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Eukaryon

Exploring possible mechanisms by which Intrinsically Disordered Proteins promote tardigrade desiccation survival

Olivia Dellomodarme, Emma Kuhr, Chisomo Mwale, Clarisse Pelaez
Lake Forest College
Lake Forest, Illinois 60045

Summary

Tardigrada, a phylum of microscopic animals that occupy a diverse range of biomes across the globe, has evolved to possess an extraordinary ability to tolerate extreme environments. One mechanism these animals utilize to survive such extremes is desiccation, in which tardigrades rapidly dehydrate and fall into an inactive state until rehydration. A family of Intrinsically Disordered Proteins (IDPs) called Tardigrade-specific Intrinsically Disordered Proteins (TDPs) have been found to be highly expressed in tardigrades during desiccation. Although TDPs have been investigated as a possible mechanism through which tardigrades endure total dehydration, the molecular mechanisms by which these proteins promote desiccation survival have yet to be elucidated; therefore, this research proposal seeks to explore potential mechanisms by which IDPs assist desiccation survival. This study predicts that IDPs play a role in promoting tardigrade desiccation survival by using a combination of vitrification, water replacement, LEA protein mediated aggregation reduction and LEA protein-mediated membrane protection. In Aim 1, desiccation survival will be demonstrated to be a result of IDP-mediated vitrification by establishing the relationship between IDPs and desiccation tolerance via RNA-interference (RNAi). The proteins’ abilities to vitrify will be established using differential scanning calorimetry on tardigrades with and without the necessary IDPs, thus illustrating the changes in heat capacity that accompany vitrification. In Aim 2, Fourier transform infrared (FTIR) microscopic mapping and spectroscopy will be used to determine if IDPs work through a protective mechanism. More specifically, this research aims to study the contribution of water replacement to the protective capabilities of IDPs during desiccation. Following this, research aims to investigate whether tardigrade LEA proteins reduce aggregation induced by desiccation to aid dehydration survival and tolerance. This will be done by assessing in vitro aggregation using citrate synthase (CS) as an aggregation substrate via heat and desiccation stress. Finally, this research will determine if tardigrade LEA proteins also protect liposomes from desiccation-related membrane damage. Liposome-drying assay will be performed on the liposomes to mimic desiccation, and liposomes will be measured before and after the assay to determine their mean change in volume diameter. The implications of illuminating the methods behind IDP-mediated desiccation tolerance have the potential to advance knowledge through insight to the role of these proteins in general organismal stress intolerance. This research also has the potential to benefit society in a multitude of ways, including medical and agricultural means, such as developing efficient storage of biological tissues and engineering drought-tolerant crops, respectively.

 

Background

Tardigrada is a phylum of aquatic, microscopic invertebrates, commonly referred to as tardigrades, that are widely distributed around the globe.1, 2 They occupy a diverse range of biomes in both the northern and southern hemispheres, including polar regions, tropical regions, and intertidal zones.1, 2 As might be expected from their wide range, tardigrades are remarkably resilient, and can even survive desiccation, an environmental condition in which the liquid surrounding the tardigrade evaporates.3 Desiccation is dangerous because it can damage cellular structures and eventually lead to the death of any organism that is not adapted to such strain.4 However, there are several species of bacteria and invertebrates, including tardigrades, that have mechanisms in place to survive desiccation.4 

When faced with desiccation, tardigrades undergo a process called anhydrobiosis in which they rapidly dehydrate themselves and enter an inactive state, contracting their bodies to form a “tun” and keeping only enough water to synthesize necessary proteins.3 One molecule that increases in concentration in anhydrobiotic tardigrades is a sugar called trehalose, and it is widely considered to be important in tardigrade survival of desiccation.3, 4 Trehalose has been shown to have a variety of protective roles in anhydrobiosis, such as stabilizing proteins and protecting lipid membranes.4, 5, 6 However, there has also been evidence to suggest that trehalose is not required for anhydrobiosis.4  This has inspired a search for other proteins that may be involved in desiccation tolerance, and recently a novel type of protein called Intrinsically Disordered Proteins (IDPs) has been discovered in anhydrobiotic tardigrades.7

IDPs are a rather peculiar family of proteins. They are globular proteins but interestingly, do not possess a persistent tertiary structure.8 Several jobs have been assigned to IDPs including roles in transcription, post-translational modifications, development, cellular organization, and abiotic stress tolerance.4, 8 IDPs are known for their strong hydrophilic properties.8, 9, 10, 11 A novel class of IDPs called Tardigrade-specific Intrinsically Disordered Proteins (TDPs) are expressed by Tardigrade-specific Intrinsically Disordered Protein genes during exposure to stresses such as desiccation.8 These TDPs are thought to promote desiccation survival in tardigrades; however, the mechanisms by which they do so are still largely unknown.8 Therefore, this grant proposal seeks to explore potential mechanisms by which IDPs assist desiccation survival. The goal of this study is to determine whether four distinct IDP mechanisms-vitrification, water replacement, late embryogenesis abundant (LEA) protein-mediated reduction of protein aggregation and LEA protein-mediated membrane protection-play a role in desiccation protection in tardigrades. Given the extensive roles IDPs have been shown to play in previous research, this study predicts that IDPs play a role by promoting tardigrade desiccation survival using a combination of vitrification, water replacement, LEA protein mediated aggregation reduction and LEA protein-mediated membrane protection.

 

Significance

The benefits for understanding these mechanisms could expand intellectual merit by furthering the path of research involving desiccation survival; understanding these mechanisms could also have broader implications such as clinical utilization and beyond, to various external applications of understanding dehydration. The current study hopes to identify IDPs as mediators of tardigrade desiccation tolerance, which in turn would give insight to the involvement of these proteins in general stress tolerance. Expanding the knowledge of the relationship between disordered proteins and desiccation could have implications for the development of medical technologies, potentially allowing the dry preservation of cells and tissues. These advancements could allow scientists to improve the efficiency and expediency of storing and transporting cells and tissues for testing or transplant. It has been found in certain bacteria that the functions necessary for desiccation survival are also those used in radiation tolerance.12 The bacterial DNA was found to be similarly damaged in prolonged desiccation compared to that in radiation exposure. The understanding of desiccation tolerance could therefore potentially lead to understanding radiation tolerance. Exploring radiation tolerance could impact people on an individualistic scale in the medical field, as well as externalize to large environmental prevention efforts. Another external application could involve the future engineering of crops that are tolerant to dehydration. Desiccation-tolerant plants would permit farmers to utilize previously-infertile land for now-successful farming. Similarly, farmers that directly or economically rely on their crops for survival would no longer be at risk should a drought occur.

 

Specific Aims

Aim 1: Do tardigrade IDPs promote desiccation survival through vitrification?

The objective of this aim is to determine if IDPs mediate desiccation survival through a process called vitrification. To achieve this goal, the relationship between IDPs and desiccation tolerance will be established by utilizing RNA-interference (RNAi) to knock down gene expression of specific families of IDPs called CAHS and SAHS proteins, and the effects on desiccation survival will be observed. To confirm that these IDPs are involved in vitrification, vitrification abilities of the proteins will be quantified using differential scanning calorimetry on subjects with and without these IDPs to illustrate the changes in heat capacity that accompany vitrification. By connecting whether the RNAi-injected tardigrades that undergo desiccation show a significant decrease in survival to whether the proteins targeted undergo vitrification during desiccation, this experimental design could illuminate a potential mechanism by which IDPs facilitate tardigrade desiccation survival. 

 

Aim 2: Does water replacement contribute to the protective capabilities of IDPs during desiccation?

The purpose of this aim is to uncover the role water replacement may have in the protective capabilities of IDPs during desiccation. To achieve this aim, FTIR analyses will be used. Differential scanning calorimetry will also be used to measure the heat absorption of the tardigrades. If water replacement does have protective capabilities of IDPs during desiccation then we predict that FTIR analyses will indicate that the anhydrobiotic animals stay in a glassy state at high temperatures allowing survival. In a highly dehydrated state, such as when tardigrades go through desiccation, the bound water molecules surrounding proteins and membranes are replaced by trehalose and highly hydrophilic proteins, which may form hydrogen bonds with them and thereby keep membranes in liquid crystalline states at room temperature. This methodology could expose the role of water replacement in the protective capabilities of IDPs in desiccation. 

 

Aim 3: Do tardigrade LEA proteins reduce protein aggregation during desiccation?

The goal of this aim is to investigate whether tardigrade LEA proteins reduce aggregation induced by desiccation to aid desiccation survival and tolerance. To achieve this goal, an in vitro aggregation assay will be performed on the aggregation of citrate synthase (CS). Light scattering will be used as a measure of absorbance to determine aggregation. Five conditions will be used: a CS only positive control, a non-stressed CS negative control, and experimental conditions containing CS with the LEA proteins RvLEAM, AavLEA1 or Em. RvLEAM will be obtained from the tardigrade Ramazzottius varieornatus, AavLEA1, from the nematode Aphelenchus avenae and Em, from the plant Craterostigma plantagineum. Förster resonance energy transfer (FRET) will also be used to determine molecular interactions between LEA proteins and their targets. This procedure could help elucidate the mechanisms IDPs use to aid tardigrade desiccation survival. 

 

Aim 4: Do tardigrade LEA proteins protect cellular membranes from desiccation damage?

The purpose of this aim is to determine if a specific type of tardigrade IDP called LEA proteins protect tardigrades from desiccation damage through membrane protection. To achieve this goal, a liposome drying assay will be used, and membrane integrity will be quantified by measuring the difference in volume diameter of liposomes before and after drying, using dynamic light scattering. Three conditions will be used: a trehalose positive control, a lysozyme negative control, and an experimental condition containing RvLEAM from the tardigrade Ramazzottius varieornatus. Differential scanning calorimetry will then be used to determine if RvLEAM significantly altered the chemical properties of the liposomes compared to the lysozyme condition. This design could shed light on yet another possible mechanism IDPs  may use to assist with desiccation survival.

 

Design and Methods

Aim 1: Do tardigrade IDPs promote desiccation survival through vitrification? 

Vitrification via intrinsically disordered protein (IDPs) is a mechanism by which tardigrades are hypothesized to be able to survive complete desiccation (anhydrobiosis).2, 8 Sakurai et al. (2008) demonstrated first that animals that undergo anhydrobiosis (extreme desiccation) replace the cytoplasm of their cells with a glassy substance upon dehydration, and second, that this glassy state positively affected desiccation survival.2 As a next step, Hengherr et al. (2009) found that different species of tardigrades had different glass-transition temperatures and therefore demonstrated a variety of heat tolerances, supporting the belief that desiccation tolerance is directly related to vitrification ability.13 Then, Boothby et al. found that IDP expression was upregulated in tardigrades that underwent desiccation compared to those that were hydrated, leading to the belief that IDPs are the key for vitrification, and therefore desiccation survival.8 This study therefore aims to determine if IDPs are imperative to the vitrification of tardigrades as a means to survive desiccation, using a previously-used tardigrade species Milnesium tardigradum as a model organism.

In order to determine whether IDPs are necessary for desiccation survival through vitrification, the relationship between IDPs and desiccation tolerance will be established by utilizing RNA-interference (RNAi) to knock down the gene expression of the proteins believed to mediate desiccation survival. The methodology for the RNAi knockdown was modeled after a previously published protocol from Tenlen et al. (2013); however, the current study will be adjusted as a means of reducing potential confounding factors that could influence the survival of the tardigrades.14 The appropriate RNAi for the chosen proteins will be made according to protocol and injected into tardigrades, which will then be put through either an experimental extreme dehydration or a hydrated control environment.8, 14 All of the subjects will be subjected to rehydration after a standard period of three days and observed for survival, quantified as coordinated movement.8, 14 The proteins targeted for downregulation will be various cytosolic abundant heat soluble (CAHS) proteins and secreted abundant heat soluble (SAHS) proteins, as these are IDPs that have previously been shown to be upregulated in desiccation.8 Like the protocol used by Tenlen et al., green fluorescent protein (GFP) will be used as a negative control protein for the RNAi injections, as GFP is unrelated to both desiccation tolerance and general survival .8, 14 CAHS94205 will be used as a positive control, as previous RNAi studies illustrated this protein to have the most significant impact on desiccation survival.8 Independent samples t-tests will be run to determine significance of the change in survival rate between the IDP-RNAi tardigrades and the GFP-RNAi controls for both the hydrated control and the desiccation environments. Finally,  differential scanning calorimetry will be used on tardigrades with and without the necessary IDPs to demonstrate these proteins’ abilities to vitrify by illustrating the changes in heat capacity that accompany vitrification.8 It has been previously found that when tardigrades are dehydrated too rapidly, they do not have a chance to express the necessary IDPs to survive the desiccation.3, 8 Therefore, the control for the calorimetry will be tardigrades that were dehydrated too rapidly to express the IDPs. 

If IDPs do promote vitrification and thus desiccation survival, it would be expected that the CAHS-RNAi and the SAHS-RNAi-injected tardigrades in the hydrated control environment would show no significant changes in survival rate compared to the GFP-RNAi control, as there is no need for vitrification in a constantly hydrated environment. Similarly, it would be predicted that the CAHS-RNAi and the SAHS-RNAi-injected tardigrades that underwent desiccation would show a significant decrease in desiccation survival compared to the negative control, as their necessary protective proteins are not being expressed. The GFP-RNAi would be expected to show no significant change in desiccation tolerance, as the presence of GFP is not related to the ability to survive desiccation. It would then be predicted that the rapidly dehydrated tardigrades would not show a significant change in heat capacity, as they do not vitrify. Conversely, tardigrades that have the necessary IDPs are predicted to demonstrate significant changes in heat capacity that tracks the vitrification process. 

 

Aim 2: Does water replacement contribute to the protective capabilities of IDPs during desiccation?

Trehalose is a disaccharide of glucose and it has been known to be an effective cryoprotectant.15 A previous study used molecular dynamic simulations on solutions of both water and trehalose. The concentrations of trehalose in the solutions varied and they were conducted at different temperatures. Results indicated that trehalose modifies the hydrogen bond network of water as well as intramolecular hydrogen bonding.15 This is a proposed mechanism for the role of trehalose as a cryoprotectant as well as in water replacement in tardigrades. Moreover, Carpenter et al. (1989) looked into the interaction between stabilizing carbohydrates and dried proteins.16 Results from this research show that carbohydrates, in this case trehalose, will bond to hydrogen atoms of dried proteins. Carbohydrate bonding is also necessary to induce the stabilization of the proteins not only during rehydration, but also during freeze-drying. This supports the water replacement hypothesis as the bonding of trehalose replaces the water hydration. The water-replacement hypothesis states that the hydrophilic molecules directly interact with macromolecules through hydrogen bonds. By doing so, the molecules then take the place of water.2 It is known that there is a large amount of sugar, trehalose, in organisms that undergo anhydrobiosis.17 Researchers aimed to study how trehalose interacts with biomolecules during this process. To do this they performed molecular dynamic simulations of trehalose-water solutions mixed with carboxymyoglobin; results show that the trehalose was excluded from the ‘protein domain’ and remains enriched with water. There was also hydrogen bonding of the trehalose to the protein. This furthers the understanding of the role of trehalose in water replacement in tardigrades.17 

A study done by Sakurai et al. in 2008 showed by Fourier transform infrared (FTIR) microscopic mapping image that when entering anhydrobiosis, P. vanderplanki accumulated nonreducing disaccharide trehalose that was uniformly distributed throughout the dehydrated body.2 With this information, they came to the conclusion that trehalose plays an important role in water replacement. This method was used to study the protective role of water replacement in trehalose-based protection, and we believe it may be useful for exploring whether IDPs work through a protective mechanism. More specifically, we aim to study the contribution of water replacement to the protective capabilities of IDPs during desiccation.  

To study any protective role water replacement may have, FTIR spectroscopy will be used. Differential scanning calorimetry will also be used to measure the heat absorption of the tardigrades. The methodology of this experiment is  modelled after that of Sakurai et al. (2008). Two samples of tardigrades with different concentrations of trehalose will be used. Tardigrades that dehydrate over a 72-hour period are “slowly” dehydrated and tardigrades that dehydrate over a span of several hours are “quickly” dehydrated.2 The animals will then be placed in heat treatments ranging from 20 degrees celsius to 120 degrees celsius as these replicate the extreme temperatures tardigrades may encounter. Absorption abilities of the slowly dehydrated animals will show a shift from baseline as they have increased trehalose.2 It is already known that concentrations of trehalose are higher in animals that “slowly” dehydrate. If water replacement does have protective capabilities of IDPs during desiccation then we predict that FTIR analyses will indicate that the anhydrobiotic animals stay in a glassy state at high temperatures, allowing survival. FTIR spectra will show sugars formed hydrogen bonds with phospholipids and FTIR imaging data would also show an increase in IDPs and trehalose throughout the animal.2 

 

Aim 3: Do tardigrade LEA proteins reduce protein aggregation during desiccation?

Late embryogenesis abundant (LEA) proteins are a branch of highly hydrophilic Intrinsically Disordered proteins distinct from classical molecular chaperones such as heat shock proteins (HSPs).9 They were first described in cotton and wheat,18 and have been produced in excess amounts during seed development.19 They have been thought to act as an ion scavenger in cytoskeletal components and have been found to be highly expressed in desiccating larvae of the African chironomid.11 LEA proteins can belong to major categories: Group 1, Group 2 or Group 3.10 Group 1 and 2 proteins are mainly found in plants, while Group 3 proteins have been found in other organisms.10 Goyal et al. (2005) studied the effect of Group 1 and 3 LEA proteins on the aggregation of citrate synthase (CS) due to desiccation. These scientists found that both proteins were able to rescue CS aggregation.10 Their role in desiccation survival with respect to reduced induced aggregation of cellular proteins has also been investigated by Chakrabortee et al. (2012). They investigated two LEA proteins, AavLEA1 and Em, from a species of nematode and plant respectively.9 Chakrabortee et al. found that LEA proteins display an activity unique to molecular chaperones, named “molecular shielding”, which affects intermolecular activity such that aggregation due to desiccation is prevented.9 The results from previous studies offer a compelling argument for LEA protein involvement in the reduction of aggregation. So, perhaps LEA proteins are critical for tardigrade desiccation survival. This study therefore aims to investigate whether tardigrade LEA proteins reduce aggregation induced by desiccation to aid desiccation survival and tolerance.

Recently, a novel LEA protein called RvLEAM was identified in the tardigrade species Ramazzottius varieornatus by Tanaka et al.20 They found RvLEAM to have characteristics specific to Group 3 LEA proteins.20 This protein, localized in the mitochondria, caused increased metabolic activity in cells placed in water deficient conditions and possessed heat-soluble properties characteristic of LEA proteins.20 RvLEAM is an excellent candidate for tardigrade desiccation tolerance investigations.

Much of the methodology for this investigation is inspired by assays conducted by Chakrabortee et al. (2012). In vitro aggregation will be assessed using citrate synthase (CS) as an aggregation substrate via heat and desiccation stress. CS aggregation will be conducted using the same assay techniques,9 but RvLEAM will be compared with other LEA proteins-AavLEA1 (from the nematode Aphelenchus avenae) and Em (from the plant Craterostigma plantagineum)-rather than with HSPs. A CS only control and non-stressed CS control will also be used to exclude external effects on aggregation reduction. In addition, to explore the possibility of interactions between the LEA proteins and their targets, mCerry-tagged LEA proteins will undergo assays described by Chakraborkee et al.9 Light scattering as a measure of absorbance at different absorbances, as mentioned by Chakrabortee et al., will be assessed in both cases to determine effect on aggregation. Statistical relevance of the aggregation assays will be determined with a one-way ANOVA and a Tukey post-hoc.9 

If RvLEAM does aid in the reduction of CS protein aggregation as a result of heat stress and desiccation, it would be unsurprising to see a reduction in aggregation due to desiccation for the CS+LEA conditions.9 Whether the same would be seen for aggregation due to heat stress is less clear, as Chakrabortee et al. found no effect on aggregation due to heat stress for both AaVLEA1 and Em.9 Additionally, if RvLEAM interacts with the target, albeit loosely, Förster resonance energy transfer (FRET) levels should reflect this.9 This procedure could help elucidate the mechanisms IDPs use to aid tardigrade desiccation survival.

 

Aim 4: Do tardigrade LEA proteins protect cellular membranes from desiccation damage?

In addition to preventing protein aggregation during anhydrobiosis, LEA proteins have also been implicated in stabilizing and protecting phospholipid membranes from desiccation damage in several anhydrobiotic organisms, including plant seeds and a species of brine shrimp (Artemia franciscana).5, 6, 21 Moore et al. (2016) found that LEA proteins conferred protection specifically to liposomes with similar lipid composition to the inner and outer mitochondrial membrane, as well as parts of the plasma membrane of the entire cell, suggesting that LEA proteins are involved in protecting these structures in anhydrobiotic organisms.6 Tolleter et al. (2007) further elucidated the mechanisms these proteins use to stabilize cellular membranes by demonstrating that LEA proteins are able to bind to liposomes and alter their chemical characteristics.21 The natural next step would be to determine if tardigrade LEA proteins also protect liposomes from desiccation-related membrane damage, and if they do so by interacting with lipid membranes and changing their chemical characteristics as plant seed LEA proteins do.

Much of the methodology of this experiment is modelled from the methodology that Tolleter et al. (2007) used in their study. Liposomes will be prepared using their method, and their liposome-drying assay will be performed on the liposomes to mimic desiccation.21 As Tolleter et al. stated, lysozyme makes a good negative control, because it has a weak interaction with phospholipids and is not damaged under conditions of low water concentration.21 Therefore, lysozyme will be added to a group of liposomes as a negative control. Trehalose will be added to another group of liposomes as a positive control, as multiple studies have demonstrated that trehalose protects liposomes from desiccation damage.5, 6 For the experimental group, the tardigrade LEA protein RvLEAM will be added.20 All three groups of liposomes will be measured before and after the assay using differential light scattering to determine their mean change in volume diameter; if the volume diameter were to increase significantly, it would suggest membrane breakage and rearrangement, indicating that the membrane was not protected from desiccation damage.21 Additionally, during the assay the interactions between the three groups of liposomes and their respective proteins will be examined using differential scanning calorimetry to observe possible modifications to the chemical properties of the liposomes.21 A one-way ANOVA will be run to determine the significance of any differences in volume diameter before and after the lipid-drying assay, and the significance of any differences in peaks of the thermograms gained from the differential scanning calorimetry.21

If RvLEAM does protect liposomes from desiccation damage, then it would be expected that the trehalose and RvLEAM conditions would show no significant difference in volume diameter before and after the drying assay.21 As the negative control, it would be expected that the lysozyme condition would show a significant change in volume diameter before and after the assay, suggesting membrane damage and rearrangement.21 Additionally, if RvLEAM interacts significantly with the phospholipids, the RvLEAM condition should significantly modify chemical characteristics of the phospholipids during differential scanning calorimetry, such as transition peak temperature and endothermic peaks, in comparison to the lysozyme control group.21 This design could shed light on the mechanisms IDPs use to assist desiccation survival. 

 

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