As part of the grant, the team led by Professor Łukasz Kaczmarek from the Faculty of Biology of the Adam Mickiewicz University and Dr. Stanislav Vinopal from Jan Evangelista Purkyně University in Czechia will compare anhydrobiosis and cryobiosis, i.e. two types of cryptobiosis in tardigrades, which are respectively caused by a lack of water and freezing. Although both are associated with severe dehydration of the body, they differ from each other at the cellular and molecular level. The researchers will check how effectively different species of tardigrades can enter them and whether there are any differences between them at the level of cytoskeletal functioning and protein expression.

'Tardigrades have quite a few mechanisms responsible for cryptobiosis, from proteins that are produced in response to stress conditions, sugars that are probably also important, to their microbiome and probably the cytoskeleton', Professor Kaczmarek told PAP.

In his previous research, the scientist studied all these aspects. Now he will focus on the role of the cytoskeleton in the ability to enter cryptobiosis conditioned by dehydration.

'It is an internal cellular skeleton that ensures the stability of cellular organelles. We will assess whether disturbances in this skeleton - and we intend to disturb it with various substances - will or will not prevent tardigrades from entering the state of cryptobiosis’, he says.

TARDIGRADE SHIELDS

The key role in the process of entering cryptobiosis has been proven. In response to extreme environmental conditions, tardigrades begin to produce a whole range of proteins that enable them to survive. The most important of these include heat shock proteins (HSPs), which stabilize other proteins and prevent their damage during thermal stress. Also important are various proteins associated with DNA repair and the so-called TDPs (tardigrade disordered proteins), which are synthesized exclusively by tardigrades and help them survive dehydration or freezing by protecting cellular structures from damage.

It has also been known for some time that sugars are also involved in cryptobiosis, specifically a disaccharide called trehalose. 'However, its role has not been fully explained. It used to seem to us that this was the key piece enabling cryptobiosis. However, recent studies have shown that some tardigrades, about half of them in total, do not synthesize this sugar at all. We currently believe that trehalose is useful, but not essential for this process', Kaczmarek says.

Recent years have also been a period of intensive research on the microbiome; there is more and more evidence of its huge role in the functioning of the human body. It affects many aspects of health, from the digestive system to immunity and mental health. Now it turns out that it may be equally crucial in tardigrades. The latest reports suggest that the microbiome may help them enter a state of cryptobiosis.

'In my previous research with Professor Monika Mioduchowska from the University of Gdańsk, we discovered that the tardigrade microbiome includes microorganisms that are very resistant to stress conditions. So the question arose whether they were not the ones that +infected+ tardigrades with this extraordinary resistance, e.g. by providing them with some substances', Kaczmarek says. So far, there has been no clear answer to this question, but - according to the researcher - the issue is very interesting and worth further research.

As for the cytoskeleton, we know that it undergoes very strong changes during cryptobiosis, and especially anhydrobiosis and cryobiosis, which are associated with strong shrinkage of the body's cells. Specific proteins and structures must be involved, and it is their role that the team will explain in the latest grant. The research will be conducted on various species of tardigrades (terrestrial and freshwater species) to see if they react in the same way and if they have developed specific cellular mechanisms.

'This is a very interesting issue, because freshwater species have quite big problems with cryptobiosis. They enter it reluctantly and it is difficult for them to maintain it. This is certainly due to the fact that they live in a permanently humid environment, so this state is not necessary for them to function. On the other hand, terrestrial tardigrades are exposed to frequent, periodic water shortages, so they must enter anhydrobiosis quickly and effectively. Our goal is to investigate whether the cytoskeleton reacts the same way in freshwater and terrestrial species', says Professor Kaczmarek.

He and his team plan to disrupt the functioning of the cytoskeleton with various substances to determine which of its elements are crucial and which seem to be irrelevant in this process. 'No one in the world has ever conducted such research. We intend to be the first to learn about, understand and describe all this. If, of course, we succeed, because we are just starting out; and since nothing like this has ever been done before, there are a lot of question marks', says Kaczmarek.

He adds that tardigrades synthesize several types of tubulin proteins, which are part of the cytoskeleton. And although they are the same as in other organisms, perhaps in tardigrades they create some connections (complexes) that will prove decisive.

The scientists will also assess whether repeated episodes of cryptobiosis will increase or, on the contrary, decrease further cryptobiotic abilities.

LIVING ON THE EDGE

When asked why the grant includes research on two specific types of cryptobiosis, Professor Kaczmarek explains that both are related to the lack of liquid water. 'This is the case both with drying, in which case we are talking about anhydrobiosis, and freezing, where we are dealing with cryobiosis', he says.

However, as he adds, the mechanisms underlying both types of cryptobiosis are different, because in one of them the water in the cell freezes, in the other - it almost completely disappears. Tardigrades must therefore 'cope' with completely different problems.

During freezing, the biggest problem are ice crystals formed in the cells, which can damage the cell membrane. Animals protect themselves from this by using the above-mentioned trehalose, which is co-responsible for the so-called glassy state of the cytoplasm (glossy matrix), which prevents the formation of crystals. 'On the other hand, some tardigrades do not synthesize trehalose at all, so they must have some other way to protect themselves against the formation of crystals and cellular damage. There may be, but do not necessarily have to be, similar mechanisms that we want to discover during this research', Kaczmarek says.

In turn, during anhydrobiosis (drying out), the greatest difficulty is that tardigrades lose 90 percent of water from their cells. 'This means that they become thin and dry like chips, and all cell organelles are located very close to each other. Any reactions in such conditions are very difficult’, Kaczmarek adds.

'Imagine this: a human who loses 15 percent of water dies. Here we are talking about a loss of over 90 percent', he says.

The remaining types of cryptobiosis, i.e. osmobiosis, which is a response to changing osmotic pressure in the environment, anoxybiosis, which protects the body in anaerobic conditions, and chemobiosis, which protects against harmful chemicals (e.g. cyanide), are probably not associated with such significant changes in the cytoskeleton. 'In these cases, the animals become rather elongated and swollen; they do not shrink as much', he continues.

Professor Kaczmarek's team includes invertebrate zoologists, molecular biologists and cell biologists. They hope that by using various research methods, they will learn what the cytoskeleton looks like during cryptobiosis and tardigrade reactivation. This is necessary to fully understand the mechanisms behind this unusual phenomenon. (PAP)

PAP - Science in Poland, Katarzyna Czechowicz

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