234 | Tobias Warnecke on Cellular Structure and Evolution

0:00:00.0 Sean Carroll: Hello everyone. Welcome to the Mindscape Podcast. I'm your host, Sean Carroll. I'm not sure if everyone knows, but I have mentioned sometimes on Twitter, I keep a list of the Twitter feeds of all previous Mindscape guests who are on Twitter. So you can, it's a public list, you can go and you can subscribe to it or check out who's on there, et cetera. It's kind of enlightening, it helps me keep track of what is being said by all the former guests, and since I follow that list, one of the people on it is Theodore Ornav, who was our guest some time ago who talked about CRISPR and gene editing, a wonderful molecular biologist, and on Twitter recently, he got very, very excited. Theodore started talking about this new result that is gonna rewrite the biology textbooks. It's very, very exciting.

0:00:47.0 SC: And that result is what we're gonna talk about today. It has to do with a little kind of protein called a histone. I think I had heard of histones before this appeared on Twitter, but I didn't really know anything about them. The point is that in a human being or other fairly mature organisms, we have DNA, the DNA carries our genetic information, and there's a lot of it; there's a lot of genetic information to be carried. So if you were to take a single DNA strand from a human being and stretch it out, it would be something like 1.8 meters long. That's very long, and we have to squeeze it into the cell, the nucleus of a cell in our body, every single cell that we have. So how do we do that? The answer is that the DNA wraps itself tightly around these little proteins called histones.

0:01:38.5 SC: So histones are the backbone or the spool, if you like, that you spin the thread around that keeps the DNA organized. And because it's biology and biology is always complicated, the individual units around which the DNA wrap to do this are called nucleosomes, and then the nucleosomes are put into these fibers that form chromatin, and these are all what makeup your chromosomes, et cetera. I could never remember all these words, that's why I became a physicist. But so clearly histones are very important; they help organize the DNA. And because it's biology and everything serves multiple purposes, they also help regulate the expression of the DNA. The histones are not just sitting there, they're not actually like spools in thread, they have a functional role to play here. So if you go to Wikipedia and you look up histone, as you can bet that I did, the first line says, in biology histones are highly basic proteins that are found in eukaryotic cell nuclei.

0:02:40.4 SC: So remember back in your biology days... I don't know, my biology days in high school were too long ago so the field has moved on since then. But these days we talk about three kingdoms of life. We talk about bacteria, archaea and the eukaryotes. The eukaryotes are the group that have nucleus in the cell. And there's a complicated history because there's, they also have mitochondria, and the pre-history of life was quite, people slept around a lot, all these little cells; there's a lot of interchange of DNA and things going on. So the boundaries back in the primitive forms between bacteria, archaea and eukaryotes are a little bit fuzzy sometimes, but you can tell the difference. So Wikipedia is telling you that these histones are a eukaryotic phenomenon, but it turns out you can find some in Archaea also, and the new discovery is, you can even find them in bacteria.

0:03:39.6 SC: What does that mean? Why is that important? Why do the textbooks need to be rewritten because of this discovery? You've come to the right place. I came into this having no idea the answer to these questions, but we found an expert; Tobias Warnecke, today's guest, is one of the authors on the new paper. And so he's gonna explain to us, and you know, because it's supposed to be educational here we get into a lot of the basic structure of the cell and the role that that plays in evolutionary biology in addition to the new discovery about histones. So for those of you like me, whose high school biology class was a long time ago, this is the perfect podcast episode. Welcome to the Mindscape Podcast.

0:04:17.6 Tobias Warnecke: Hello. Thanks for having me.

0:04:18.3 SC: I'm gonna start very basic, as a more of a physics person than a biology person, I hope you'll forgive me how basically we start, and we'll get into some deep waters here, but remind us what eukaryotes are, for example.

0:04:32.8 TW: Nice start with a false modesty of physicist. I am always intimidated by having to talk to a physicist 'cause clearly they know much more than I do. But eukaryotes are one branch of the tree of life, if you want, and they are set apart from other organisms like bacteria, mainly by the fact that they have what is called a nucleus, right? So a compartment within the cell in which the genetic material, so DNA, is stored. That's the main distinction.

0:05:02.3 SC: And so as opposed to things without nuclei.

0:05:06.7 TW: So that would be bacteria, which many people will be familiar with. And another group called Archaea that if you think about morphology often look quite similar to bacteria. So they're single celled organisms, they have typically circular genomes unlike eukaryotes, but they don't have a nucleus. What's interesting in evolutionary terms is that Archaea are actually more closely related to eukaryotes than they are to bacteria. So although they look a bit different, the machinery that you find in the cell, the machinery that does the copying of the genetic information that keeps the cell going is in many instances more closely related to what we see in eukaryotes, like me and you and plants and flies and fungi.

0:05:58.1 SC: So if they're all unicellular and have no nuclei, what is the difference between archaea and bacteria? And it was a fairly recent historically speaking development that we even distinguished between these, right?

0:06:13.2 TW: Yeah, it's true. We only really learned about the sort of fundamental distinction between the two from sequencing their genomes. Because they're morphologically so similar people just thought that archaea would be weird bacteria. So really it's just by looking at the genome sequence and trying to map out the evolutionary relationships that people realize that the sort of key machinery, including ribosomal RNA... So ribosomes are the machines in the cell that make proteins. So the ribosomal RNA is actually more closely related to eukaryotes than to bacteria. And that you're right, it's very recent. Oh, like, well, in historical terms, quite recent observation, so in the '70s, and then people started to work it out in the '80s.

0:07:02.3 SC: Anything while I was alive counts as a recent scientific advance.

[laughter]

0:07:07.1 TW: Fair enough.

0:07:10.8 SC: I think that's the objective truth. So is it possible, I'm skipping way ahead here, but given that we only identified an entire kingdom of life within my lifetime, what is the chance that we're not done yet, that this whole idea that there are three kingdoms is not even the final answer?

0:07:28.4 TW: That's a super interesting question, and I don't quite know how to answer that. So the one thing that we haven't mentioned is viruses and things that many people wouldn't count as organisms because they need some other cell to replicate in. But people, again, quite recently, a bit more recently even than the archaea, people have started to find viruses that are actually really quite large, encoding thousands of genes. So how they exactly fit in between archaea and bacteria and eukaryotes and whether you can call them organisms, I guess you could ask that question. The second point I think that's worth making is that, especially with the onset of sort of large scale sequencing of genomes, we've started to characterize the diversity of life much better, and it can still be allocated to bacteria, archaea, eukaryotes. But, obviously, we only know what we are... We only recognize what we can see empirically through the methods that we use. So if there's a life form that for some reason is resistant to us seeing it through the sequencing approach, then maybe there is something out there that maybe not defies the classic categorization but maybe just blurs the boundaries a bit.

0:08:47.7 SC: And it might be something that even we already know exists; we just don't understand how different it is. That sounds like that's the case with the archaea, that we discovered something about them after we discovered their existence.

0:09:00.5 TW: So that's true, but I think it's also fair to say that if we know it exists based on sort of visual observation, we are now very good at obtaining the genetic material in those cells. So I'm not aware of any, if there had been a case where that was true, I think I would've heard of it. But yeah, so I think, what's maybe possible is that there's something that has been unobserved both at the morphological level or just visualizing the cell, and at the genetic level, but sort of the unknown unknowns, if you want.

0:09:36.1 SC: Good. Yeah. Okay. So we have eukaryotes and prokaryotes and the prokaryotes are divided up into bacteria and archaea. The other piece of very elementary information I wanted to get on the table is how we carry our genetic information. Back in my day there was a central dogma of how DNA information went through RNA to proteins, et cetera. It seems to be more complicated than that now.

0:10:00.9 TW: I'm not sure it's that much more complicated. Bacteria, archaea, eukaryotes all encode their genetic information using DNA. There are some viruses that use RNA, but when you talk about the central dogma, if you mean that the information when it comes to constructing the cell flows from DNA to RNA to protein, that is still largely true. And then there's some feedback mechanisms that maybe mean that if you have a protein that binds to DNA, that might affect how the DNA operates. But the genetic information when it comes to heredity is still very much based on the DNA to DNA.

0:10:44.2 SC: I guess what I was thinking of, and maybe this is the wrong way of thinking of it, when I say more complicated, the two things that I did not learn when I took my last biology class, which was in high school, is, number 1, that there's DNA not only in the nucleus of our cell but also in the mitochondria and elsewhere for we eukaryotes, and number 2, that the expression of these DNA is actually really important. Maybe you're making the distinction that's a functional thing, not a sort of design of the cell kind of difference.

0:11:17.3 TW: Yeah. So totally true. So we have, in many organisms, eukaryotes specifically, multiple parts of the cell that can hold DNA. So you have the genomic DNA that's encoded in the nucleus, then you have a contribution from mitochondria. And mitochondria used to be bacteria that became associated with an archaea ancestrally but have retained, in most instances, some of the DNA and that DNA gets independently replicated and passed on. And then a similar thing happens in plants where you have chloroplasts, which you need for photosynthesis. They are also ancestrally a different type of bacterium, and they also have their own DNA that they independently replicate and pass on. So, yeah, there's multiple streams of genetic information that are happening in eukaryotes. So eukaryotes are really symbions, if you want, ancestral symbiosis between archaea and bacteria.

0:12:21.1 SC: Well, good. That leads into exactly where I wanted to go because I'm curious about the evolutionary relationship between these three kingdoms we have here. And my impression is that this is a very active area of study; it is not settled like we might have hoped it would be by now.

0:12:38.4 TW: It's certainly very active, and the activity comes from a discovery that happened around the 2000, early, mid 2000s, of some archaea called Asgard archaea. And those Asgard archaea intrigued people because they happened to encode many proteins that were associated with eukaryotes, or many, sorry, many genes that were associated with eukaryotes that you wouldn't otherwise find in bacteria or the archaea. And if you, again, try to reconstruct the ancestral relationships between eukaryotes, archaea and bacteria, it turns out that these Asgard archaea seem to be more closely related to eukaryotes than any of the other archaea that we previously knew about. So people got excited about that because they were always hunting for what might be the closest archaea organism that you could find that might inform you about how eukaryotes evolved.

0:13:44.5 TW: And very recently, people actually have taken, managed to grow these in the lab, and the first group that accomplished that took years to do that because we have to figure out the growth conditions, make them grow, make them happy, isolate them from a complex mixture of microbes. And I guess the most fascinating aspect of this was that they were not sort of round or rod like cells but had long noodle like almost appendages. And some people had predicted that the closest relatives to eukaryotes would have those appendages because they thought that bacteria might actually nestle in with the archaeal cell and over time, this association between bacteria, that would eventually become mitochondria, and archaea, that would become the remainder of the cell, would become increasingly intimate until they started forming one cell.

0:14:40.7 SC: Okay. So we think, so people predicted that; is this evidence that it's true? I didn't quite get the connection.

0:14:48.1 TW: So people have tried to make models and tried to understand how this initial association of an archaeon with a bacterium would lead to what we now recognize as a eukaryotic cell. And to put it sort of simply, there's different models of how people try to square the fact that mitochondria and the nucleus and the outer membrane of eukaryotes are quite different. So how would that have evolved? And one of the models that explained that is that actually the archaea cell started to envelope bacteria that were initially maybe more loosely associated exchanging nutrients, but not in a totally obligatory style.

0:15:37.0 SC: Okay. I guess something that I should have asked, maybe you've already answered, but it slipped by me. Is there a way of explaining what the difference is between archaea and bacteria if they're both prokaryotes? You say that it's a genetic difference. Am I gonna have to just trust you on that, or is there a way to explain what the genetic difference is?

[laughter]

0:16:00.1 TW: So it's, I mean...

0:16:00.2 SC: I'm happy to trust you.

0:16:00.5 TW: If you just looked at them, there's no obvious differences. There's some components that are different. So, for example, bacteria have a chemical element called peptidoglycan on their cell wall. Archaea don't have that. There are elements that some archaea have bacteria don't have. But the distinction really comes from the differences at the genetic level, at the level of the sequence. So, basically, all I can... The easiest way to explain it is maybe that some of the machines are recognizably different, particularly the machines that are key to running the main processes in the cell, such as transcription, translation, replication.

0:16:48.0 SC: And those machines on the archaea side are closer to what we see in eukaryotes than the bacterial version.

0:16:56.9 TW: Yes. Correct.

0:16:58.1 SC: So some plucky archaea back in the day swallowed a bacteria, and was that the birth of eukaryotes, or is it something we're a little sketchy about still?

0:17:09.9 TW: Well, that's the model; how the details happened is borderline impossible to say. You can make a case about whether something is consistent with the evidence that we have, whether something is parsimonious, but there's so many variables that we don't know about that I think that we'll never be able to reconstruct the precise nature of the event that happened.

0:17:33.2 S21: Okay. That's fair. And the cell nucleus itself, when was the first cell nucleus? Is that... I mean, it's not, I guess it's not quite clear to me the relationship between this archaea swallowing of bacteria, that helps me explain why there are sort of substructures like mitochondria in a eukaryotic cell. I'm not sure exactly where the cell nucleus itself originated.

0:18:00.9 TW: Yeah. And I'm not an expert in that at all. So rather than getting something wrong, I'll decline on that question.

0:18:07.0 SC: Okay. That is perfectly fair. Let's go back to the Asgard archaea, which are, number one, hilarious because I was reading up about them, and they're named after, you know, Heimdallr and Loki and Thor and everything, so people took the the consistency of the metaphor very centrally, and they're the ones that are, we think, most related to, we, eukaryotes, so in some sense, almost a missing link between regular archaea and eukaryotic life.

0:18:35.0 TW: Yeah. You could, I'm not sure whether you could use the word missing link, but they're the closest that we currently have to what we think in ancestral proto eukaryote might have looked like prior to the association with bacteria.

0:18:47.6 SC: And there's something about the amount of complexity in these particular Asgardian archaea that helps us think that, oh, yeah, I can see how maybe... I'm saying things as if they're statements, but you can correct me if I'm completely crazy.

0:19:03.7 TW: Yeah.

0:19:05.5 SC: That allows for a kind of phase transition to more complex structures that is compatible with what we might want out of the original eukaryotic cells.

0:19:15.3 TW: Well, so initially when people looked at these archaea, all they had was genomes. So from the genomes you can reconstruct what proteins are encoded in those genomes, and then you have to make a leap of faith to say, actually the protein I find here that I also found in eukaryote does exactly the same thing as it does in eukaryote.

0:19:38.6 SC: Okay.

0:19:39.6 TW: And it might not.

[laughter]

0:19:40.7 TW: So what, I think, would've emboldened people, and rightly so, is the fact that now that we can visualize what those cells look like, directly visualize and can grow them in the lab, that some of those predictions actually appear to have become true. So, for example, they were predicted to have complex, arguably more eukaryotic-like cytoskeleton, so a structure within the cell that gives it's shape and allows it to change it's shape, and that was indeed observed.

0:20:20.6 SC: Okay.

0:20:21.0 TW: But I should stress that these results are so recent and one of the key papers here was literally published a few weeks ago, that we are really at the beginning of this journey to understand to what extent the similarities that we see at the level of the genome really translate into similarities that we see at the level of cell architecture, cell function and interaction with say, bacteria, but which we know very little at the moment.

0:20:49.8 SC: So please do tell us what the dates of these things are. Because part of me thinks that one learns things in high school or college, and then a typical person who is not an aficionado of the field just sort of gets their knowledge stuck there. So I'm rewriting a lot of what I thought was true about the evolution of life as I talk to real biologists about these kinds of things.

0:21:14.5 TW: Yeah, and that's... When I was at school, I also got a very misleading question that basically all the big questions have been answered.

0:21:20.9 SC: Oh, no.

[laughter]

0:21:21.3 TW: And you could just, all you did was scrape around ornamentally at the surface a little bit. But no, so the one of the papers I mentioned was published, I think in January. So yeah, it's a second visualization of one of those, a second successful culture of one of those Asgard strain; it's called Lokiarchaeum Ossiferum. And now that people can grow them and can grow them a bit more efficiently than having to wait for 5 to 10 years to get something that you can visualize, now people can start doing interesting things. Biochemistry, look at the sort of internal processes of the cell. How are genes transcribed? How are they made into proteins? So really we're just starting to get to all those questions through our ability to grow these creatures.

0:22:20.3 SC: Are the actual archaea all over the place? Or is it bacteria where, there's bacteria in me, et cetera, or are archaea things that you have to really go to exotic locations to find some?

0:22:33.9 TW: So that's what people used to think, and the reason for that, so initially they were found in acidic lakes, hydrothermal vents.

0:22:44.8 SC: Right.

0:22:46.4 TW: Places where many other things don't survive. So archaea were thought to be in the main extremophiles. So Yellowstone, for example, was a big source of archaea. As I mentioned, hydrothermal vents, acids, drainage from mines. So weird places. And it turns out that actually now that we have the technologies to look much more sensitively in all kinds of environments, archaea are pretty much everywhere.

0:23:20.3 SC: Okay.

0:23:21.7 TW: But often in very low abundance. So that in the past we weren't sensitive enough to detect them or because our technology was maybe biased towards detecting what we already knew, so bacteria. So you find archaea in the soil, you find vast amounts of archaea in the sea, you find archaea in the human gut.

0:23:43.9 SC: I was gonna say, are there archaea in me?

[laughter]

0:23:47.1 TW: They are, chances are they are, in your gut, on your skin. And again, probably in quite low abundance. And for most of them we don't know what role they play in interacting with bacteria or yourself but they're there. So we know, for example, that they're quite abundant in the gut of termites and also in ruminants. And so they're probably playing important roles there in processing the food stuff that these creatures eat.

0:24:25.1 SC: So.

0:24:25.5 TW: Or benefiting.

0:24:27.3 SC: To the extent that we human beings like to classify things and keep things simple, is it more accurate to say that the eukaryotes evolved out of archaea or that it's better to just separate them off because of this symbiotic thing where both archaea and bacteria were involved?

0:24:45.8 TW: So if I had to pick between the two, I would say the latter, because I think eukaryotes should really be seen as a merger of different domains of life. Only then will you probably truly understand what makes a eukaryotic cell tick.

0:25:00.9 SC: Right.

0:25:01.0 TW: But where the current model is different from what it was before is that previously people used to think you really had, in terms of genetic similarity, three domains of life; you had bacteria, you had archaea and you had eukaryotes. And that sort of tripartite view implies that all the eukaryotes are more closely related to each other than they are to any of the archaea, and all the archaea are more closer related to each other than they are to any [0:25:31.2] ____ and so on. What we know now is that actually some archaea are more closely related to eukaryotes than they are to other archaea, so in terms of their genetic similarity of they, the eukaryotes nestle within a broader archaea diversity. So that's really what's changed over the last 10 years or so in terms of our appreciation of how eukaryotes and archaea relate.

0:26:02.8 SC: And is it... Am I correct in remembering that it was the development of eukaryotes that took so long in the evolution of life, that life itself began not too long after the earth sat down and got formed, but then for a long time it was all prokaryotes?

0:26:19.2 TW: Yes, although, again, that's not really my area of expertise. And I have to say, if you go billions of years to the past, you have no fossil record to rely on; you're just making inferences from the genomes that you can observe today. So you're looking at current genome sequences, reconstructing relationships and trying to understand how these relate to each other in terms of time. And often you have to make some assumptions. So for example, how many changes do you see in a genome over time? And we know very little about how regular those changes in the DNA really are and whether what you can see at the moment you can extrapolate 2 billion years backwards. So there's, in terms of how long bacteria existed before eukaryotes emerged, people have been debating, but the margins of error are probably quite large.

0:27:09.0 SC: Okay. That's fair. I do apologize for asking you these questions outside your expertise, but they're all, it's all biology to me. I can't see the fine distinctions there, so.

[laughter]

0:27:18.4 TW: Yeah, I know.

0:27:18.7 SC: We'll get to your real expertise.

0:27:20.3 TW: I'm sure I'll get angry emails from people who know what they're talking about but that's fine.

[laughter]

0:27:26.1 SC: Yeah, that's just part of the bargain here. And therefore I'm gonna ask you one more question, which I think might not be in your area, but then we'll move on. It's...

0:27:36.2 TW: I'll stop, no commenting you, so.

0:27:38.0 SC: You could do that, that's fair. Be a good politician. Is it correct that multicellular organisms are always eukaryotes?

0:27:47.4 TW: So in terms of multicellular organisms that, so I'm trying to define it for myself, because I know that there are bacteria for example that can occur in, I wanna say, assemblages of multiple cells, and you can also have assemblages where you have defined roles of individual cells within that assemblage.

0:28:18.2 SC: Okay.

0:28:18.3 TW: Right.

0:28:18.9 SC: Yeah.

0:28:19.0 TW: So that does exist in bacteria but it's... So what makes a multicellular organism, I'm having to think here. So I think I'll stop with the fact, with the observation that bacteria can form assemblages with specialized cell types, but I'm not sure to what extent that answers your question.

0:28:41.7 SC: Yeah, no, that's okay. This is why biology is messy and it's much better to be a physicist because the boundaries are a little bit fuzzy. The overwhelming majority of what we think of as multicellular organisms are in fact eukaryotes but there are...

0:28:56.4 TW: Yeah, I think that's fair to say.

0:28:58.4 SC: Yeah.

0:28:58.7 TW: That's in a way what makes biology interesting, right? It's a science of exceptionalism.

0:29:04.2 SC: Exactly. Right.

0:29:04.6 TW: So you can always find something that violates your perceived wisdom.

0:29:09.2 SC: Okay, good. So thank you for indulging my, fairly elementary, but still difficult questions 'cause biology is hard. But let's get into something that I think is more in your specific domain of expertise. What's going on inside these eukaryotic cells in particular? And we'll get to some of the cool ways in which they show up in the other cells also. But I guess as an open-ended question, what does the person on the street need to know about the substructure within a typical eukaryotic cell? We mentioned that there's a nucleus, that's obviously important. We mentioned mitochondria, there's a cell wall. I'm sure there's many other things going on, but I'll let you suggest which ones are the most important.

0:29:56.7 TW: Yeah, I think there's a number of compartments that do dedicated things. So, for example, what's called the Golgi structures that are responsible, if you want, for producing proteins that get secreted. Structures in which proteins or other things get digested. And depending on the eukaryote you at you might find different structures. So in plants you have chloroplasts, which are key for photosynthesis. And then if you look around, you'll find some structures in some organisms that you don't see in others. But that structures, that sort of compartmentalization, if you want, is also present even though you don't have a nucleus in prokaryotes. So you also have different structures and those that do specialize things.

0:30:52.9 SC: And where it gets really interesting to me is the structures that, shepherd, I guess is the right word, and protect our DNA. We always like to brag or amaze ourselves by, if you took a human DNA and you stretched it out, it would be very long. Do you know the actual number? I don't think I do.

0:31:15.7 TW: Oh, I don't know, so in the meter range...

0:31:19.3 SC: Meter, yeah, something like that.

0:31:21.4 TW: Yeah. So from a single cell, that is, if you took the DNA from all the cells, you'd stretch, I don't wanna give a number, but it's a long, it's a long journey.

[laughter]

0:31:31.8 TW: Yes. So I think that the first thing to mention here is that DNA has to do a number of different things, right? One is just as a material for passing on your genetic information to the next generation, right? The other is to decode the genes that you need at the right time in the right place. And throughout that journey, you wanna protect that DNA from damage, from... If you're single cell organism, you might care about UV damage, for example, or most all organisms care about water damage, so oxidative stress; you wanna protect the DNA from that.

0:32:13.2 TW: So there's an intrinsic trade off here between making all that information accessible to the machinery that transcribes it but also protecting it and allowing that information to be accessed at the right time in the right place while suppressing other bits of the DNA, which is why the DNA doesn't float around nakedly in the nucleus or in prokaryotes, just in the cell, but it's associated with a number of different proteins that impart some organization onto that DNA, to pattern access as you want. I think about it mostly as access control.

0:32:56.3 SC: Well, when you see some of the relationships between the different pieces of substructure, and I was looking at complete maps of the metabolic pathways and some of the archaea, it's almost enough to make you believe in intelligent design, isn't it? I mean, there's a lot going on there that is very intricately connected.

0:33:15.8 TW: I often have the opposite feeling.

0:33:25.3 SC: Good.

0:33:26.0 TW: That if somebody had designed this intelligently, it would've been much simpler. [laughter] There's so many things that, especially in metabolism, that make no obvious sense to me. But I have no training in that, but.

0:33:33.5 SC: That's fair enough. Yeah.

0:33:37.0 TW: There's so much historical contingency in many parts of genome evolution. It's often so convoluted that it's often hard to dissect what really is going on.

0:33:45.3 SC: Yeah. Okay. That is fair enough. I guess the right thing to say is there is complexity there, right? It does, it actually doesn't seem very designed, but there are different pieces that manage somehow to work together.

0:34:00.1 TW: Yes, correct. It's often extremely complex. And we understand the sliver of the complexity, I think, especially if it comes to the things, and it's going back to the point I made earlier, that maybe we just haven't measured. So people, again, only recently have started to systematically ask, what are the small molecules that are present in the cell? Like, okay, we can look at DNA, we can look at RNA, we can look at proteins, but there's so many other things in the cell. Ions, metabolites. What do they all do and how do they interact? How do they interact with proteins and the DNA and how does it all hang together? And again, technological advances driving our understanding of what the cell's doing, only with the ability to measure those different metabolism, are we getting a clearer understanding of what might be happening.

0:34:44.8 SC: And that raises a question that's been in the back of my mind actually. We say that the DNA encodes information that governs the structure of the cell, et cetera, and even the function, I suppose, later on down the line, how exclusive or exhaustive is that, in the sense that, are there aspects of the cell that are just spontaneously self organizing? Like, the shape of the cell walls or whatever, or does the DNA really like micromanage everything that is going on in all the different parts?

0:35:21.4 TW: No, it's not. And there's obviously also an aspect of physics here, right? So for example, when tissues form the way they form is very much patterned by how they arrange against each other, how they arrange against the surface of other cells. And that building plan is not necessarily directly encoded in the DNA, but it's part of the history that the DNA has come to work with. So if you... 'Cause you have to have the DNA and transplant it into something. If you took DNA from a bacterium and put it in the context of a eukaryotic cell, what would that actually do? And people have asked that question to some extent, not quite over that distance, but say, take a human chromosome, put it in a mouse. How does that mouse recognize this new DNA? Can that DNA instruct the cell to do certain things? And it works to some extent if this information is not critical, but you cannot just remove the entire genome from a human cell, put a bacterial DNA in there and suddenly the human cell will turn to bacterial cell. So there's this, if you want, historical patterning; the cell has to be there for the DNA to act on this, provide the instructions that are needed to build another type of the same cell.

0:36:44.8 SC: Yeah. Okay. Good. Yeah. The DNA is tailor made or vice versa for the cell. I don't know. They're, it's not one size fits all.

0:36:47.0 TW: Yeah, exactly.

0:36:49.3 SC: Okay, good. So let's start addressing this question of how we take a meter long molecule of DNA and squeeze it into the nucleus of a cell. Is this something that we understand?

0:37:02.3 TW: Yeah, I think we understand that reasonably well. So I think that the first thing to think about is DNA is, think about it as a sort of rubber tubing, right? So it's a bit stiff. It's a bit stiff. You can fold it over but not quite as a very sharp bend angles. Second thing to understand is that it's highly negatively charged 'cause there's phosphate in there. So if you now have it, a molecule that's negatively charged throughout this whole rod and try to bend it over, there's actually repulsion, right? So you have to neutralize that charge. How can you neutralize that charge? First answer is ions, so you can have positively charged ions that neutralizes the charge.

0:37:45.3 SC: That was my guess. Yeah.

0:37:46.9 SC: Yeah. And it turns out that because of the geometry of the DNA, ions with a valency of one don't really do it for you. So you want something that has a higher valency, so more positively charges. So two, you're getting to the point where you might be doing something. But if you really want to compact DNA, you go three and higher, and you can provide those charges either through some ions directly or through proteins that also can be charged. So, for example, proteins that encode a lot of arginine are positively charged, and they're really good at compacting DNA. So, for example, one cell where DNA is extremely compacted is sperm. So sperm cells have a protein called protamine, and proteins are extremely highly charged, almost just arginines. And they can really clump together the DNA really nicely. The trick here is that if you're a sperm, if you have DNA in a sperm, you don't really have to do all that much. All you wanna be is compact...

0:39:00.2 SC: You wanna reach your destination.

0:39:02.2 TW: Exactly. Metabolism is still there, you can still swim, but you don't need to encode any new information; you don't have to be responsive to an environment, just swim, swim, swim, swim, swim until you reach your target, fingers crossed. Right? So the key for other cells that need to either dynamically respond to their environment or replicate or do something else is that just compacting doesn't work for you. So you need to be compact and provide access to [0:39:32.7] ____ in the right time at the right place. So our cells in our body, sperm apart, don't have protamines, but they're packaged in the DNA using a different type of protein, it's called a histone. So you have histones that assemble into a complex called the nuclear zone, and they wrap the DNA around that complex. And again, the histones have positively charged amino acids so that they partly neutralize the charge.

0:40:02.9 TW: So in eukaryotes histones are universal, as far as we know, with a quirky exception, which I can tell you about if you want. So all your eukaryotes, except in one case that we know of, organize their chromosomes at a basic level using those histone proteins. And the exception is called dinoflagellates; they live in, they're sort of unicellular creatures, live in the sea. They're responsible for algal blooms, for example, in some instances. And they still encode histones, you can find histones if you look at their genomes, but they don't seem to be using them for packaging; instead it appears that somehow they acquired a protein that comes originally from a virus and that protein has taken over the packaging function. They packaged their DNA, most of the DNA very tightly in this sort of almost crystalline state. And how exactly gene expression works in these organisms I think is something people still try to figure out.

0:41:09.7 SC: Okay. I mean, I really love that 'cause I think I understood something and probably 'cause it was sounding pretty Physicsy there for a while, which...

[laughter]

0:41:18.5 TW: I should talk more about ions.

0:41:19.7 SC: Yeah. As long as there's electrical charges repelling each other, I'm very much on board. So the DNA by itself doesn't want to be compacted 'cause it's, like charges repel and it's negatively charged, so you can shield that with some positive charges. But in some sense, unless you're a sperm cell, like you said, you don't want to be too efficient at that because the DNA has a role to play. You wanna protect it and you want to shield it and you want to compactified it, but you also want to let the cell access it to figure out what to do about which proteins to build. Right?

0:41:53.6 TW: Exactly. Yeah. And so you have some special instances like sperm or say spores, bacterial spores or where you are sort of a dormant state, you wanna survive some harsh conditions, you initiate a spore relation program, you package up your DNA, you're fine. You just wait until some trigger, maybe water, maybe some suitable conditions activate you and you can restart your normal growth cycle again.

0:42:22.0 SC: So these histones seem like kind of a big deal. I'll be very honest and confess that before 2023 I never heard of a histone, or at least I didn't remember what histones were. So do we know where they came from? Do we know, what were the first cells that had the idea of using these particular... Histones are proteins, right?

0:42:45.0 TW: Correct. They're proteins.

0:42:45.1 SC: Kind of a protein, yeah.

0:42:47.8 TW: And as I said there, you find them in all eukaryotes, but you also find them in many, many archaea, including the archaea we talked about earlier but many other archaea so they're quite widespread. And I think it's fair to say that most people assume that they are being used for the same purpose, so in part compacting the genome and controlling gene expression, but actually we don't really know that yet. And in some archaea they might be used in a different way to others. And I'll tell you that for the following reason. If you look at archaea genomes, you have many where you find the histone, but then if you actually look at how many of those proteins you find in a given archaea cell, that can vary quite a lot. So some archaea express minute quantities of the histone, so there's no way they're using it in the same manner that eukaryotes do. So they're just too lowly abundant to be used as a sort of packaging agent, whereas other archaea might actually be doing just that; they might have histones that coat or wrap most of the DNA in.

0:44:01.5 SC: So maybe some archaea way back in the day first hit upon the idea of making histones and they put them to various uses, and then at some point they realized, oh, this is really, really good for packing DNA and the eukaryotes borrowed that.

0:44:20.0 TW: Yeah. Or if you think about how we think eukaryogenesis happened, the common ancestor of all eukaryote might have started out from a ground state where most of the DNA was parceled up in a similar manner than it's now.

0:44:36.4 SC: I said borrowed but inherited would've been a better word.

0:44:40.9 TW: Having said that, there's a big gap in our knowledge, in our understanding of not histones themselves but the transition between archaea and how they use histones and eukaryotes and how they use histones. So in eukaryotes, in all eukaryotes, you have a very well conserved complex of different histones. So they all fit together. There's four different types; they all fit together in the same manner in all your eukaryotes. They're always arranged, eight histones, four different types, two of each assemble into the same complex. In archaea you also have histones, but they seem to be combining much more freely. There's no fixed size of what we call a nuclear zone, so this eukaryotic histone DNA complex seems a bit much more sort of versatile in a way. So how exactly archaea eukaryotes got stuck with this particular architecture is something we still don't understand.

0:45:36.9 SC: Is it possible that we can think of the archaea as little experimental laboratories trying out different things and once they hit upon this particular configuration of histones that is used in eukaryotes, it was both very useful and even helped the initial evolution into eukaryotic life.?

0:45:57.4 TW: It's a possibility. So people have certainly suggested that histones might have been beneficial for eukaryote genesis.

0:46:03.4 SC: Yeah.

0:46:05.3 TW: To what extent the specific architecture of the histone DNA complex is useful I don't know., but it's certainly been suggested. What I think is interesting, an interesting distinction between eukaryotes and prokaryotes when it comes to histones is that in eukaryotes histones give you something like localized access control. So imagine you have your genome and most of that genome is wrapped up in histones. So now these histones have flexible protein tails and those tails can get modified. So other enzymes, other proteins can put information on those tails and they can do that in a locally specific manner. So, for example, I can put a, what's called a post-translational modification on the histones associated with a chunk of DNA that codes for a gene of interest and that modification will be recognized and shut down that gene. That gives you a flexibility of sort of localized control because you can modify some histones in some places but leave other others untouched. And that, as far as we know, doesn't really exist in our care.

0:47:21.1 SC: Oh, okay.

0:47:21.2 TW: At least it hasn't been demonstrated. So we can maybe change the overall expression of the histones or change different types, but the default assumption is that if we do that, we affect the state of the, what's called chromatin, so the DNA protein complex, we change that globally rather than locally.

0:47:40.8 SC: I see. So that's great. So the histones are not only an efficient packing matrix for the DNA, but they offer some measure of control that we didn't otherwise have. 'Cause we were talking before about gene expression and how it's not, the DNA is not just a passive thing there; we sort of pick and choose how it is expressed and the histones are playing a role there.

0:48:04.2 TW: Exactly. And that's why I think arguably most people are interested in histones and eukaryotes because they... You could see them as a platform almost for the integration of different types of information. So something happens in the outside world, signaling cascades and the cell get triggered, that end up in histones being modified, recognized by the proteins, and then that particular gene starts getting expressed or shut down.

0:48:31.8 SC: So a couple of weeks ago, a few weeks ago, I was reading a tweet by Theodore Ornav, who's a previous Mindscape guest, an expert in CRISPR and gene editing and so forth. And even through the medium of Twitter, I could tell that he was jumping up and down in excitement. He said, oh my goodness, we're gonna have to rewrite all the textbooks; this is really crucial. And what he was pointing to was a paper of yours that says, okay, now we found that there are histones in bacteria as well, not just archaea and eukaryotes. And it seems that they're even important to the bacteria; they're functional in some way. Tell us about that.

0:49:08.5 TW: Well, I still have to get past the point of imagining Theodore Ornav jumping up and down in Joy, so yeah. So I mentioned before, you find histones, or eukaryotes, in many, many archaea, but I think it's fair to say that most people would think that histones are absent from bacteria, and that is true for the vast majority of bacteria; about 98% or so of bacteria don't have histones. But it turns out that if you systematically look through a collection of bacteria genomes, you find sequences in those genomes that comparatively looked like a histone. And people had observed that two, three years ago just through bioinformatics, and maybe at the time that paper didn't get the coverage it maybe deserved. What was unclear is whether these things that looked like histones really were histones, so whether they would fold up into the same three-dimensional structure, and what they did, if anything.

0:50:14.8 TW: 'Cause sometimes you have instances where you find genes getting passed around, so what we call horizontal transfer from bacteria to bacteria or archaea and maybe they pick up some genetic material from eukaryotes, and maybe they just hang around in a genome and don't end up doing very much. But a postdoc of mine, Antoine [0:50:37.5] ____, started looking at that more seriously and found that actually if you look at certain types of bacteria, you find them quite well conserved. So they're not just present in a single genome but in closer related GMs as well, and if you measure their abundance, they're extremely abundant. And if something is manufactured in large quantities by the cell, it's probably useful because otherwise the...

[laughter]

0:51:05.0 SC: You get rid of it a lot.

0:51:05.5 TW: The cell doesn't tend to churn out any old crap it doesn't need.

0:51:09.6 SC: Yeah.

0:51:12.2 TW: So then we went on and with collaborators in the US and more locally tried to, A, test whether those histones were useful to the cell, and it turns out that they are, so we couldn't get rid of the histone gene. So normally if you have a gene that is not vital to cellular functions in the laboratory environment, you can go in, cut that piece of DNA out of the genome and the bacterium would be fine, just keep on growing. We tried doing that, it didn't work, and we tried multiple times and it always failed. And that tells you that it's essential for growth under the conditions what we have. That makes sense. It's extremely highly expressed, it's gonna be useful for something.

0:52:00.3 SC: Yeah.

0:52:03.6 TW: And then our collaborators in the US, Sean Lawson and Karen Lugar, showed that indeed that piece of sequence that looked like a histone when you compare the sequence actually folded up into something that looks remarkably like a histone from your eukaryotes with some important but subtle tweaks. So it's a shorter version of the hill, basically.

0:52:29.4 SC: Okay. So what is the moral of this story? So you said that not all bacteria have these histones, but the histones do seem to cross the lines of the kingdoms. They're in all the eukaryotes, I forget whether it's all or some of the archaea, and some of the bacteria.

0:52:47.3 TW: Yeah. Many many, the majority of archaea have histones or eukaryotes and a few selected types of bacteria. I'm not sure what the overall moral of the story is, except to say that biological perceived wisdoms are meant to be broken. So some people when they comment on this saying, "Oh, it's a dogma defying discovery." I wouldn't go that far, 'cause a dogma to me is something that you postulate cannot be different. And nothing in biology would've made the case that histones cannot exist in bacteria. What people were surprised about that they hadn't been observed before but they're actually there. And I think that it's probably too, for many enzymes which were once thought to be unique to group A and then are found in some of group B, and also tend to transpire, to play an important role in that group.

0:53:55.4 TW: And second moral of the story maybe is that if parts in one organism turn out to be very useful and can be passed around through this horizontal transfer process, they might well be used in other genomes, not necessarily for the same purpose, but what's sort of intriguing in a way is that the bacteria that we found have those histones. They also in many instances have a life stage that to me is almost a bit like sperm.

0:54:26.5 SC: Okay.

0:54:27.6 TW: So one bacterium, for example, is called Bdellovibrio, and it's a super interesting bacterium in that it's a predator of other bacteria. So it swims around, in an almost like a sperm like fashion, swims around hunting for prey. It attaches to another bacterium, drills a hole into the outside of the bacterium, sneaks inside, seals the neck behind, and then it starts to eat the bacterium from the inside.

0:54:56.3 SC: Wow.

0:54:57.0 TW: By secreting enzymes that chop up it's prey. So the key point here is that you have two different life cycle stages, the sort of sperm-like free-swimming attack cell, as they're called, and the thing that replicates actively inside the host. And the small attack cell is really small but the highly condensed chromatin. So initially we're thinking, well, maybe the histone is useful in bringing about that compaction. We don't think that's the entirety of the story, but it's certainly an intriguing lead into trying to understand why the histone was acquired by bacteria like that.

0:55:39.0 SC: I never knew that they were bacterial predators; I thought the bacterial were all vegans, essentially, but I guess it's a bacterium eat bacterium world out there.

0:55:48.9 TW: Yeah. To be fair, like before we started working on this project and discovered that histones were present in this particular bacterium, I hadn't truly heard of it before. But that's, again, a pleasure of biology that's sort of endlessly fascinating novelty.

0:56:06.1 SC: I mean, maybe we should pause to talk a little bit about this whole big idea of horizontal gene transfer or just more generally the idea that we've been taught, again in our high school education, that there is a tree of life and, you know, one organism evolves from another one and then they branch off and they're separate. But down there in the unicellular world it seems a lot more sociable. People, cells share genetic information they pick up tricks, substructures, proteins, what have you. Is this something we're beginning to discover, or do we have a good handle on how it's working? Is it just amusing or is it crucially important? What's going on?

0:56:53.7 TW: I think it's, in many instances crucially important both to understand how evolution happened in bacteria and in unicellular organisms in particular. It's important to understand because one of the key aspects of that is that pathogenic elements, genes that help in pathogenesis get shared around.

0:57:18.3 TW: So often bacteria acquire certain genes and then become pathogenic and can pass them onto other bacteria. So it's interesting from a human health perspective, if you want.

0:57:31.3 SC: Yeah. [laughter]

0:57:32.1 TW: And I think overall we have a good handle on the mechanisms of how this happens. What we are still beginning to understand is how common really that phenomenon is, not only in bacteria but also in eukaryotes, and what the implications of that are for our understanding of early evolution. 'Cause the further back you go, the harder it is to envisage the sort of tree-like bifurcating structure that you just mentioned.

0:58:06.6 SC: So I guess, I was going to say that it seems like, that grown up eukaryotes are a little bit more settled in their ways and less likely to do this kind of transfer, but you just said it happens. Is it less common or are there good examples?

0:58:21.8 TW: So it happens. It happens frequently in some species. So, again, maybe in a way...

0:58:31.8 TW: The way to understand it is that as humans, for example, we have a large part of our body dedicated to just running around doing things, and only a small part of our body is dedicated to making genetic material that gets passed on to the next generation. So if you are a piece of DNA and you wanna get into that specific population that sells, that gets to the next generation, you have a whole lot of body to get through. So our germline, if you want, is sequestered away and quite protected in that way; much more protected than yeast, for example, single celled organisms. If you think about acquiring naked DNA from the outside, much harder to see that happening for the human germline than it is for yeast cell.

0:59:31.2 TW: So that's coming to the mechanisms of how this happens, right, so acquisition of DNA from the environment is one of them. DNA doesn't tend to be super stable in many environments, so often it gets degraded. The other is direct bridges between different bacterial cells, for example. So bacteria do a thing called conjugation where they link up neighboring cells and then they can pass genetic material through those cells.

1:00:01.8 TW: And then I think one of the key mechanisms is actual viruses or what you call phage in bacteria. So they can pick up, when they infect a bacterial cell, they pick up a fragment of the genome, phage replicates, a new infection cycle starts and then it might actually inject that piece of DNA that it previously picked up into a new host that might then, if the infection fails, integrate that bit of DNA into its genome. So yeah. Different ways of sharing things around.

1:00:33.1 SC: And my impression is that not only can little bits of DNA get shared around but they could even discover new uses for them when they're in a different kind of cell. 'Cause like you mentioned before, like a DNA out there in the wild is not doing that much; it depends a lot on the particular kind of cellular environment it's in.

1:00:50.8 TW: Yeah, it depends on what sort of protein, for example, we're talking about. So something that might be almost immediately useful is a single enzyme. So say your quiet enzyme and the enzyme can degrade some antibiotic and you're in an environment where you have to resist being killed by the antibiotic, immediately useful. If you are part of a large complex of several dozens of proteins and you get transferred as a single gene, you might find yourself in the context where actually you don't quite know what your purpose is, to put it in a the sort anthropomorphic way, right?

1:01:32.6 SC: I felt that way. Yeah.

1:01:36.9 TW: The genetic context in which those transfers happen, the environmental context, whether that bit of DNA is gonna be useful or just selected out, obviously matters a lot. So that explains in part why organisms that are closer to each other, both in terms of their genetics, sequence similarity but also in terms of their environment, tend to exchange genes more frequently because something that has proven useful to a bacterium that lives at 80 degree might also prove more useful to a bacterium that, an archaeon that lives at 80 degrees than an archaeon that lives at 30 degrees.

1:02:14.8 SC: So since we're near the end of the podcast here, I will once again be very unfair and and ask you questions that are not necessarily within your professional wheelhouse. But given all these, given what we've learned about the sharing of different genes and different mechanisms and the appearance of histones where they weren't predicted, is there any implication of this for the origin of life, how the whole thing got started or just a better understanding of the nature of life which hopefully someday will lead to a better understanding how it might have originated? And the answer could be no.

[laughter]

1:02:51.6 TW: You mean in relation to horizontal gene transfer?

1:02:54.8 SC: Well, just everything we've been talking about. Yeah, horizontal gene transfer but just also the mixing and matching of different parts of the genome or the repurposing of proteins, which I think is fascinating.

1:03:08.5 TW: So I think when it comes to understanding the origin of life, if anything, it complicates matters. Because our ability to reconstruct those very deep ancestral states currently depends on comparing genome sequences and making assumption about what those genes did in the past. And those assumptions, as we just highlighted, might be wrong. What I think, where I think this sort of knowledge of repurposing horizontal gene transfer, what some people call moonlighting, so a protein having maybe one main function but also being able to do something else and then eventually it might focus on that secondary task, will be interesting in a synthetic biology context, so when it comes to engineering desirable properties into a microbe, so...

1:04:00.4 SC: I see, yeah.

1:04:02.2 TW: So you could say, how do I build the smallest genome I possibly can that can do X, Y and Z, survive at 80 degrees but replicate very fast? So I think a better understanding of the toolkit and how it can be shared around and how it can be repurposed actually gives us a much larger selection of potentially useful molecular components that we can use to build those synthetic organisms that'll be...

1:04:33.4 SC: That's a very very good point. I mean, that's clearly gonna be one of the growth areas in the decades to come, not just editing existing DNA to tweak blue eyes versus green eyes versus brown eyes but making new functions, right, making kind of dramatically new organisms maybe.

1:04:54.7 SC: Yeah. And I think ultimately that ability, and maybe also playfulness in creating those new organisms will then enable us to go back and and say, well, actually now I understand much better about the rules in which I can combine those different elements. What does that tell me about what I think went on 2 billion years ago? It's maybe sort of what do you call it, learning by building, right? So you try to assemble a system, maybe use parts from archaea and bacteria and eukarya to see how they can or cannot work together and then end up with a much clearer understanding of the rules of life, if you want, and how they might have emerged ancestrally.

1:05:39.7 SC: You know, I teased a biologist, 'cause it does seem very messy and and hard to understand to me, but it is also very clear that really important questions are being not just asked but answered in real time. So it's great to get a little view of the front lines on that. So, Tobias Warnecke, thanks so much for being on the Mindscape Podcast.

1:05:57.6 TW: Thanks for having me again.

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