great ones like Brown and Goldstein, Messelson and Stahl, Watson and Crick. This was actually much longer lasting than Watson and Crick, but this was really one of the great team partnerships in science that lasted many years. And you can actually, um, learn about it. There's a, a, about 11-minute lecture by Tom, uh, on the Heuser-Ries experiment on eye biology. And there's also, um, fantastic, uh, interview between Tom and John Heuser where they kind of talk about their scientific partnership. But the essence of the experiment was that, you know, one of the foundational things in neuroscience is understanding how two neurons communicate with one another and what the mechanism of chemical release from one neuron is to the other. And there were indications that that chemical release phenomena occurred as kind of quanta that stimulated the postsynaptic cell. But what Heuser and Rhys did was really to kind of solve the cell biological mechanism of how that occurred. And these sets of experiments that kind of evolved over a period of at least 5 years involved showing that synaptic vesicles indeed fuse with the plasma membrane. And that once released, there's a recycling mechanism involving clathrin-coated pits that retrieve that membrane again, allowing the synapse to, you know, re-engage in repeated rounds of chemical release through these synaptic vesicles. And what they did to do that is also the latter part of the work to really demonstrate that synaptic vesicle fusion occurred at the plasma membrane was that they had to build a special device. So you could not order this, but what they realized they had to do was to stimulate a neuromuscular junction and then rapidly freeze the sample within milliseconds. So, because after that, the release mechanism already occurred, and they wanted to catch it in the action. So, what they did was a truly heroic experiment with this device over here, where they would stimulate the nerve. And within literally milliseconds, and exactly around 4 milliseconds after stimulation, they could time the time when the sample hit this frozen helium-cooled block and rapidly freeze the sample so they could do EM. And this experiment took them years to really perfect, develop the apparatus. But they knew that this was an incredibly important experiment to do. They didn't— they worked on this experiment together. It wasn't a team of graduate students and postdocs. It was like Tom and John kind of working. It was actually even a long-term relationship. Tom flying to San Francisco to do this experiment with John. But, um, uh, at, at, well, yeah, I mean, two things to know about, um, uh, both John Heuser and Tom Rees is, um, first of all, they're both incredible, uh, scientists. Um, But they, they also are the most driven. They're probably two of the most driven people I've ever met, both of them together. And when they wanted to do this experiment, there was just nothing that was going to get in their way. And they worked on this, uh, with such amazing dedication until they were able to solve this problem. And What I love— this is actually a quote from Tom as he ended this short 11-minute talk that he gave for iBiology. And this really just came out of his mouth at the very end. I don't think it was even planned. But, you know, he said at the time, because they were so dedicated, they almost had no they kind of shut out a lot of other things of their life just to work on this experiment. And Tom said to John, "John, why are we doing this?" And he said, "Well, you know, Tom, the most important thing in science is to be together with a colleague at the moment when you discover something, when some little piece of nature unfolds and you see something new." And I just think this is incredible. And this is also just, you know, almost this quote of why we do science and why it is really so important, you know, to have this passion for really understanding what nature is and to try to— all of you as students or postdocs or faculty, whatever, you know, to have this hunger of really discovering something new about how nature works. And, you know, that is all I could say is pretty much Tom Reese. He had that passion. Now, now I'm going to transit a little bit more into work that I did with Tom, but I should also say that in general, from that Feynman quote, there are two ways to look at the thing. And one is the snapshot. Single image. That single snapshot is often very powerful if it's done at high resolution. It's a static image. And the other way to look at something is with a movie, which is more obviously involving light microscopy. And, you know, the two together really complement one another, and that's what I'd like to illustrate the high-resolution snapshot, you know, together with the dynamic information of light microscopy in combination is often what lets you tease apart how living organisms work. Okay. So this was my time with Tom at the MBL. I think well before many of you were born. Nevertheless, Mike Sheets, who's on the left, and I came from Stanford. I was a graduate student at the time. And we hooked up with Tom and Bruce Schnapp, who's next to me. Yeah, that is me. Hard to believe. And like many people, Tom actually kind of took us into his laboratory. Was excited to work with us. And as I'll show you in a second, you know, Tom's lab with Bruce kind of also opened up my world to a whole new way of doing science as well. So here was the problem that we were interested in working on, and that is how material is transported down an axon, so called axonal transport. And as you know, this is a neurobiology class, so excuse this basic drawing, but, you know, nerves are— a neuron can be the long— you know, the largest cell in your body by dimension. The cell body, for example, of a motor neuron is in your spinal cord, and nerve terminal could be at your foot. And there's a— could be a 1-meter axon in between. And for many years, this process was studied by injecting radioactive material into the cell body. Ray Lassig did a lot of the seminal work. And then what you could see then— of course, these are static snapshots. If you did in a rat, you have to take the— wait a day, for example, take the nerve out of the rat, chop it up, and figure out how far that radioactive material, which was, for example, amino acids, radioactive amino acids incorporated in protein, how far it moved down the axon. So as you can imagine, those snapshots were very laborious work to do. And, but it was known that material could move down this axon. And in a person, for example, it would take about 2 days to go from the spinal cord down to your foot over that kind of dimension. Okay. So making a long story short, but like many things in biology, you have to pick a good model organism for a question. And a good model organism for this question of axonal transport was a squid. Slightly different reasons, but for the same reasons that Hodgkin and Huxley used the squid. It was possible to dissect out the squid, the axon, the giant axon from the squid. Squid have this axon that's about 50 times larger in diameter than a human axon. You can actually see it with your eye. You can dissect it out of the squid and you can study it. Now Hodgkin and Huxley were more interested in the membrane and not— they kind of threw away the inside of it. But at this time in the early '80s, people were interested in what was happening inside the axon for how material moved. And there were many people here working on this problem. And a particularly exciting thing that happened in this era was really a revolution in light microscopy, which was quite amazing because all of you take it for granted now that you're using microscopes and their fancy cameras and computers and robotics and all of this kind of stuff. Some of the microscopes even now look like black boxes. You don't even know what they look like. They do everything for you now. But But there was an era, believe it or not, when you actually looked through your eyepiece of the microscope. And there— and at this era, there were two groups, Shinya Inoue, you know, two real giants of microscopy, Shinya Inoue and Robert Allen, who And Rudolph here was from Shinya's lab, worked with Shinya for many years. So you could talk to him about Shinya's many achievements. But— The microscope is in the Lilly lobby. Oh, the Shinyascope? Oh, it's in the Lilly lobby. OK. Oh, that's great. I have to check that out. So you can see one of the historic microscopes that Shinya built in the lobby of Lilly. Anyway. What these two individuals— they did many things, but relevant for this, what they did is to put, instead of looking at the eye, they put at that time analog cameras on a microscope and also developed ways of increasing with some like kind of simple even at the time analog-based electronics to amplify the contrast level from the camera images in ways that you could see much higher contrast in the sample than you could ever see by eye. So what you see here is a movie made by Robert Allen with one of these video-enhanced contrast microscopes. And if you look at the squid giant axon with your eye with the same microscope, you would see almost nothing. But with the camera and the contrast enhancement that could be achieved, this is what Robert Allen saw, which was the inside of the axon was full of life and things were moving up and down. Now, this basically in like 5 minutes completely replaced all the dynamic information that you would get from a radioactive tracer experiment. Because here was this rich movie image where you could see everything that was going on in the axon. Now also compare, you know, the static image which also Schnapp and Reese were working on at the time. And this shows an electron micrograph which they were looking at what the inside of an axon looks like. And you can also see from this that, you know, realization that the inside of the cell is not a bag of liquid at all. In particular, the axon is absolutely packed with material. Most of this, what you see here, is neurofilaments. Some of it is microtubules. But it's an incredibly dense environment of fibers. This was the movie that you saw, you know, from the Allen Group. And, you know, I still think it's a fascinating— I still think it's a fascinating problem today, even to understand how material can move through this, you know, jungle of filaments right now. And there are probably even dynamics of how these filaments are, you know, moving and how what you see as static cross-bridges between filaments here must have some dynamic flavor as well to let material pass through it. Okay, so after that, one thing that Robert Allen's group did and we also did with Tom was to begin to try to tease apart this system in a test tube. And what you could see here is now the squid giant axon out of the squid and out of the axon and the material kind of mixed up and put on a microscope slide. And now here with this video microscope, which now Bruce Schnapp built in Tom's lab, you can see these now discrete filaments, and that's a mitochondria that's moving its way down the middle of the screen over there. These long sausage-shaped things are mitochondria and a whole variety of small filaments. And now I'll take a pause because we, we— some of that work of how we made that prep, but I want to tell you something else about Tom. Tom was the perfectionist of an image. I've never known anyone like it. Joanne's laughing. But, you know, like, he had a full-time person that produced photographs in the darkroom for all of the electron micrographs that he published, or even the light micrographs. And he would have this person work in the darkroom. Now, this is, you know, pre-digital, right? Work in the darkroom just getting the contrast right. And would produce like literally, like, could be 100 prints to get the one that showed, you know, the kind of contrast and detail that he felt he could see even in the negatives or with the image. So, So every image that Tom produced in print, well, it was kind of like a work of art. But now going back to the science, this— what we saw under the video microscope at the time was some filament. We had no idea what the filament was.

It—

by the light microscope, there is no information there. All these filaments appear at the resolution limit of the light microscope, which is about 250 nanometers. They could have been bundles of actin. They could have been microtubule. It could have been a bundle of microtubules, combination of actin and microtubules. We didn't really know. So here's my second, uh, uh, heroic experiment to tell you about. And this is an experiment that Bruce Schnapp and, and Tom did, and largely I was an extra pair of hands and watching in admiration along with Mike, and they really did everything that I'm going to tell you about. But the heroic experiment was to first shadow with carbon on a glass coverslip a grid pattern. Now this grid pattern was like a little map. So you could find any corner or any square of that grid would have a coordinate system that you can go back and find later. And the experiment started by taking the squid giant axon and putting it on that glass coverslip, putting it in the light microscope, and then watching it. And then you can see things moving along filaments. What I should say is a lot of these preparations, you know, necessarily didn't work. But then ones that worked well, to transfer that from the light microscope to that freezing apparatus that I showed you to rapidly freeze it and then etch it with metal and then look at it under the electron microscope. And the goal was to find a filament that we could see with a clear pattern under the light microscope and then go to that same coordinate system and find the exact two same filaments under the electron microscope. And in my memory, although I'm trying to remember if Bruce said it was even a larger number, in my memory it took 43 attempts to get this experiment to work. And, you know, just one thing or the other prevented it from working. It was— there were many, many steps in the sample preparation to get to this final point. But then, as in a perfect Tom Riesen experiment, if you do the experiment right, you get a definitive answer and there's no question about it.. And, you know, that again was, you know, I told you about synaptic vesicle release and recycling. Here the image again was absolutely definitive. The first sample, and then of course we did more than that as well, showed that these filaments were single microtubules. And also what was interesting about that was that single microtubules support recorded organelles moving in two directions. So that indicated that even a single microtubule filament was a two-way highway. So anyway, that was an incredibly beautiful experiment, an important experiment. It told us what the substrate for movement was, and that information allowed then me to use some of my expertise that I learned at Stanford as a graduate student, where I learned how to also purify proteins and reconstitute things. I learned a lot of biochemistry there. I learned microscopy at the MBL, and, you know, that combination allowed the discovery of kinesin in Tom's lab. And that also proved to be a very— this combination of biochemistry and microscopy basically has driven kind of a lot of my research ever since. But this just shows the nature of the experiment and why that EM was so important. What you're seeing on the left here is that same image. Here we didn't know what those filaments were, but when we knew that they were microtubules, now with biochemistry we could get microtubules actually from another source, from a pig brain. Purify the tubulin, and reconstitute microtubules that are absolutely clean. They don't have anything else on them. There are no other associated proteins. Clean microtubules. And what you see on that right image is some of those purified microtubules in lines over there. So that's part of reconstitution. You want to purify things if you can. But the other thing interesting about this experiment that I'll show you is— a long story, but we were able to get rid of the native organelles, all those mitochondria and small vesicles, and instead of organelles, replace the organelles with plastic beads, and found that these plastic beads picked up some motor in what was a kind of a soluble fraction, has a lot— basically a fraction that had lots of free soluble proteins from the squid axon. Some of those proteins would stick onto those beads and they would move these beads, plastic beads, just like organelles. And this reconstitution basically then led to biochemical fractionation of finding what protein caused those beads to move. So, and that led to the discovery of kinesin in Tom's lab. I'll say a word about how motor proteins produce motion. They basically take a high-energy chemical, ATP, they hydrolyze the gamma phosphate bond, then they release phosphate, they release the ADP, and during one of these chemical cycles, as I'll show you in a movie, what the motor does is it takes a step, and it hydrolyzes another nucleotide, it takes another step. So this chemical cycle gets coupled to the motor stepping along tubulin subunits along the microtubule. But the question is, how does a motor protein do that? And to solve that problem, I will still use the phrase, you just look at the thing. And here, for example, just looking at the thing is an image of a horse galloping. This was a famous high-speed videography made in the 19th century. Of watching a horse gallop, and there was a debate about whether the horse had its feet off the ground or always had a foot on the ground. And anyway, this movie illustrated how the horse moved. So again, solving this problem was a combination of— oh no. I have a power problem here, or there will be about to be a power problem. No, oh wait, I think we're good, we're good, we're good, I think we're good. So You know, the two approaches here basically to solve how a motor protein takes a step was to look at the thing. And you had to look at it in two ways. One is looking at it at very high-resolution snapshots. And that was either cryo-EM, which was not as high resolution at the time as it is now, but that provided some information, or getting X-ray crystal structures. That gave us static images. And the second element was getting dynamic images of what this motor protein was doing at a single molecule level. And I'll, I'll talk to you about that later. But the combination of that high-resolution information allowed one to actually make— this is based upon atomic resolution information— know exactly what the microtubule looks like, how the motor binds to the microtubule, what all the details of the chemistry that is happening in the active site, could understand that. So this animation is really based upon high-resolution snapshots, and this dynamic information here was based upon putting probes on this motor protein to understand some of the dynamics of the steps that, that you see here, and also some of the conformational changes that occurred. Now, I'll tell you one last story, and then I'll, I'll try to move into modern times, although I'm somewhat slow here, but tell you a little bit about some recent work. But here's another story that if Tom is watching this, he probably will remember this, and that is, I've kind of reenacted this in my own house with a paper napkin. But for a while, we could only get unidirectional motion from that, that bead preparation that I showed you. You could see all those beads were moving in one direction, and that was the kinesin direction. One day, by a long story, by a mistake, we kind of made a mistake in the way we prepared those soluble proteins from the giant axon. And that was actually a time that Tim Mitchison was visiting the lab at Woods Hole. But sometimes accidents or mistakes or when you do something not quite right in the lab can turn out well. We made a kind of an error in that preparation. And we made a preparation that showed something different. The beads were now moving bidirectionally. And, um, uh, I was so excited about this because we were looking for a possibility of another motor. And I put this sign on Tom's door, um, and then Tom was like overjoyed and thrilled. And he also kept that paper towel on his door, I think, for years and years and years. But it is kind of these joyful moments where, where, you know, you, you can share some discovery. And, um, it was also fun that Tom kept that paper towel for quite a long time, uh, on his door. Now, so we knew that there was another motor. It moved in the opposite direction. Uh, it had characteristics of dining. And a year later Richard Vallée showed that indeed it was dynein that from cytoplasm that there was the minus end-directed motor. So kinesin goes in one direction, dynein goes to the other. And what I should say is that dynein was actually a known motor. It was discovered many, many years ago, and it was involved in in ciliary beating. So dynein has a form that's present in your axon and a form that's present in cilia. And I'm looking at the time now, and I think I'm just going to tell you one story. But the reason I showed that is our lab shifted from working on kinesin. We were understanding the details of how that worked. We then shifted our lab to work on dining. It was also a very exciting time in the lab. Had a chance to work with another one of my great heroes and mentors, Ian Gibbons. Ian discovered dining in 1965. And he moved to the Bay Area, and we had a very nice chance to work with him. And also a great group of postdocs joined the lab to work on these problems. I don't need to work on dynein anymore because they're all off on their own labs doing amazing things. But also, I just want to show you, this is what dynein looks like in comparison to kinesin. So it is a massive motor protein. It's one of the largest proteins encoded by the genome. So it's a monstrous machine. The ATPase site is in that ring structure in the head, and it has this long coiled coil stalk, almost like a stilt, going down to a foot. And it's this tiny foot that's actually stepping along the microtubule track. And I did have two stories here, but I think I'm just going to show you— I'll skip the dynamic story and I'll talk to you about this recent work that we're doing because it involves EM, which is Tom's obviously favorite thing. So I'm going to skip through this. This is a bunch of data of dining, how we measure dynamics of dining, but I am going to Skip that. This, believe it or not, could have gone very fast. I'm just going to show you the movie. So, uh, interesting thing about how we think dining works is in Kinesin, the two heads are coordinated. You can see it and it steps in this regular pattern. Here, best of our understanding is that the two heads of dynein work very independently. They're somewhat chaotic. In fact, sometimes dynein moves backwards as well as forwards. The heads— dynein can move to lateral protofilaments. So it has a very, very different kind of walking mechanism than kinesin. But let me just tell you a problem that I think is really exciting in this field right now, and one that I think electron microscopy is already providing many answers. And I think maybe in the next few years, this is a time when this problem may actually be solved. And that is how cilia and flagella work. So dynein is a motor that is moving cilia and flagella. This is a Chlamydomonas that you see here that shows the beating pattern of these cilia. And this beating pattern is produced by this incredibly interesting and complicated machine. It's really one of the most elegant and to some extent still mysterious structures in biology, which is this axoneme. And it's made up of microtubules. You probably have seen this from biology classes, but it has these 9 outer doublet microtubules surrounded by a central pair. And this is conserved through evolution from ancient unicellular organisms to man. And it's made up of hundreds of proteins. And How this machine works is, is still very poorly understood, but I, I could illustrate what, uh, the dynein motor is doing. The dynein motor is located on these outer doublets, and these dyneins are interacting with and walking along the neighboring outer doublet. Um, now we know that's true because Gibbons did an old experiment many years ago of taking the membrane away from the axoneme and lightly trypsinizing all of these bridges between that connect these outer doublets. And what he found is he added ATP and these microtubules would slide past one another. So the microtubules would just— neighboring outer doublets would just undergo this linear motion and just slide. Is it plugged in? God, it's—

yeah.

Oh, it looks like it's working. Oh, there must be something wrong with my power cord. Okay. Thank you for saving the day there, Ellen. Appreciate that. Um, so back to electron microscopy, and this is what I'll talk about. I think, you know, you've probably seen drawings like this in your textbook, and now is a time where really electron microscopy is shining, that we can now see, uh, what these structures really look like, uh, approaching now, even in the whole axoneme, close to atomic level resolution. So I'll give you a couple recent pieces of work, both involving electron microscopy, of trying to understand the structure and how it works. So In addition to the dynein that I showed you that's interacting between these outer doublets and causing them to slide, there are these interesting radial structures here that point to the center of the axoneme, and they are called the radial spokes. And somehow these radial spokes are required for converting this sliding motion into a, a beating pattern. Because you can imagine if all the dynines are walking at the same time, no one's going to be getting anywhere, and you can't create, you know, a beating pattern. Dynines have to be active on one side of the waveform and inactive on the other, and somehow these radial spokes are responsible for converting dynine between active and inactive states. To allow this beating pattern to occur. And if there are mutations in the radial spoke or they're absent, the flagella is completely immotile. So there's something about this particular structure that is required for creating these kinds of mechanical-based oscillations. So to understand the radial spokes, what Iris did was to reconstitute 12 proteins, express them in baculovirus, purify the proteins— this is a pretty monumental experiment just to purify 12 proteins— and then was able to do a single particle cryo-EM and get a structure of what this radial spoke head looks like. So that's that little ball that you see at the very end of that structure. And we're right now looking at it head-on. If you were the radial central pair there, this is what you would see, that particular surface there. And One thing that's very interesting about that surface— so this is the surface that's contacting the radial spoke to the central pair structure— is the end of this radial spoke head, first of all, is amazingly flat. It's pretty actually hard to create even a structure that is so molecularly flat. That's one interesting thing. So this is looking at it like the head of the anvil and this is like the side of the anvil. And the second thing is that this face there is highly negatively charged. The red is negative charge, blue is positive charge. That whole face there is negatively charged. The reason why— I should give you the punchline. The reason why we think that is probably true is that when the axoneme is bending, we think that these radial spokeheads are actually mechanically hitting this central pair, and that mechanical, like, collision is involved in some kind of mechanical feedback that is controlling the dynein. Um, and if you want these mechanical collisions to be elastic We suspect that there, although we don't know what the structure is like in the central pair, that there may be a matching surface of also negative charges on the central pair that prevent these protein structures from basically sticking to one another and elastically recoiling. So these negative charges are highly conserved. And one experiment that we did, then Eurus did, in Chlamydomonas was to change some of those negative charges to either neutral charges or positive charges and test this theory whether these negative charges are important, put this mutant back into the clammy, and would see what happened. And this is a wild-type clammy over here. You can see it's swimming just fine. And this is the mutant clammy now, where these mutant radial spokehead protein that had the charge changed at the surface, you can see that the swimming pattern of these— it still swims, but not swimming as well. And you can just see that if you analyze the images over here. So Iris also looked at mutations in the radial spoke head. So it turns out that there are human diseases that are called primary cilia dyskinesias. They often have problems with respiratory cilia, so have bronchitis, infections. Sperm in males are often dysfunctional, and there are a lot of families right now with these point mutants. And Iris also then began to put these human point mutants into her recombinant protein and ask, can she see any difference of what these point mutants, these human disease mutants, would affect the protein complex. And the bottom line is that a lot of these point mutants here, even a single point mutant here, can by EM entirely disrupt the particular structure. And that was a dramatic mutant. But some of the other point mutants that still produce protein structures, if one does what's called a thermal denaturation, which tests how stable the protein is— this is what a wild-type protein looks like. It melts at about 42 degrees, or it starts to unfold. Protein structure starts to unfold. But these human disease mutants push the denaturation of the protein to lower temperature. So the bottom line is we think a lot of these human disease mutations are really protein stability mutations and are somehow affecting the stability of this entire radial spokehead structure. And lastly, and I'll make this very short because I realize I've run a full hour, but we're also using cryo-EM to look at the mammalian sperm. Mammalian sperm, a lot of people have studied Chlamydomonas and these ciliated epithelial cells, but mammalian sperm have been very poorly studied by cryo-EM. And the sperm structure is also very different. That's to scale. That is a mouse sperm. It's about 100 microns long. And compared to a Chlamydomonas, that little flagella is only 10 microns. So it's a very, very different structure. And Zhen Chen in the lab with a group at Janelia have been asking, are there any differences in the axoneme structure between mammalian cilia and flagella? And what they've been using is a technique called cryo-EM tomography. That's a— I'll just go to this slide— basically a technique where you can create a very thin structure of the axoneme on ice. You take pictures at multiple angles, and then you can use those multiple angle pictures to reconstruct a 3D image of what that axoneme looks like. And that was the image in great detail. And what Jen has found so far, and this is all unpublished work, is that there are structures in sperm flagella that have never been seen before in other kinds of cilia. So this is just one example here. This is an outer doublet over here. So these are looking at the inside of the two outer doublets, and this is the same structure in a cilia. This is from Tetrahymena, but they all look fairly much the same. And what Jen has found is that the sperm has a completely new structure, an additional structure, that is sitting inside of one of the tubules. And I'll just show you what that looks like in this, in this image over here.

Oh, shoot.

So this is what this filament looks like. So it's an entire mysterious second filament residing inside the microtubule. We think it may have something to do with the mechanical needs of this mammalian sperm being so long. Maybe this adds extra rigidity to it. But it's a new structure that you know, hasn't been seen, and we're going to have to figure out what kind of proteins make up that structure. And I'll try to be brief here, but he's actually found kind of a gold mine of additional structures in mammalian sperm that do not occur in axonemes from cilia. So here's just another example. These are the radial spokes that I showed you. Here are the flat heads.. And in the sperm structure, there are these, you know, quite large additional structures here that we actually don't understand what they are, what they're made out of, or what they're for, but they have something to do with the very specific movement of cilia. And I think I'll just skip this. And I'll just tell you what I think right now is the big problem in this field, which is, you know, what I alluded to. This is the radial spoke head that I showed you before. It's contacting the central pair. And these are the dynamotors in purple here that are somehow being turned on and off based upon some kind of mechanical interaction between this radial spokehead and that big central pair structure. And, you know, this is the big question in the field right now, which I don't have the answer to. But somehow there must be some signal being transmitted, probably even mechanically, from this central region where these collisions are occurring to change the structure of this dynein so it's being turned on and off during this mechanical wave beating. And so I think that's the next question that we'd like to know, probably again using electron microscopy to see if we can see that kind of structural change. That must be occurring. And probably some, all of these structures along this journey to this final dynein molecule are changing in subtle ways to relay some signal to turn the motor on and off. And I think if we understand that just by looking at the thing, then maybe we'll understand how cilia and flagella work. So no answer as of yet. But I'll just close by saying, first of all, thank you, Tom. I owe a lot of my career to Tom. He took me in along with Mike Sheets as a graduate student to be able to work on that problem. I really learned the beauty of microscopy from him. At Stanford, I was trained as a biochemist. And I came to MBL, and I became a microscopist.. And I really owe that to Tom. And I've learned a lot of how one can study biology just by looking at the thing. And I'd like to— I didn't have a chance to talk about Stefan's work, but Jen and Iris are doing this recent work on sperm and the axoneme and trying to understand their structures. And sorry for going a little long, but anyway, thank you for your attention. So any questions? Yeah. Is that Tony? I can't tell anyone here.— you know, wearing masks. Yeah. Yeah. The question is, is that filament the The sperm is very long, the axon in sperm is very long and it has even kind of different parts to it. That is a good question. I don't know whether that is kind of in the middle region. It's not, I mean, there's a region that's closer to the head, you know, the midpiece where there are mitochondria. I mean, maybe Jen knows the answer. I don't. I think it's present for much of the length of the axoneme. You know, whether it extends all the way to the very base of the axoneme, you know, close to the head, I don't know the answer to that. [Speaker:JOSHUA_ELKINGTON] I was thinking maybe it might not extend all the way to the tip. [Speaker:MICHAEL_STRANGE] Oh, I see, to the tip. Yeah, that's a good point. I don't know the answer. I don't know the answer to that or whether he's really looked at the far ends of the axoneme. Actually, great. That's a great point about the tip. So I'll pass that along. So thanks, Tony.

It's good.

Good suggestion.

Yes. So the structure is to have kind of a radial tube creating a linear motion.

It's something that's in mechanics today. Yeah. The one question I had was about the— what did you say about the ATP going— Well, I mean, the dynein itself is creating a linear motion upwards. It's trying to push one of the— I mean, interesting thing is they're connected at the base, at a basal body, but the dynein is actually sliding microtubules towards the tip. So it— the dynein itself is not really creating any kind of circular motion. It's just trying to push its neighboring microtubule towards the tip. And that is what happens. So if you proteolyze everything, what you see is these microtubules just sliding to the tip. What I should say, there's also a very nice piece of work from Danielle Nicastro's lab, which I didn't describe, but which I showed you in that movie of dining. I showed that animation of dining and walking. I didn't really go into it, but part of that dining was colored yellow and red, and there was part of that dining that had an arm and it was changing its state, the position of that arm, you could tell whether the dynein is kind of in a waiting state or an active state. The work by Nicastro suggests, interestingly, and this was surprising to many people, a lot of people thought that dynein may be inactive, and only on the side that is creating the bend would the— would on one side, would the dynein be active? It looks like the opposite is true, that most of the dyneins are in— around the axoneme are in an inactive state. So they're all trying to move upward towards the tip of the flagella. They're all trying to move their neighbor, which doesn't really get you anywhere. But her study suggests that dynein on one side to create a bend, dynein is actually being inactivated. So something is inactivating the dynein. The dynein on the rest of the circle is active, and the dynein that's inactive kind of loses out, and basically a bend gets created there. But anyway, the motion is actually trying to move, move towards the tip. Yes. Hello. That's very nice. Well, Tom, you created plenty of magic for many people, not just me, but rooms full of students, just like we have here today. So anyway, that was— anyway, hello Carolyn, and hi Tom. And it's— I know it's still a little bit of a, obviously, an unusual summer here, but I do hope all the students will also come back to MBL on another year too, because I'm sure you're getting your dose of magic this year, but especially compared to last. But this is an amazing place where, I don't know, just magical Conversations happen and everyone is, is especially in the summer, just kind of remembers why they fell in love with science in the first place. You know, not necessarily trying to get the next figure of your paper done or get your grant out or whatever it is that keeps us busy in our other lives and home institutions. Everyone just, you know, comes here kind of for the raw pleasure of talking about science, or getting a crazy experiment to work, or spending the time to talk to people, which, you know, we don't do enough, I think, at our home institutions, but somehow does happen here. So it is a magical place. And I think Joanne was right. I mean, Tom, like, would be in there in the Nobel Court, you know, holding court over, you know, an electron microscope with both working on the scope but also educating students at the same time.

And, you know, that's what education is all about.

Do you have any more questions from the live audience, particularly our students? Well, in that case, I will thank you, Ron, for an amazing lecture and invite everyone outside to have a reception and continue the conversation.

Okay, thanks everyone. I want to thank everyone for attending. We hope you'll consider registering for other lectures in the series. Also, our Friday evening lecture series has gone virtual once again this year, and we hope you'll consider registering for Clifford Brangwynne's lecture this Friday, July 9th.

Thanks again, everybody.