Tardigrades - nicknamed water bears - are millimetre-long aquatic micro-animals. You can boil them, dry them, freeze them, irradiate them, dump chemicals on them and so on - yet, they just keep coming back to life. Their high durability means they are classed as extremophiles - they can survive in environments that are too extreme for most other organisms to handle, such as intense heat and high pressures.
Tardigrades can be found in a wide variety of habitats, ranging from the deep sea to the highest mountains, and from tropical rainforests to the freezing Arctic. There is also variety in their diet, which consists of plant fluids, algae, small invertebrates, or even each other. Tardigrades feed by piercing the cell walls and membranes of their food using piercing mouthparts called stylets.
The ability of tardigrades to survive extreme conditions was tested further than ever before in 2007 when astronauts brought around 3,000 water bears into space on the European Space Agency (ESA) orbital Foton-M3 mission. Without space suits to protect them against sub-zero temperatures, harmful solar winds and oxygen deprivation, they were put to the ultimate test. The title of the article probably already hinted at whether they survived or not.
Of course, they did.
There were no effects on tardigrade survival, reproduction or DNA integrity from microgravity and radiation. In short, they were absolutely fine and there were only minor changes to their morphology, behaviour and gene expression levels.
How do they do it?
Tardigrades have evolved several mechanisms to keep their proteins, nucleic acids and membranes ‘alive’ and stable. They can enter a dormant state called cryptobiosis, whereby their metabolic activity is reduced to an undetectable level. This consequently pauses energy-extensive processes such as reproduction, development and repair until favourable conditions return.
The most studied type of cryptobiosis is anhydrobiosis, meaning ‘life without water’, because water is the fundamental component of all living organisms. Tardigrades undergo different morphological, physiological and biochemical changes in response to desiccation - the state of extreme dryness and one of the most severe stressors for most organisms.
Morphological changes include retraction of body extremities into a compact body shape called a “tun” (Figure 1), and a decrease of cuticle permeability to prevent internal water from evaporating. When conditions are hospitable, tardigrades can revert to a normal active state by absorbing water. Anhydrobiosis can occur in any stage of the tardigrade life cycle, including the eggs.
Figure 1: Scanning electron microscopy images of a tardigrade species, Milnesium tardigradum, in the active and tun state during normal conditions and desiccation respectively. This figure was taken from Schokraie et al. (2010).
As early as 1989, Jonathan Wright made an interesting observation: different tardigrade species survive drying at different rates, but all of them die when dried too quickly. This gave scientists a hint that the key molecule or protein that acts as a bio-protectant takes some time to be produced. In an attempt to find out more about these key molecules or proteins, a straightforward transcriptome gene expression analysis was carried out in a 2017 study to identify tardigrade transcripts that were enriched during desiccation. Three families of proteins were identified: cytosolic abundant heat soluble (CAHS) protein, secreted abundant heat soluble (SAHS) protein and mitochondrial abundant heat soluble (MAHS) protein.
These proteins have one key similarity - they are all intrinsically disordered proteins (IDPs). This class of proteins lack persistent secondary and tertiary structure, and IDPs generally do not aggregate or precipitate when exposed to high temperatures or extreme pH levels. Since they are highly disordered anyway, there are not many large-scale changes in structure happening in the first place.
In contrast to this, precisely-folded proteins depend on covalent or non-covalent linkages for their structure, and these linkages are sensitive to such extreme changes. A well-known example of an IDP is α-synuclein, an unfolded protein that accumulates into clumps of protein in the brain, leading to a variety of neurodegenerative diseases such as Parkinson’s disease.
Figure 2: Example structures of intrinsically disordered proteins (IDPs; left) and structured proteins (right). Sometimes, the entire IDP or regions of IDP can take on increased secondary structure when interacting with protein partners (middle). Green represents disordered regions and red represents structured alpha or beta helical regions. This figure was adapted from Weis Research Group.
Most tardigrade-specific IDP (TDP) genes possess no sequence homology to non-tardigrade genes. RNA interference (RNAi) knockdown of TDPs significantly reduced the survival of tardigrades after desiccation, confirming their role in desiccation tolerance. However, the survival of tardigrades after freezing is not affected by RNAi knockdown, suggesting that tardigrades have different defensive and protective mechanisms against different stresses.
Side note: If you ever want to isolate IDPs from your organism for your project in the future to look at their stress tolerance characteristics, you can simply use trichloroacetic acids (TCAs) to uncover the ‘unfoldome’. The addition of TCA to proteins disrupts hydrogen bonds, causing proteins to lose their secondary structure and denature. IDPs can be separated from structured proteins by specific denaturing conditions due to the lack of tertiary structure.
Cytosolic abundant heat-soluble (CAHS) proteins
Scientists have suggested that hydrophilic molecules form a glass-like matrix within cells during desiccation, consequently encasing and immobilising all proteins to prevent denaturation. To investigate whether highly-hydrophobic CAHS TDPs contribute to the glassy state during desiccation, a type of thermal analysis was used called differential scanning calorimetry (DIC).
DIC is a powerful experimental technique for determining thermodynamic properties of biomacromolecules, polymers, nanomaterials and many more. A sample is heated or cooled in a differential scanning calorimeter, and the changes in the heat flow are tracked (Figure 3). It allows the detection of transitions such as glass transitions, defined as the temperature at which polymers undergo a transition from glassy to rubbery state. This is similar to heating a solid past its melting temperature, turning it into liquid.
The slowly-dried tardigrades were shown to produce a glass-like matrix, while quickly-dried tardigrades did not (Figure 3). This was in line with the previous observation by Jonathan Wright that tardigrades die when dried too quickly, and emphasises the importance of CAHS TDPs’ glass-forming ability.
Figure 3: An example differential scanning calorimetry thermogram that shows the difference between slowly and quickly dried tardigrades. Slowly-dried tardigrades can form a glass-like matrix, evidenced by a drop in heat flow that indicates the glass transition temperature at which polymers undergo the transition from glassy to rubbery state (an endothermic reaction). However, quickly-dried tardigrades are not able to form glass-like matrices. This figure was adapted from Boothby et al. (2018).
What scientists did next was to insert tardigrade CAHS genes into other organisms and see if they could also make CAHS proteins that would protect them from extreme desiccation. Amazingly, heterologous expression of CAHS proteins in yeast and bacteria significantly increased their desiccation tolerance. This indicated that the CAHS functioned normally in a foreign host and proved yet again that the genetic code, transcription, and translation are universal and can be used by all living organisms. However, the scientists who did this research missed the chance to test the TDP-expressing bacteria and yeast against other types of extreme stressors such as heat tolerance or radiation.
Engineered yeast that expressed heterologous CAHS proteins also produced a glass-like matrix during desiccation.
Mitochondrial abundant heat-soluble (MAHS) proteins
The MAHS family of TDPs is likely responsible for protecting the powerhouse of cells - the mitochondria. It produces adenosine triphosphate (ATP) - the energy currency of life - via aerobic respiration and simultaneously generates reactive oxygen species (ROS) as a by-product. ROS are highly reactive chemical molecules that can cause DNA damage or modify some cellular components due to oxidation, potentially leading to apoptosis.
Under normal circumstances, there are ROS detoxification mechanisms in the mitochondria that can counteract ROS generation. However, ROS accumulates under stressful environments such as desiccation and dehydration. Amazingly, the mitochondria in tardigrades have their own TDP bodyguards - MAHS proteins - that will localise to their aid during desiccation. The TDPs are speculated to regulate the mitochondria’s ability to produce harmful ROS.
In addition to having a protectant function, MAHS proteins could also be involved in maintaining the cell membrane of the mitochondria. Tardigrade mitochondria were observed to shrink during desiccation, but their level of function remained the same after rehydration. A recent paper beautifully hypothesised how MAHS proteins preserve cell phospholipid membrane integrity in tardigrades (Figure 4).
Phospholipid membranes can exist in different phases: crystalline (high fluidity) and gel state (low fluidity). During desiccation, the loss of water volume causes the membrane lipids to become closely packed into the gel phase. Rehydration does not occur uniformly across the membrane, resulting in a mixed gel and crystalline phase. This eventually causes transient holes to form in the membrane and potential bursting of cells. MAHS proteins are speculated to help prevent bursting by maintaining proper spacing between phospholipids during desiccation and aid even membrane rehydration.
Figure 4: Cross-sectional view of a phospholipid bilayer at hydrated, dehydrated and rehydrated states with and without proposed stabilisation of mitochondrial abundant heat-soluble (MAHS) proteins. Phospholipid membranes can exist in different phases: crystalline (high fluidity) and gel state (low fluidity). The loss of water volume during desiccation causes the lipids to become closely packed into the gel phase. Rehydration does not occur uniformly across the membrane, resulting in a mixed gel and crystalline phase. This eventually causes transient holes to form in the membrane and potential bursting of cells. MAHS proteins are speculated to maintain proper spacing between phospholipids during desiccation and aid even membrane rehydration. This figure was taken from here.
Most functions of MAHS proteins have not been experimentally confirmed; speculation about proposed function is based on structural predictions by software, or homology to existing proteins. Because TDPs are generally made up of disordered regions, protein crystallisation studies are impossible. Despite this hindrance in understanding the exact functions of MAHS proteins, we can still try to understand the impact of these proteins in heterologous systems. Tardigrade MAHS proteins have been introduced into human cells which surprisingly resulted in improved hyperosmotic tolerance. However, no one knows how these proteins confer osmotic tolerance in human cells. Again, the experiment proved that TDPs are transferable to other organisms and they might have protective roles during water stress.
Secreted abundant heat-soluble (SAHS) proteins
SAHS proteins are TDPs secreted by tardigrades into the environment. They were identified using mass spectrometry of proteins found in the growth media with cultured tardigrades. A simple protein similarity search using BLAST (Basic Local Alignment Search Tool) against the protein database found weak hits against mammalian fatty acid-binding proteins (FABPs). SAHS proteins could be related to FABPs, but they contain a secretory signal peptide at the N-terminus, while FABPs are cytosolic proteins. Little is known about the functions of SAHS proteins; scientists have speculated that these proteins might serve protective functions for extracellular components or secretory organelles.
Scientists made a breakthrough in 2017 in determining the structure of a SAHS TDP. Unlike CAHS and MAHS proteins, some regions of SAHS proteins have less predicted disorder, which allows crystallisation and thus, structural insights. SAHS proteins have structural similarities to FABPs, however, scientists do not know what ligand SAHS proteins bind to yet. The current hypothesis of SAHS protein function is the recapturing of fatty acids that are lost during anhydrobiosis and compaction of the tardigrade body into a tun state.
High-dose radiation tolerance
Apart from TDPs, scientists also wanted to understand the mechanism behind the tolerance of tardigrades against high-dose radiation (proven by our water bear friends in space during the Foton M3 mission). Using mass spectrometry and subcellular localisation assays, In 2016, Hashimoto and his team found a protein named dDsup (short for damage suppressor) that binds to DNA. A DNA-binding function of a tardigrade Dsup hinted at a potential DNA protection role from irradiation stress and consequent damage. To prove this, they expressed tardigrade Dsup in human cells in a heterologous manner and exposed the cells to X-rays. Amazingly, D-sup expressing human cells had ~40% fewer DNA breaks in the genome compared to untransfected cells.
This is a very important discovery for future biotechnology applications to confer radiation or stress tolerance to other organisms and prepare them for future bouts of extreme climate changes. A good example of these applications was shown in a 2020 study, where scientists expressed Dsup in plants and the tardigrade-specific protein protected the plants against DNA damage. However, we have to be cautious that there may be other factors in tardigrades apart from Dsup that could contribute to the extreme radiation tolerance, such as an efficient DNA repair pathway. More research should also be done in understanding the side effects of overexpressing Dsup in other organisms, such as potential interference with transcription, DNA replication or endogenous DNA repair pathways.
Where do the extreme stress tolerance genes come from?
These tiny animals were initially reported to contain ~17.5% foreign genes in their genome. This means that tardigrades obtained at least one-sixth of their genes from other organisms such as bacteria, fungi, plants and Archaea. For comparison, the proportion of foreign genes in most animals’ genome is less than 1%.
Horizontal gene transfer (HGT), a natural process of genetic transfer between different species, plays a huge role in the evolution of organisms in addition to genetic transfer from parent to offspring.
But how does HGT occur in tardigrades? The scientists who did the study speculated that when the cell membrane is rehydrated after desiccation, it becomes temporarily leaky which allows foreign DNA to pass into the cell, and the tardigrade’s DNA repair system integrates the DNA fragments into the genome.
Figure 5. Diagram showing horizontal gene transfer (HGT) in red and lateral gene transfer (LGT) in blue. HGT and LGT are processes of genetic transfer between and within domains respectively. This figure was adapted from Kado (2009)
However, inferring horizontally-transferred genes using HGT event prediction software does come with technical flaws and biases. A team of scientists from the University of Edinburgh who sequenced the same tardigrade species, instead found just 2% of genes were foreign. They claimed that the previous study’s massive HGT was due to undetected contaminants in their tardigrade culture, and the debate of HGT contribution to tardigrade’s extreme tolerance remains open.
Despite the technical challenges, a group of catalase proteins involved in anhydrobiosis and oxidative stress tolerance was very likely to be acquired from HGT from bacteria. Apart from HGT, stress tolerance proteins are likely to have evolved independently in tardigrades, evidenced by Dsup being unique to the Tardigrada phylum.
Why do we care about water bears?
Cuteness aside, exploitation is the answer. Our lives with nature are intertwined. Humans have developed many technologies that are inspired or exploited by biological organisms, such as the polymerase chain reaction and the green fluorescent protein marker. Scientists are particularly interested in proteins that are made by extremophiles. Because they live in such extreme environments, extremophiles are predicted to produce some of the most stress-tolerant proteins that could be exploited for biotechnology.
For tardigrades, one idea includes large-scale production of recombinant TDPs in a bacterial system. The TDPs can be used to stabilise sensitive pharmaceuticals, vaccines or cells at room temperature by dehydrating these products. This could replace freezing and thawing heat-sensitive proteins that could potentially affect their biological activity. Vaccines for instance, currently rely on liquid nitrogen for freezing and low-temperature storage.
According to Dr Thomas Boothby - a researcher whose work focuses on tardigrades - during a New Scientist interview, “This could help us break dependence on the cold-chain, a huge economic and logistical hurdle for getting medicine to people in remote or developing parts of the world,” he says. “We are pursuing these applications".
Tardigrade-specific proteins could also be engineered into heterologous systems, like the aforementioned examples. Dsup could be used to protect DNA for long-term survival and viability. Applications can include increasing the survivability of stem cells for cell-based therapies, storing genetic material from eggs or sperms for longer at room temperature in fertility programs, or potentially preserving fossil DNA.
There are many other creative ways we could exploit and apply tardigrade proteins in our everyday life. For example, we could potentially incorporate tardigrade proteins in sunscreen products to protect our skin from harmful ultraviolet rays from the sun. There is so much more to discover about tardigrade biology and their responses to other extreme stressors not covered in this article (e.g. extreme heat and cold), using different omics technology and molecular techniques. The sky's the limit.
So, are tardigrades immortal?
There will be old papers in literature or news articles claiming that anhydrobiotic tardigrades can survive for more than a century. However, this claim is too good to be true. A closer look by studies over the years has concluded that tardigrades cannot survive in anhydrobiosis for indefinite periods. Being dormant has its downsides; despite having the amazing protective ability of Dsup for DNA, damage done by ROS over a long period of time could prove too much for tardigrades. Survivability of tardigrades vary widely between different species and conditions, and decreases over time; tardigrades recovered from the Antarctic after eight years of storage at -22°C can still be revived, but a study showed that dried tardigrades can be revived only up to three to four years of storage. Sadly but realistically, tardigrades are not video game characters!
PhD in Molecular Plant Sciences
The University of Edinburgh