Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

BACKGROUND: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. PRINCIPAL FINDINGS: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. CONCLUSIONS: The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades

Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades

Many limno-terrestrial tardigrades live in unstable habitats where they experience extreme environmental conditions such as drought, heat and subzero temperatures. Although their stress tolerance is often related only to the anhydrobiotic state, tardigrades can also be exposed to great daily temperature fluctuations without dehydration. Survival of subzero temperatures in an active state requires either the ability to tolerate the freezing of body water or mechanisms to decrease the freezing point. Considering freeze tolerance in tardigrades as a general feature, we studied the survival rate of nine tardigrade species originating from polar, temperate and tropical regions by cooling them at rates of 9, 7, 5, 3 and 1 degrees Ch(-1) down to -30 degrees C then returning them to room temperature at 10 degrees Ch(-1). The resulting moderate survival after fast and slow cooling rates and low survival after intermediate cooling rates may indicate the influence of a physical effect during fast cooling and the possibility that they are able to synthesize cryoprotectants during slow cooling. Differential scanning calorimetry of starved, fed and cold acclimatized individuals showed no intraspecific significant differences in supercooling points and ice formation. Although this might suggest that metabolic and biochemical preparation are non-essential prior to subzero temperature exposure, the increased survival rate with slower cooling rates gives evidence that tardigrades still use some kind of mechanism to protect their cellular structure from freezing injury without influencing the freezing temperature. These results expand our current understanding of freeze tolerance in tardigrades and will lead to a better understanding of their ability to survive subzero temperature conditions

Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state.

Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state

Comparative expression analysis of selected proteins.

<p>Total emPAI is the sum of emPAI of each protein member. Large lipid transfer protein (LLTP) superfamily belong to major components in all three states. This protein family is highly expressed in EES compared to adults. Semi-quantitative analysis of proteins contributing to the structural integrity of ribosome indicates a significant up-regulation of ribosomal proteins in EES. In contrast proteins involved in cytoskeletal-, muscle- and vitellin membrane structure are not highly abundant at this state compared to adults in AS and TS. Proteins with antioxidant activity such as GSTs are approximately 3 fold higher in adults compared to EES. In contrast Cu-Zn SODs are upregulated in EES.</p

Semi-quantitative analysis of selected proteins associated with diverse processes such as response to stimulus, protection and development.

<p>BP: biological process; MF: molecular function.</p

Major protein components in early embryonic state (EES).

<p>38 proteins were found as major components in EES, 10 of which belong to the large lipid transporter superfamily. Other members are the heat shock protein family, structural constituent of ribosome and cytoskeleton. In addition, 20 proteins are without annotation. The contig description is indicated with asterisk, in case we found putative candidates by DomainSweep analysis. For one contig DomainSweep analysis delivered a significant candidate (indicated with #). Proteins with annotation are ordered by their biological function.</p

Major protein components in adult tardigrades in active and tun state. 53 proteins were found as major components in adult tardigrades in AS and 49 in TS.

<p>Comparing the annotated proteins in AS and TS we found the same two major functional groups, protein members of structural constituent of cytoskeleton/muscle and protein members of large lipid transporter family. The contig description is indicated with asterisk, in case we found putative candidates in DomainSweep analysis.</p