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Show Notes
This is the first of several interviews we will do with significant people in the history of developmental bioelectricity. Richard Nuccitelli is a legend, having invented (with Lionel Jaffe, in 1974) the Vibrating Probe - a technique that allowed the non-invasive sensing of ion currents around living tissues. This is about a ~1 hour conversation in which he talks about his background, the history of the field, and his current clinical work on the bioelectrics of cancer.
Richard Nuccitelli: https://scholar.google.com/citations?user=2JirSrEAAAAJ&hl=en and https://www.linkedin.com/in/nuccitelli/
Aastha Jain: https://www.linkedin.com/in/aasthajs/ and https://www.livelongerworld.com/
CHAPTERS:
(00:00) Rich's bioelectric beginnings
(09:12) Cells reading electric cues
(21:41) History to pulsed fields
(34:46) Cancer therapy and development
(43:32) Life in Lionel's lab
(52:53) Challenges and future directions
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Transcript
This transcript is automatically generated; we strive for accuracy, but errors in wording or speaker identification may occur. Please verify key details when needed.
[00:00] Michael Levin: I'm excited to have you here and have a chance to talk about some of this stuff. I'll let Aastha go first and then I have a bunch of questions to ask you.
[00:08] Richard Nuccitelli: Okay.
[00:09] Aastha Jain Simes: Mike, feel free to just jump in and ask questions as well. So Rich, I know you started talking about the history of it, but let's just take it back. Maybe you can get started by telling us a bit about your background and how you got interested in bioelectricity in the first place.
[00:30] Richard Nuccitelli: My background: I started out in physics. I got my bachelor's degree in physics and then went to graduate school at Purdue. In the physics department they had professors give us a lecture about their work. It was a rotating thing. We'd be able to hear all the professors, what they're up to, and decide if we wanted to work with them. One of the professors that came on to do that was Lionel Jaffe. He was actually in the Department of Biological Sciences, but he liked to go to the physics department because he found the students there were better and more appropriate for his interests. He told us about his work in measuring electrical currents in germinating seaweed eggs, and I found that fascinating. At that point I had never taken any biology in my career. To think that you can measure currents in living cells—I thought that was fascinating. I went and did a rotation with him, fell in love with the whole idea of measuring these currents, and I did my PhD under his guidance. That really was a turning point in my life because instead of studying solids and solid-state physics, I was studying living cells and we were measuring currents in those cells. It was fascinating. We developed a whole new technique at the time called the vibrating probe. It was basically an electrode that vibrated back and forth, and we used a lock-in amplifier to measure the voltage between the two points. If current is flowing in a medium, that will generate a voltage in the medium. It's very small because you're in seawater; it's very conductive. The voltage you generate is in the nanovolt, 10 to the minus 9 volt, range. It's very small, but with some fancy electronics and averaging many measurements, you can get down into those regions. We did. We measured currents around a single fucoid egg, a Pelvetia egg. That's how I got into it. Ever since then I've been looking at those kinds of things—fields and currents in cells and their response to imposed fields—because that's so critical for development of organisms. It's really been fun.
[03:25] Aastha Jain Simes: So when you first got into this research, you developed a vibrating probe with Lionel Jaffe as well. What was the next step? What was the significance of bioelectrics back then? Was the field even called bioelectrics?
[03:40] Richard Nuccitelli: Not really. Bioelectric was a term that was coined much later. There it was simply called "Ionic Currents in Development." We had a whole book called "Ionic Currents in Development." And that was because, when people talked about currents, they thought about electron movement in wires. But we aren't talking about that. We're talking about sodium, potassium, chloride movements through cells. So that's why we emphasize the ionic current aspect. Because that's a whole new way of thinking of current with ions carrying it. That's the whole way we started describing it. It was fairly new. People didn't quite know what to make of it at the time because they didn't think of cells as driving currents for themselves. It was a whole new way of thinking. As Mike will attest, it's still somewhat difficult to get people to realize that's what's going on. I think it's largely because it never quite made it into the biology literature, into the biology textbooks. They teach all kinds of things about genetics, physical chemistry, and biochemistry. But you don't get much biophysics taught in the biology realm. So even doctors, growing up, don't learn about this stuff until they get into cardiology and realize that their heart is controlled by electrical voltages and currents, and they start realizing how important it is then. Before that, in their basic training, they just don't hear much about it. And that's unfortunate because I think the basis of life is bioelectric and it's just not completely understood and taught that way. When you think about it, every single aspect, every organ in your body has a voltage across it, across the outer boundary, the epidermis or the outer layer of the organ. There's a voltage across each one. And that voltage is important for the function of that organ. So we are electric beings, that's for sure. And it's something that's very, very slow to be realized by the medical community or anybody else.
[06:46] Aastha Jain Simes: So this is one. Go on, Mike.
[06:49] Michael Levin: I came across the first paper on the vibrating probe. I first read it in 1987. It was obvious that this was a breakthrough technology because for the first time we were able to see this living pattern of electric activity around, and spatialized too, around all of these things. Can you mention the range of things that it's since been applied to? It's been applied to a million different things, right?
[07:19] Richard Nuccitelli: It's been picked up by about 20 to 30 scientists around the world. They've used it to measure the currents through germinating pollen tubes and through the gastrula stage of many embryos. We used it to measure the current through a blastomere. A mouse blastomere has current going in the apical end and out the basal end, a constant steady current. We measured that many years ago, but it's been shown that in the chick embryo and in the frog embryo, during gastrulation, the currents are coming out of the neural tube region, and those currents are generating a voltage within the embryo itself that's very important for its polarity. If you disrupt those voltages, change the voltage inside the embryo, you'll completely mess up development. They don't develop properly. You get all kinds of monsters created. That's one of the things that Mike's been working on as well, looking at the molecular aspects of those pumps and leaks to see how that's involved in pattern formation. And it's critical, absolutely critical. The probe is used quite a bit. It's not currently being used by many people. I don't know of any current labs that are using it every day, but there are about 100 or so papers out there showing different organisms in which the probe has been used to determine the current patterns.
[09:12] Aastha Jain Simes: When you first got into bioelectricity and started recognizing that ourselves are electric, what was the thinking in the field around what the significance of this is? What was the main research interest around what's going to happen with understanding that ourselves are electric?
[09:34] Richard Nuccitelli: The first thing we did when we measured the current in this fucoid egg — it's a seaweed egg that germinates a rhizoid at one end. It starts out as a perfectly symmetrical sphere, and it has the capability of growing anywhere on its surface. It grows by secreting material that softens the wall; it builds up pressure on the wall, and as the wall gets softer, it expands, and that's how it elongates. With this particular organism, it looks at the cues around it, mainly light and temperature, and it grows to the dark to form a holdfast until it sticks to a rock, and it could grow up from that. That current is critical for determining the axis of growth, and you can change that by changing the current flow. Normally it controls the current by the light being absorbed by photoreceptors, which change the permeability of channels in that area and open calcium channels that let calcium in and stimulate secretion. But if you take that same cell and put an electric field in a different direction, it'll grow towards that electric field. It listens to what's out there. If light is the main factor, it'll listen to that. If an electric field is the main factor, it'll listen to that. It depends on the different environmental variables to make a decision. I think that's the case with many cells in the body. When they are developing they're surrounded by other cells, and they get information from that, often electrical, so they have a current nearby or a voltage gradient that affects the way their ion channels and pumps are oriented or localized on their plasma membrane. That will in turn generate a current through them that polarizes them. Basically, the electric fields that any cell experiences will influence the way it grows, in both its direction and its differentiation — the genes that are turned on. That's one of the things that Mike has been finding: genetics is listening to the electrical signals and making decisions based on these external inputs.
[12:28] Aastha Jain Simes: I have a question for both you and Mike. Mike, when you first read about the vibrating probe... This field remains underrated, even though there's a bunch of fascinating research happening. Why do you think biophysics or bioelectricity has not made its way into the textbook? Rich, you want to go first.
[12:58] Richard Nuccitelli: No, I think you should answer this one.
[13:00] Michael Levin: Well, here are my thoughts. One of the things that make biochemistry and genetics a lot easier is that you can study them in the dead state. You can fractionate your cell. You can isolate the DNA, the RNA, the proteins, the lipids, the sugars; you just spread this stuff out. And then you can analyze them. The amazing thing about bioelectricity is that, as a number of people have said, it's the spark of life. It's only there when the system is alive and functional. That means it had to really wait for technologies like what Rich created and various things since then, the fluorescent dyes that we've used, before you could actually get these data. Molecular biologists and biochemists have been at this for decades, long before that, because you can break up your cell and that's good enough. The first thing is, I think it's more difficult and the technology needed more time to catch up. Also, I think as Rich said, you need a different mindset for this. It's not as simple as the kinds of things that you normally would learn in biology and chemistry to really comprehend it. I don't know if Rich agrees, but to me, not only do you need physics, but you also need a lot of what's now basically neuroscience. You need a lot of computation to really understand what's going on. It's a fundamentally interdisciplinary field. And I think that's harder.
[14:34] Richard Nuccitelli: I think that a lot of the problem is that our classical education divides things up. A biology student doesn't get enough physics to really understand electricity and magnetism to a depth that makes them comfortable with it. They typically get one, maybe one semester, at the most two semesters of physics. It's just enough to hate it and not to enjoy it and have it become a tool. So they're afraid of it. Because of that, they don't go beyond it into the biophysics needed to appreciate the ion currents and those kinds of things. I think that's a large part of it. I don't know how to fix that other than having that be changed in the mindset of our education system to get more physics to all the biologists out there and all the pre-med students.
[15:47] Michael Levin: It's in the Gilbert Developmental Biology textbook. It has two bioelectric stories in it now. So it's getting there.
[15:57] Richard Nuccitelli: Yeah. that's great to hear.
[16:00] Aastha Jain Simes: Have you noticed, then, that most of the scientists looking into the field have a bit of a physics background?
[16:10] Richard Nuccitelli: Yes, in general, that's the case. When thinking of the ones that I know of that have used the vibrating probe, for example, those guys all had to have some pretty good feeling about physics to understand how to interpret the data.
[16:33] Michael Levin: There's another way in which people get dragged kicking and screaming into this field, which is that somebody who does regular genetics will do a screen of some sort and they'll get their mutant and they say, this is fantastic. This is the thing I need to study. Then they find out it's an ion channel. And so now there's been amazing work by Emily Bates in the Harris lab at Harvard, where they do these purely genetic screens where nobody was out to find anything bioelectric, but then you find that it's a channel. And so now they need to understand how this voltage change gives rise to an altered wing or size control. There's probably almost three dozen good developmental channelopathies now that people have studied that bring them into the field even though they weren't really trying to study them.
[17:29] Richard Nuccitelli: That's a great way to get in, to be pulled in unsuspectingly. I love that.
[17:38] Michael Levin: I would often, when I give talks to those kind of audiences, I would say at the beginning, "How many of you have done either a microarray or an RNA-seq for your..." and they said, "So how many of you have found ion channels in your top list?" "Oh, absolutely, the first 10 things for IonJet." And they said, "Well, what did you do?" "Oh, we skipped them and went to the transcription factors, of course."
[18:02] Richard Nuccitelli: Exactly.
[18:03] Michael Levin: Because the tools were there. That's one of the things that we worked on pretty hard is to develop tools and protocols that we would share. I would say everybody who had that, here's a how-to manual and here's some reagents. Now we can study these things.
[18:19] Richard Nuccitelli: Yeah, fantastic.
[18:22] Aastha Jain Simes: Now is the reason they skip from the ion channels to say the transcription factors, is that because they don't recognize the significance of the ion channels? They don't at that point recognize that it's doing much or they just don't have any knowledge of it.
[18:38] Richard Nuccitelli: The bottom line is they don't have that basis of every cell has a transcellular current. They don't think about that. They aren't taught that. It's farthest from their mind. But it's absolutely true. We haven't found a cell yet that doesn't have a transcellular current, which is intimately involved in its function. Think of your epithelial cells in your gut. They're polarized. They have sodium channels on the apical side, potassium on the basal side. They have current; they're generating a voltage across themselves. That voltage is intricately involved in digestion. So things as basic as eating and digesting require these transcellular currents, but they don't know about that. It's not part of their education.
[19:33] Michael Levin: There's also a subtlety here, which is that when you have a transcription factor, you can tell a story about a gene that does a particular thing. That's a very popular story. But what you find with these ion channels is that it isn't actually the ion channel gene or protein that's doing it. It's the physiological state that ends up being the critical factor. You can get the same state by different channels, and you can get multiple states from the same channel, depending on whether it's open or closed. Seeing physiological patterns as a causal factor is not what people are used to. It goes back to what Rich was saying before about these different fields. I remember in 2007 we had this paper on a particular proton pump that was required for tail regeneration in tadpoles. Then we showed that if you get rid of that pump, you can rescue it back with a yeast proton pump that basically has no sequence homology but does the same physiological job. We sent this paper out and we got two reviewers. The first reviewer says, "Good, you found the gene for tail regeneration. Get rid of all this voltage stuff. You don't need it. You got the gene for tail regeneration. This other stuff is junk." The other reviewer says, "Clearly the gene doesn't matter because you've replaced it by this thing from yeast. Get rid of all of that stuff and keep the voltage." This was Danny Adams, the first author on this.
[21:08] Richard Nuccitelli: Unbelievable.
[21:09] Michael Levin: We wrote to the editor saying, "So what do we do?" And the editor said, "I don't know, but make it shorter. Make it shorter." That's the thing. You have to think a little bit differently; it's not the gene, and then it gets progressively worse after that, because then you end up with patterns — it's actually the voltage patterns that determine what happens next.
[21:34] Richard Nuccitelli: Exactly. Yeah. even more difficult to explain.
[21:41] Aastha Jain Simes: I'd love to understand a bit of the progression of the field and the research interest from, say, the 1970s up till now. Maybe a good way to do this would be a comparison. You could talk about what was the predominant thinking in the field in the 1970s, 80s, and 90s, or you could tell me how it compares to today and how the thinking was different back then.
[22:09] Richard Nuccitelli: Wow. You're asking a lot there.
[22:12] Michael Levin: If you don't mind, I want to make it even harder. I'm even interested in going back further before that, because there was some great work in the 20s and 30s and 40s. Anything you want to say about all of that stuff and going forward, we'd love to hear it.
[22:34] Richard Nuccitelli: If you go back to the early 1900s, people like Burr and a variety of very well-known biologists were looking at these kinds of things, measuring voltages with cotton wicks soaked in saline and putting it on different regions of embryos. That work was almost lost. It was really groundbreaking and so unique that I think it was just not quite appreciated. The main reason, I believe, is that in the 1950s the whole DNA story came out. So molecular biology took over and the body of work measuring electric voltages and showing that there are fields in these embryos was forgotten in many respects. But that was sad because certainly the molecular revolution, the DNA revolution, was huge. We thought that it would explain everything. We now know that it doesn't, but it made us believe that we could understand life by knowing the sequence of our DNA. I think even molecular biologists now agree that that doesn't tell you how things work, that it gives us some idea of how proteins are made and what the structure of proteins is, but it doesn't help us understand life completely. That's where Mike's stuff is much more important. In looking at the evolution, the molecular revolution pretty much, in my opinion, completely overshadowed all the physiology. The electrical developmental physiology that was going on went away and was not even looked at anymore. I think when Lionel's generation came in, he was fairly unique.
[25:12] Richard Nuccitelli: I don't really know of a lot of comrades for him that he could work with. He was a very unique person and he started looking again at these electrical currents in developing embryos and plants, mainly in these pelvisia. He raised the awareness again of this, but it never took off to a huge extent. He didn't have that many followers. His students all followed him: Ken Robinson, Bill Rich Borgens, and myself all continued doing his work. We had a dozen other scientists who started using the vibrating probe in Germany, France, Italy, and Canada, and made some progress in the field. In the 70s and 80s, and in the 90s and around 2000, I found the role of ultra-high voltage, ultra-short fields in cell signaling. That's a whole different area of bioelectricity that I've been exploring for the past decade, two decades really. That's another aspect of bioelectricity that is fascinating. I started out measuring nanovolts, 10 to the minus 9th volts, and now I'm applying a 10 million times larger field of 50 kilovolts or 20 kilovolts. In a way that's still physiological. It produces very little heat, fractions of a joule, but it communicates to the cell in a way that gives it a message. The message is to undergo regulated cell death and to start doing something that it would normally do at the end of its useful life, but start doing it now. It's a fascinating area that I've been exploring and enjoying.
[27:53] Aastha Jain Simes: Can you talk a bit more about the ultra-high voltage field? Sure.
[27:58] Richard Nuccitelli: Those are called nanosecond voltage pulses. The whole idea is that, back in the 1950s—1954 was when the first discovery that a pulsed electric field could permeabilize cells occurred. It was called electroporation; it is still called that. And what happens is you make an electric field that's strong enough to push water molecules. Water is a dipole, H2O, two hydrogens and one oxygen. So when you put a voltage, it moves along the field. If you put up to about 400 millivolts, maybe half a volt across a lipid bilayer, which all of our membranes are made of, it's strong enough to push water into that lipid and form a water-filled pore. That is permeabilization. Now this lipid bilayer, which normally separates inside from out, has a little leak, and that leak lets ions pass through it if there's a gradient of force. A typical cell is maybe 10 to 100 microns, and if you put about 1,000 volts per centimeter across that, that gives enough voltage across the membrane to permeabilize. If you want to permeabilize organelles like the mitochondria and ER, one kilovolt per centimeter is not enough because you're looking at a much smaller distance across that organelle. You need a larger field to generate the same half volt across that membrane because the membranes are much closer together. So now we have to have 20 or 30 kilovolts per centimeter, and that's possible only with ultra-short pulses. One kilovolt per centimeter you can use with a microsecond; it doesn't generate enough joules or heat to cause significant heating. But if you go up to 20 kilovolts per centimeter, at a one-microsecond pulse that would be way too much heat; it would boil the cell. The joules you're introducing are too much, so you have to shorten the pulse. Instead of a microsecond, you can do a nanosecond, which is one thousandth of that, or ten or 100 nanoseconds; as long as you're less than a microsecond you can introduce it. Now the trick is not only do you permeabilize the plasma membrane, you permeabilize the mitochondria. That's important because mitochondria function by generating a 200 millivolt inside-negative potential across their inner membrane. That voltage is critical for making ATP from ADP: it helps add a phosphate onto ADP. It's also a proton gradient; that's important. So it's a combination of a proton gradient (pH) and electric field that make ATP. If you permeabilize the mitochondria, make them leaky so they can no longer generate a voltage gradient, you don't make ATP. That's been shown. The effect is that we not only permeabilize the plasma membrane but also permeabilize mitochondria; water moves in, the mitochondria swell, and they stop producing ATP.
[32:49] Aastha Jain Simes: Did you lose Rich as well, Mike?
[32:50] Michael Levin: I did, yes. Sorry, Rich. Hang on a second. Your voice is off. I don't think he can hear us either.
[33:02] Aastha Jain Simes: Rich, can you hear us?
[33:05] Michael Levin: We can't. We can't hear you.
[33:07] Aastha Jain Simes: Yeah, I think he.
[33:11] Michael Levin: Say something.
[33:12] Richard Nuccitelli: That's better. Now I can hear you.
[33:14] Michael Levin: You're back. We lost about 15 seconds.
[33:18] Richard Nuccitelli: Where did I stop?
[33:26] Aastha Jain Simes: You were talking about how the mitochondria would lose the energy in the ATP.
[33:32] Richard Nuccitelli: Yes.
Aastha Jain Simes: You can permeilize it.
[33:33] Richard Nuccitelli: When that happens, not only does a cell lose its energy source of ATP, but messages typically are released by the mitochondria, for example cytochrome C, which is one of the messages required to initiate regulated cell death. It starts the pathway that all cells have when they find that they're not functioning well, they become leaky or they have been damaged. They release cytochrome C, they make a message go from the ER to the plasma membrane — calreticulin. When calreticulin is expressed on the surface of the cell, dendritic cells that are always looking around for problems bind to that and that's a message to phagocytose that cell. It's a way for our body to go after cells that are damaged, clean them up, and rescue any problems that are happening.
[34:46] Aastha Jain Simes: So does this have significance for cancer research and killing cancer cells as well?
[34:52] Richard Nuccitelli: We've been using it to treat different cancers. So in a mouse, for example, we're able to make mouse models of all sorts of cancer, from pancreatic cancer to basal cell carcinoma to a whole variety. In a mouse, if you can make a tumor, we treat it by putting our electrodes around the tumor. We simply give it a few pulses and it dries up and goes away. In literally a couple of weeks, the tumor is completely gone and it never reoccurs. When I found that with these nanosecond pulses, I was absolutely blown away. That happened in 2007. Ever since that, I've been working on that. We now have Pulse Biosciences, where I'm working. It has generated a machine, a pulse generator for this. It's been approved for use now for treating benign skin lesions. We've just had a percutaneous electrode approved that we're using to treat thyroid nodules by putting the electrode into that, and it causes the nodule to shrink and completely go away. So we're starting to use it to go after problems in our medical field.
[36:25] Aastha Jain Simes: Is there any damage to the good cells in chemotherapy you might have?
[36:32] Richard Nuccitelli: Yes, there can be. You just have to be careful because every cell will respond to these fields. And that's what it's a good thing and it's a bad thing because it's a biophysical phenomenon. You can't prevent getting holes in membranes when you pulse with these fields. It's a physical property of the lipid bilayer. So when we apply it, we're always careful to try to limit the energy to the tumor so we don't damage other cells. But the fact is that in the skin, for example, we've done a lot of treatment of skin lesions. Skin cells are pretty good about recovering. For example, when we treat a wart, which is embedded, it will typically get a few of the skin cells damaged as well. But skin is amazing for regeneration. When you cut yourself, it heals so beautifully and recovers. So that skin can regenerate and we damage a few of those skin cells and it's not a problem. But the main thing is we get rid of the wart. So that's typically how it works.
[37:49] Aastha Jain Simes: Going back a little bit to the progression you mentioned, early 1900s there were people doing some experiments with cotton wicks and realized that there might be electrical forces in our body, but a lot of that research was lost. In the 1950s, because of DNA research, the entire field focused on that. Then Lionel Jaffe came along, you and Ken Robinson, and you had the vibrating probe, which made it much easier for people to detect these electrical signals in the body. Around that time, when people started detecting electrical signals, when did they start realizing that these electrical signals play a very significant role beyond the DNA? In other words, when did they start realizing what the significance of the electrical signals in the body could be?
[38:51] Richard Nuccitelli: That's a great question. As I think about it, the field really was concentrated for a long time on a technique called patch clamp. This is what electrophysiologists use to study ion channels and the way the channels operate, the gating of channels. I think when the patch clamp technique was invented, that was again around, I would say that was in the 1980s, early between 75 and 80, that really made electrophysiology all concentrate on that technique. That's how they decide how they were able to explore ion channel opening and closing, the timing of it. But when you think about the polarity or development of the asymmetry in channel activity, it's really hard to put your finger on that as a phenomenon that people really appreciate. I think it's really the work that Mike has done that has made more progress in people understanding the importance of that. One of the things that we showed over the years in the 1980s and 90s were currents leaving growing limb bud stumps in regenerating limb buds and in tails. Those currents were certainly published and people recognized that they were happening. But I think that the sole phenomenon of developmental currents and voltages is something that people are still trying to get their head around. Would you agree, Mike?
[41:06] Michael Levin: The earliest paper that I have, you could go back to Volta and then so on, but the earliest developmental paper I have is 1892 from Wilhelm Ruh, and he's writing in German about the effect of electric fields on chicken embryos.
[41:27] Richard Nuccitelli: Exactly, yeah.
[41:28] Michael Levin: One of my old favorites is Burr, Harold Burr; this is the 1930s and 1940s. He basically had a good voltmeter. That's all he had. He went around measuring everything from embryos to maple trees to various psychiatric patients — dozens and dozens of papers. In his book, he was extremely prescient. He said things that he had no way of being certain of, but that in the end turned out to be quite correct, because he saw a lot of these pre-patterns and these subtle voltage pre-patterns. Another researcher I liked is Clarence Cohn in the 1970s. He showed that if you depolarize neurons, they re-enter mitosis. He was really pushing the resting potential, this idea of resting potential.
[42:24] Richard Nuccitelli: Absolutely, yeah.
[42:25] Michael Levin: Right.
Richard Nuccitelli: And yeah.
[42:28] Michael Levin: In the 2000s, what we started doing was really tying this stuff to the molecular genetic downstream consequences. So our first paper with Ken Robinson was on left-right asymmetry in the chicken. There was this very important gene, Sonic Hedgehog, and everybody wanted to know about Sonic Hedgehog. It turns out if you want to know why Sonic Hedgehog is turned on the left side and not on the right side, you have to know about the voltage gradient that precedes it. Otherwise it's a big mystery.
[43:05] Richard Nuccitelli: I know that's fascinating. It's all coming together when you look at the way you've integrated the molecular biology with the electrical field understanding, pH gradients, potassium ion currents, potassium pump location, so critical. It's amazing.
[43:32] Michael Levin: Rich, do you want to talk about what was it like working with Lionel? Because I knew him a little bit and I always thought he was a very interesting character and you obviously knew him much better. What was that like in his lab in those days?
[43:47] Richard Nuccitelli: He was quite a character. Not only did he have incredible physical intuition; he just had a way of thinking about these things that I hadn't encountered before him. He was also very much into politics and very strong in his political beliefs. He was very liberal. Almost every afternoon when we had our coffee or any time to sit and chat, it would be about politics. He and Ken Robinson and I were also very liberal, so we were able to get along pretty well. The thing about being in his lab is that he worked very hard. He was there typically around eight in the morning and would often stay till eight or nine at night. He had no problem staying late hours or overnight when we had an experiment that had to run 24 hours. He would share the nighttime with me. I would take the first shift and he would come to take the second shift. He was a very hard worker and very fastidious. When he started doing his work, the first thing he did was clean everything. Everything gets removed; you only have the instruments you need. He was very particular about that, and it was important because the work we were doing—the development of the vibrating probe—required very fine work making the probe itself, which was a very fine needle with a platinum ball on the end of about 30 microns in diameter, which you make under a microscope. You watch it grow in front of your eyes with an electric field to attract the platinum to the surface. That is all pretty fine work. We typically had Bach in the background, his piano concertos to keep us calm while we were doing this fine work. It was a fun place to be. Everyone in the lab really enjoyed being there.
[46:20] Michael Levin: Would you say that it's always interesting, the motivation that great scientists have. And would you say that he and you and the rest of you, were you motivated by a specific outcome or capability or an endpoint that you wanted to get to? Or was it more just the curiosity of whatever showed up in the moment, more joy of the experiment? How did you guys see that?
[46:47] Richard Nuccitelli: It was the joy in the moment. None of us knew; we didn't have an expectation of where we were going. This was all new. The whole idea of currents around cells was a completely new concept. We didn't know where it would take us. One person in the lab would put a field across the cell. Another person would try to measure the normal current. Ken was trying to figure out what ions were carrying the current. He actually put the eggs in a metal screen separating one side from the other and used radioactive tracers to determine what was going in and what was coming out. We had all these different approaches to understand the mechanism; it was a fascinating place to be. It was all discovery of completely new things that no one had heard of before. It was an exciting time, but we were there just to try to understand how it worked: what was carrying the current, why there was a current at all, what was driving it, and that was fascinating. We could show that the electric field could change the current direction and polarize the cells. You can make the cells grow in a certain direction by the electric field. And how does that work? What was going on there? We showed that it was affecting the calcium permeability, which then affected the chloride permeability. It was a remarkable time. I still have great memories of it.
[48:32] Aastha Jain Simes: Mike, what was your impression of Lionel? You said you interacted with him briefly.
[48:36] Michael Levin: I met him a few times, also when he was at Woods Hole. I would go up there and meet with him. He was an iconoclast, he was always thinking in new directions, wasn't particularly concerned with what anybody thought, had all kinds of wild ideas. He played tennis. He was quite advanced in years and he was still playing tennis. He complained to me about not being able to find enough partners at that point.
[49:11] Richard Nuccitelli: That's true.
[49:12] Michael Levin: Yeah, I played tennis with him.
[49:14] Richard Nuccitelli: I remember that. was great.
[49:19] Michael Levin: Yeah.
Aastha Jain Simes: Mike, when you first entered the field, what was your understanding of bioelectrics?
[49:27] Michael Levin: By the time I had gone through undergrad and grad school, I had been reading what Rich and Lionel and Ken and Richard Borgens and those guys; they were my heroes. I ate and slept and dreamt all of this stuff, but I also knew it was pretty clear that at a certain early stage of the game I better keep my mouth shut about all this, so I got my degree in genetics and didn't say anything about any of this straight up, just learning the molecular genetics of chick development with Cliff Tabin and so on. During my postdoc, I started sliding into it because I figured that pretty soon, if I had my own lab, I would be able to really get at this. Around that time I started being able to meet you guys in person. I went to the Gordon Conference, and eventually I had some actual data to show. I was just so thrilled to meet everybody; they were helpful and excited. I found it amazing because over the years you guys did not have it easy, to do all that work against the mainstream of genetics and molecular biology and to hold on to that—that's what I remember. I remember the excitement about the field, not at all diminished by all the years of hard work and arguing with people and having to break through with this stuff. I just remember thinking that this is great: if I was ever able to do this, the key thing to hold on to is that sense of wonder and excitement about the work and not let the difficulties get you down. I was amazed at how welcoming they were to this new young kid who didn't know anything but was also excited about the field. I had really good experiences there.
[51:35] Richard Nuccitelli: We were excited to see a molecular person come in with interest in our stuff because we had just not seen much of that. So you were the first one to come along and show an interest and understanding the connection between the ion currents and the molecular biology. That was fantastic. We were so happy to see that.
[52:03] Michael Levin: I couldn't believe I got to meet all you guys in person. I first ran into this field in 1986. This was before the internet. There was no searching you could do online. It was like in a dusty basement of a library somewhere, Boston Public Library, trying to find these things. The catalog was barely functional. Each one of these papers had citations to other papers, and you had to go chase them down. Then you made photocopies, and sat there and read all of it. That's literature that I was eating and dreaming of for years.
[52:51] Richard Nuccitelli: Phenomenal.
[52:53] Aastha Jain Simes: Rich, maybe you can talk a bit about the criticism that the field received when you, Lionel, Ken were coming up with all this research?
[53:05] Richard Nuccitelli: Well, when I say criticism, I'm not sure that we had much problem getting it published. We always did experiments with good controls and a large data set so that we published in good journals, Journal of Cell Biology, Developmental Biology, without very much problem. So I don't think that there was any criticism in that regard. I think the main question is why more people weren't excited by it, and why we didn't have a lot more followers than we did. Again, that was a molecular biology aspect. Most of the biologists out there found our stuff intriguing, but didn't get excited by it as we were and didn't quite appreciate the importance of it. And I think that's still the case. I really don't think that the importance of the role of these ion gradients and currents in development and in physiology is really appreciated by the bulk of the community of biology. Would you agree with that, Mike?
[54:26] Michael Levin: I think it's gotten a lot better. I think once again, people publish this stuff now in Cell and Development. I think we're back there, but still a lot of people have never heard of it. That's always an issue.
[54:44] Richard Nuccitelli: Right.
Michael Levin: And there's a sister feel to this, which has had a similar journey, which is biomechanics. All the physical forces, the stresses, the strain. There were all these people in the past decades, left Balusov and all these other people that were talking about the importance of all these things and writing these papers and completely ignored until some of the technology came along, people like Don Ingber and other folks who made molecular tools where you can now make high-resolution quantitative models of what the actual physical forces are doing, not necessarily the genes, but the actual physical forces. The fact that if you culture cells on a square peg versus a triangular peg, you get different genes turned on and this kind of thing. I think they went through the same sort of thing. It was a very difficult time until the technology showed up. I think technology in these cases, two things make a huge difference, the technology and then what you might call killer apps in the computer science field, something that now you can see, you can do something now that you couldn't do before. And that gets people's attention. Then the young people come up with knowing what it's about.
[55:56] Aastha Jain Simes: What's the technology that you think is missing now or would be helpful to advance the research further? Question for both of you. Mike, you can go first.
[56:09] Michael Levin: I think the biggest thing that's missing now is the physiomics data sets. So we have these massive — just think about trying to do molecular biology before we had genomic data and transcriptomic data. What we still don't know is, if you wanted to know in some model system, some organ under some disease state or at some stage of development, what is the bioelectric pattern? We only have a tiny number of these things mapped out. Rish has done a ton of it with the vibrating probe. We've done some with the voltage dyes. But there's very little bulk data available. That's pretty critical because modern tools like AI and other techniques require a lot of data to extract the signatures and functional target endpoints. So that's one of the things we're focused on now: trying to develop technologies where, in a high-throughput way, we can generate these data sets so that we know what the native states are for these things. What do you think, Rich? What's the...
[57:12] Richard Nuccitelli: I think you're absolutely right. I remember that beautiful paper that you guys came out with when you labeled the frog embryo with the dye that measured membrane potential in the different regions of the embryo and showed a visual picture of the gradient of potential in the blastomere stage of the frog embryo. That is what we need. We need to have a visualization of the electrical properties that we're seeing. It's so hard to do because the organisms that we want to study are multicellular, thousands and thousands of cells, but those cells are working together in a very intricate way. The ideal would be to be able to image the field, the whole electric field everywhere in that organism. That would be a dream. It's not obvious to me how to do that, but I'm sure that one day we will have some molecules that will show the gradient in a way that we can image them. There's quite a few different tools that are working towards that, this whole optogenetics business where you control gene activity with light. It would be nice to be able to reverse that and show light come out with the bioelectric activity. That would be fantastic. I hope that happens someday. I don't see it on the horizon yet, but I wouldn't be surprised.
[59:02] Michael Levin: We and others are now generating transgenic mice where every cell is expressing one of these optogenetic voltage reporter proteins. I think in the next couple of years we'll have model systems where every cell is advertising its electric state. We still have the issue that it's hard to see through tissue, and so what's going on — we still have all that, but at least the cells will be trying to tell you what their voltage is, as much as you can see them.
[59:34] Richard Nuccitelli: Exactly. That would be fabulous if we can get that to work. I love it.
[59:42] Aastha Jain Simes: Do different humans have different electrical states around the cells?
[59:50] Richard Nuccitelli: Different humans.
[59:53] Aastha Jain Simes: Are your and my electrical states around tissues, organs, and cells going to be different? Or do all normal cells have the same type?
[1:00:03] Richard Nuccitelli: I think it would generally be the same. It depends on the disease state. As you get older, the patterns and the voltages get lower. I've shown that in a skin wound, for example; as you age, the amount of current flowing, the wound current, goes down. You just don't have quite as much energy to drive the current. I think you would find a difference as a function of age and any kind of disease state. In general, a healthy individual will have very similar patterns. It really depends on what you're looking at. Typically, you'll find a voltage across every epithelium, every organ, and that voltage will drive currents depending on what that organ is doing.
[1:01:10] Michael Levin: There have been stories in the cancer field about sodium- versus potassium-rich diets. One can hypothesize that if what we're talking about is a change in resting potential due to sodium, you might expect people with different diets to have shifted Vmems that would predispose them to different conditions like cancer.
[1:01:37] Richard Nuccitelli: It's been shown pretty well that depolarization is required for a cancer cell to be very active. They're typically, when you look at their membrane potential, more depolarized than a healthy cell. So obviously you want to avoid that to avoid the cancer state.
[1:02:00] Aastha Jain Simes: Last question for you, Rich. What would you like to see developed in the field going forward?
[1:02:10] Richard Nuccitelli: I think we just answered that. This whole idea of getting cells to tell us what's going on in their electrical activity would be fantastic. This thing that Mike just mentioned, having a reporter gene that expresses a signal such as a light emission or a fluorescence that we can detect, that would tell us if that cell is depolarized or hyperpolarized, or if there's actually a current going through it, would be fabulous. Then we can have an image of the electrical properties in different areas of the organism.
[1:02:54] Aastha Jain Simes: Thank you so much, Mike. Do you want to add anything? Any last questions?
[1:02:59] Michael Levin: This was great, Rich. I just want to say thank you so much for taking this time with us. It was awesome to get your thoughts on everything. Thank you.
[1:03:05] Richard Nuccitelli: My pleasure. It was fun to talk about. I'll be glad to talk anytime.
[1:03:11] Aastha Jain Simes: Thank you so much. Yeah, this was wonderful.
[1:03:13] Richard Nuccitelli: All right, guys.
[1:03:14] Michael Levin: Thanks, Rich. I'll see you in the Bioelectricity Journal. Plenty more.
[1:03:19] Richard Nuccitelli: Absolutely. Good to meet you. Bye bye, Astrid.
