Thomas Boothby: Surviving at the extremes of life

Thomas Boothby, Image Courtesy of Thomas Boothby

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Sally spoke with Professor Thomas Boothby, a molecular biologist at the University of Wyoming whose lab researches how organisms survive at the extremes of life.

To start off - what actually is a tardigrade?

Thomas: Tardigrades are microscopic animals. So, being animals, they're multicellular, they have complex organs and tissues, they have a brain, they have sensory organs like eye spots and, despite being microscopic, they're extremely robust. They can survive a number of environmental extremes that we typically think of as being incompatible with life.

Thomas: You can freeze tardigrades down to about a degree above absolute zero, the temperature at which all molecular motion stops. You can heat some species up past the boiling point of water. They can go for days or weeks with little or no oxygen. They can survive thousands of times as much radiation as you or I could.

Thomas: They do this really neat trick that we're particularly interested in, which is that they can dry out - so they can lose all the hydrating water inside their bodies and cells and survive. If people have heard of tardigrades before, they've probably heard about them because they're the only animal that's been shown to survive prolonged exposure to the vacuum of outer space.

Sally: How unique are these adaptations to tardigrades? Are there other species that can cope with such a high degree of drying out or radiation or extreme temperatures?

Thomas: In terms of drying out, I think people think this is a very rare, exotic trait. But actually, throughout the kingdoms of life, desiccation-tolerant organisms are found on every branch of the tree of life. If you think about plants for example, nearly every land plant has some sort of desiccation-tolerant life stage, whether that be seeds or spores or pollen. Many bacteria can do this too. Where this trait is rarer is within the animal kingdom. Right now we only know of four groups of animals that have members that can perform some of these tricks. So those include tardigrades, some arthropods like brine shrimp, some nematode worms and then some other little animals known as rotifers, which are aquatic very small little animals. 

Thomas: What I think makes Tardigrades very unique is their ability to survive so many different stresses, not just drying out, but also very cold temperatures, very hot temperatures, extreme radiation. And to do this seemingly at any life stage. Eggs and adults from tardigrades are very radio tolerant or very resistant to cold temperatures. So I think in that case it makes them quite unique. 

Sally: Where do they normally live? I'm guessing they don't normally live in the vacuum of space?

Thomas: No, they don't normally live in the vacuum of outer space. But here on Earth, you can find tardigrades in almost any environment. They've been found on every continent, including Antarctica. There are marine species that live in the ocean. There are terrestrial species that live on land. There are freshwater species that live in lakes or rivers. This heartiness, being so tough, has probably allowed them to colonise all these different environments. 

Sally: How do they do what they do? Because what you've just described to me is a list of superpowers and not even one superhero has all of those powers. Normally, each superhero only has about one power. But going down to just above absolute zero, that's not an environment they will have been adapted to. That's not a temperature that we experience in the natural world. How on earth is that something that they adapted to?

Thomas: I think that is a really interesting question. How did an organism evolve to survive under conditions that it would never have experienced before? The best explanation that we have for that right now is this idea of cross-tolerance; meaning by evolving to tolerate one sort of extreme condition, the animals may have as a byproduct now been able to survive other extreme conditions. 

Thomas: I think a really good example of this is desiccation and radiation tolerance. So, tardigrades being so small and diminutive, at least the terrestrial species, probably every day undergo desiccation or drying to some degree. And when that happens, we know that that imparts a lot of different stresses on their cells. For example, their DNA fragments. And when they rehydrate, they're able to stitch it back together. 

Thomas: Now, similar things happen when tardigrades are irradiated. Radiation does a lot of damage to their DNA. So just as a byproduct of being able to repair their DNA that's been damaged due to this drying process, they probably have very robust mechanisms in place that when their DNA is damaged by radiation, they're still able to cobble it back together. Even though in nature, they're never going to be exposed to the thousands of gray of radiation that they've been shown to survive.

Sally: When you say they're desiccating, how much water are they losing?

Thomas: When tardigrades dry out - just air dry naturally - they're probably getting down to around five to 10% water content. 

Sally: That seems ridiculous.

Thomas: By comparison, if we lose about 30% of our water content, we will die. A typical house plant, if it loses about 40% of its water content, it will die. But tardigrades are losing 90 to 95% of their water content and they can stay in that dry state for years or even decades. When you place them back in water, they rehydrate and reanimate and pretty soon you'll be seeing them running around, feeding and reproducing like nothing happened to them.

Sally: Do they have any physical barriers to drying out? Or are they just like "sod it, we're just going to let the water evaporate away because we know we can deal with it"?

Thomas: Well, no. So tardigrades, like many species, need to dry out relatively slowly. This allows them time to one, sense that they're drying out, and two, build up the protectants that they're going to make that are going to allow them to survive that process. To help themselves dry out slowly, tardigrades do this cool trick where they pull their eight legs and head inside their cuticle, which you can think of as their exoskeleton. They curl up into this little ball-like structure known as a ton and that comes from the German word for cask or barrel - I guess the original German scientists looking at this process thought they looked like little barrels of wine when they were drying out. 

Thomas: What that does is it allows them to bury the tips of their claws and their mouth, which are the points of their body where they lose water very readily from. It allows them to bury those parts of their body inside this little ball-like structure, which helps slow down the drying process even more. So just giving them more time to respond to this harsh environment that they sense is coming on.

Sally: Now you mentioned that they have some protections that they can put in place as they're drying out slowly, and presumably it's those protections that you are most interested in. How on earth can an animal survive drying out that much? What is it they're doing so that they don't die? 

Thomas: One thing that we've found is that when these animals are drying out, they start making a whole bunch of a very specific protein. These proteins have a long, cumbersome name, which is cytoplasmic abundant heat-soluble proteins, or CAHS proteins. It's a mouthful, so let's just call them the tardigrade proteins. They're unique to tardigrades. So if we look at the level of sequence conservation and we look and see if any other organisms out there have these, it doesn't look like they do. This is a unique adaptation that tardigrades have come up with.

Sally: Do they look like any other protein that we see in nature?

Thomas: So adding more intrigue to this question, right? These tardigrade proteins, they're what we call intrinsically disordered proteins. If people remember back to their biochemistry 101 course, they probably learned that the structure of a protein is very important for its function. However, these tardigrade proteins turned that idea on their head, because disordered proteins lack a stable three-dimensional structure, and yet many are known to play very important roles in different cell biological processes, including stress tolerance.

Thomas: If we look at what sorts of shapes these proteins like to adopt, although they are very dynamic in general, they appear to exist in a dumbbell-like ensemble of shapes. They have two collapsed ends, and those ends are held apart from each other by this long disordered linker between the two of them.

Sally: How does that protein help them survive drying out? I should say, what is it about drying out that normally kills us?

Thomas: When cells dry out, a lot of bad things happen. It's important to understand that the stress of drying out isn't an all-or-nothing stress; meaning at the early stages of desiccation, there are damages being incurred to the cell that are different than the damages that are incurred at the later stage of desiccation.

Thomas: For example, imagine a cell that has just started to dry out. Water is leaving the cell, the cell is starting to shrink down and reduce in volume. And what that does is it makes the cytoplasm, the inside of the cell, extremely crowded. Why this is bad is you have a lot of different proteins and cellular components that when they get very crowded, they can start to aggregate, they can start to clump together, and this makes them non-functional. It's just a big conglomeration of junk at that point. 

Thomas: Now, when you get to even lower water contents, what can happen is you can get down to the level where now there's insufficient water to actually hydrate things like proteins. One of the things that keeps proteins really well folded and behaving nicely is that they have this shell of water around them that's making hydrogen bonds with the exterior of the protein. When those hydrogen bonds are lost, a lot of the impetus for the protein to remain well-folded is lost, and proteins start to unfold and then become non-functional. 

Sally: It's not just a concentration thing. Everything just goes haywire.

Thomas: Exactly. A really efficient mediator of desiccation protection or desiccation tolerance is going to contribute to preventing multiple things from going wrong. So in the early stages of drying, it'll be preventing proteins from aggregating, and at the latter stages of drying, it will be preventing proteins from unfolding.

Thomas: To that point, what we found with these tardigrade proteins is that depending on their concentration and hydration level, they either act really well at preventing aggregation, or they act very well at preventing unfolding. So they're tuned to deal with these different physiological conditions and chemical conditions, depending on water content and crowding within the cell.

Sally: So do they form the hydrogen bonds as if they were the water themselves surrounding the proteins?

Thomas: That's a really interesting idea and one that's been put forward in the literature before, not particularly for these tardigrade proteins, but for other protectants. What we've actually found and how we think that these proteins are working to help maintain those hydrogen bonds, is we see that the exterior of these tardigrade proteins contain amino acids that are very hydrophilic, meaning water-loving.

Thomas: Remember I mentioned tardigrades don't become truly dry, they still have about 5-10% of their water content left. What we think is happening is that the exterior of these tardigrade proteins is very attractive or sticky to water. And so those tiny little residual amounts of water are sticking to the tardigrade proteins.

Thomas: What we've seen is actually that the tardigrade proteins, they start to self-associate and they form this higher order structure that's actually a gel. It has all the properties of jello essentially. But you can imagine this gel is being made up of fibres that are interconnected, almost like a spider web. Those fibres are again, as I mentioned, very hydrophilic. So they're attracting and concentrating water. What we think is happening is the tardigrades aren't retaining any more water, but they are organizing the small amount of water that they do have into these local areas of hydration, which are helping to keep things within that gel hydrated.

Sally: This is fascinating. I can see why you would want to study tardigrades just for the sheer hell of it, because they're incredible. But you are also then applying this research to use for human health. Can you tell me more about that?

Thomas: One of the ideas we have is that right now a lot of biologic pharmaceuticals, so that is pharmaceuticals that are made from or derived from living organisms, are very, very effective. This would include things like vaccines, blood products, cell-based therapies, antibodies, et cetera.

Sally: All the cool new tech that we hear about on this podcast.

Thomas: Exactly. Now, despite being really effective, a lot of biologic pharmaceuticals have a major drawback, and that is that they're inherently unstable. The current way that we get around this is by using the cold chain. So the cold chain is essentially just a series of refrigerators and freezers that the pharmaceutical industry uses to keep biologics cold, from the onset of their production through transportation and storage to being used in the clinic. 

Thomas: In the United States or in the UK or other more developed parts of the world, keeping things cold may not seem like such a big problem. But in many remote or developing regions, maintaining a stable electrical system or even having those refrigerators and freezers can be a major economic and logistical hurdle to getting medicine to people. 

Sally: I remember that at the start of the COVID pandemic. It was why there was such a huge push for the Oxford AstraZeneca vaccine over the Pfizer one because that one didn't have to be refrigerated as much.

Thomas: That's a great example where you had this great new technology, this mRNA-based Pfizer vaccine. The issue with that was to maintain mRNA in a really stable and effective state, it needs to be frozen at negative 80 degrees Celsius. A freezer say in your house will go down to about negative 20 degrees Celsius. So people certainly don't have this equipment in their houses, hospitals would, but some pharmacies wouldn't. Certainly in developing parts of the world or very remote regions, having a negative 80 freezer can be a huge issue. 

Thomas: Our idea is that tardigrades, when they dry out, they stabilize and maintain the integrity of all their cellular components, which include proteins, mRNAs, DNA, and membranes - all the things that biologic pharmaceuticals are made out of. They do this in a dry state, at ambient conditions, or in some cases even at elevated temperatures. I mentioned before that tardigrades when they dry out, they can tolerate temperatures well above the boiling point of water. I think the record is 151 degrees Celsius.

Sally: That's absolutely bonkers to me!

Thomas: Right now what we've been doing, and we recently published a paper on this, is we've been taking both natural products from tardigrades, these tardigrade proteins, as well as engineered versions of those proteins that we've tuned to either promote this anti-aggregation property that they have, or to tune them to promote this anti-unfolding or stabilising property that they have.

Thomas: The first biologic pharmaceutical that we've targeted is one called human blood clotting factor eight. Or just factor eight. This is a protein component that we have in our blood that normally, if you get a cut, it's essential for stopping bleeding quickly or causing a blood clot.

Thomas: In cases of extreme trauma or in cases of genetic disease, for example in Hemophilia A, which affects about one in every 5,000 men, physicians will administer factor eight to help speed up clotting in the case of a haemophiliac, or somebody that's incurred sort of an extreme trauma. And this is a very effective treatment. The problem is best practices dictate that factor eight be maintained at negative 20 degrees. So you can imagine in cases of natural disaster or on the battlefield or in a clinic in a remote area, having a freezer running efficiently at negative 20 degrees may not be an option.

Thomas: In this first work that we've published, we've taken this factor eight and we formulate it or basically mix it with these different versions of the tardigrade protein that we've engineered. We've shown that not only can we stabilise this factor in a dry state at ambient conditions, but now we can actually take it through some real-world field-like conditions.

Thomas: So for example, one test that we did was to look at if we can rehydrate and desiccate the factor multiple times. Will the tardigrade protein stop damage that's incurred through these multiple desiccation cycles. When you do that, the normal damage that's incurred is the aggregation that I mentioned before. What we saw was that using certain engineered variants that we had, we were able to very effectively stop that damage from taking place. 

Thomas: On the flip side, we also wanted to look at thermal stress. You can imagine if we have vaccines or this factor eight being transported in the bed of a pickup truck through a very hot region, not only do we need to stabilise those medicines in a dry state, but they need to also be able to deal with very high temperatures. So we also use some different engineered versions of this tardigrade protein that we have to dry out factor eight, and then we actually left it at 95 degrees Celsius for days and then looked at whether or not we were able to still have functional factor eight. We saw that with certain versions of our tardigrade protein, we were. 

Sally: Along with desiccation and drying out and how that affects the proteins, you also mentioned that tardigrades can cope with immense amounts of radiation and their DNA falling apart. How?

Thomas: That is a little bit less well-understood. If I had to put in some conjecture, I'd say all organisms have to deal with DNA damage to some degree. Just in normal replication processes, mistakes are made just through normal exposure to less extreme environments damage is incurred. So we and essentially every other organism have DNA repair mechanisms. It may be that tardigrades have just evolved to basically turn these on at full blast when they experience damage. 

Thomas: There have been some reports, from other groups, of a tardigrade protein known as D sup or damage suppressor, which is thought to actually work not to repair damage, but to actually prevent damage to DNA. It's thought to work by essentially associating with DNA and then wrapping around it and acting almost as a shield to absorb the radiation damage that would normally just be directed at the DNA.

Sally: How are you using this or how is this going to possibly be used as a technology in the future?

Thomas: I think one thing that my lab is particularly interested in is thinking about how we can take lessons from tardigrades and how they protect themselves from radiation and other extreme conditions, to try and advance humanity's presence in space. When you're in outer space, of course astronauts are exposed to quite a great deal more cosmic radiation than here on Earth because we have our atmosphere here on Earth, which protects us from a lot of cosmic radiation. Of course, we won't be engineering humans with tardigrade proteins to send them into space!

Sally: Oh, are you not? We're not going to have special human tardigrade astronauts? I mean it sounds like tardigrades could do it themselves to be honest.

Thomas: So one of the impetuses for this study is tardigrades can survive in the vacuum of outer space. They've been shown to also survive and reproduce under normal space flight conditions. So the idea isn't to engineer an astronaut with a tardigrade gene, but rather to look at maybe when tardigrades are in space and they're being exposed to all this cosmic radiation, maybe they're upregulating production of antioxidants. Things that are going to scavenge reactive oxygen species that are generated from radio damage. And so maybe we can augment astronauts’ diets, or give them supplements that are going to increase their natural production of antioxidants and are going to help them survive this better.

Sally: How many tardigrades are there flying around in space right now?

Thomas: I'm not actually aware of any active research going on with tardigrades in space. Last year we just wrapped up a mission to the International Space Station, where we were culturing the animals on the space station to look at how over multiple generations those animals cope with being in those space flight conditions. Those are studies that we're still teasing apart the data that we got back here in the lab now.

Sally: But they were kept within the confines of the space station? You're not just firing them out into space and there are just poor little tardigrades floating around?

Thomas: It was a very well-controlled and contained experiment, yes.

Sally: What do you think it's going to look like 50 years from now? What's the goal for tardigrade research? If we were to give you all the money, where do you hope we'd get to?

Thomas: I hope that we will continue to understand more of the fundamental biology of these organisms. But I think too, my hope would be that we could continue to take these lessons from nature, lessons from tardigrades, and hopefully branch out beyond tardigrades to other organisms. And that people would begin to appreciate this bridge between fundamental research and applied research, and see that folks studying tardigrades or fruit flies or roundworms aren't necessarily just messing around and it's all just an intellectual pursuit, but that doing fundamental biology can really result in real effective treatments or solutions to societal problems.