Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation
Authors: Gary J Pielak
Lorena Rebecchi
Alexandra H Brozena
Ilaria Giovannini
Austin E Smith
Samantha Piszkiewicz
Bob Goldstein
Doug Koshland
Hugo Tapia
Thomas C Boothby
Journal: Molecular Cell (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: CAHS proteins; anhydrobiosis; cryptobiosis; desiccation tolerance; freeze tolerance
Affiliations: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: ., Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA., Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA., Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Department of Life Sciences, University of Modena and Reggio Emilia, Modena 41125, Italy., Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Abstract:  Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance. 

Retracted: No

PubPeer comments:
 We published a letter pointing flaws of this paper recently. [Arakawa and Numata (2021) u201cReconsidering the u201cglass transitionu201d hypothesis of intrinsically unstructured CAHS proteins in desiccation tolerance of tardigradesu201d, Mol Cell. ](http: www.cell.com molecular-cell retrieve pii S1097276520308996?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1097276520308996%3Fshowall%3Dtrue) This paper was submitted right after the publication of Boothby et al. (2017), but publication was significantly delayed even though accepted on 26th June 2019 and many aspects that we originally expressed were asked by the editor to remove. Therefore, here I detail them below. Firstly, while this paper is entirely about CAHS proteins, but [Yamaguchi et al. PLoS One (2012) ] , which is the first and only paper about CAHS protein at the time of publication of Boothby et al. 2017, that described the identification of CAHS protein, heterologous expression of CAHS in human culture cells, and the intrinsically unstructured nature of this protein, is never cited in the Introduction. , 
 Fourteen sequences of CAHS and SAHS used in this work is listed in Supplementary Data S2 , but 1.tRegistered sequence of CAHS 89226 in UniProt is mistypes as u201cCASHu201d https: www.uniprot.org uniprot P0CU47 2.tThere are two sequences for CAHS 94205, which differs in two amino acid positions. Which of these are used in the analyses is not specified. 3.tSequence named CAHS 68135 is not CAHS, but is a completely different gene LEAM. ![file] , 
 Conservation of MAHS and SAHS in M. tardigradum was denied by the genome analysis in [Bemm et al. (2017) bioRxiv] ![file] . The manuscript (Boothby et al., 2017) method section u201cIdentification of TDP-encoding Transcriptsu201d reads u201cTranscript sequences were used as BLASTx queries and searched against NCBIu2019s non-redundant protein database. Reciprocal best BLAST was performed with an E-value cutoff of 1E-10.u201d However, 1.tRunning BLASTX search against nr database with NCBI online BLAST service with the claimed M. tardigradum SAHS gene (comp6097_c0_seq2) is clearly below 1E-10 threshold. 2.tRegistered M. tardigradum TSA assembly lacks claimed MAHS sequence (comp5259_c0). I have tried to recover this sequence by 1.tassembling 454 data used by Boothby et al. (SRR057381) with newbler v.2.5p1, -cdna option resulting in 95.05% Complete+Partial BUSCO v.3 completeness (Eukaryota DB) 2.tassembling all 454 and Illumina data following Boothby et al. (SRR057381, SRR1046127, SRR1046358, SRR1046360, SRR1046361) with Trinity v.2.10.0 (no version was given by Boothby et al.) resulting in 97.03% Complete+Partial BUSCO v.3 completeness (Eukaryota DB) Both assemblies included one contig matching MAHS protein of R. varieornatus (UniProt:MAHS_RAMVA) that are 99.9% identical, but these sequences, when included in the M. tardigradum TSA assembly to control for the database size, only matched with 4e-06 with tblastn search. Reciprocally, BLASTX search using this sequence in nr database with NCBI online BLAST service was 6e-06. Therefore, overall, MAHS is not detectable with the written threshold. ![file] ![file] ![file] , 
 The authors give a single example of u201cvitrification of Tardigrade CAHS Proteinsu201d u201cshowing a novel glass transition in yeast induced by the expression of a CAHS proteinu201d, with CAHS 77580. The data shows shift in DSC curve, but the introduction of CAHS 77580 does not increase desiccation tolerance in yeast (Figure 6A) or in bacteria (Figure 6B). RNAi of CAHS 77580 in tardigrade neither affects survival under dry conditions. ![file] In [Boothby (2021) Mol Cell rebuttal letter] 30901-1), the author argues that u201cFurthermore, in our original paper we did not infer protection from vitrification, but rather observed vitrification and then went on to perform experiments aimed at empirically correlating vitrification with survival in keeping with norms in the field (Sakurai etu00a0al., 2008).u201d and u201cHowever, because RNAi is not fully penetrant, it is possible that CAHS_77580 RNAi was simply less efficient than RNAi targeting other CAHS genes that did show loss of tolerance.u201d However, 1.tCAHS genes with significant RNAi results on anhydrobiotic survival (CAHS77611, CAHS94205) are NOT used in the u201cvitrificationu201d experiment. 2.tCAHS genes used in u201cvitrificationu201d experiments were either NOT tested for RNAi (CAHS94063, CAHS106094) or NOT significant (CAHS77580). ![file] , 
 Heterologous expression and yeast tolerance assay is replicated within the work, in Figure 6 and Figure 7. Here, the value for Control (Empty vector) is different by one order ( 0.001 in Figure 6, 0.001 and 0.01 in Figure 7), and that of CAHS77580 is different by two orders ( 0.001 in Figure 6, 0.01 and 0.1 in Figure 7. Values for significant samples (CAHS 68135, which is LEAM to be correct, and CAHS 94063, CAHS106094), are 0.1 in Figure 6 but 0.1 in Figure 7. Therefore, this experiment seems to have 10~100 fold error rate, but used to discuss ~100 fold change. ![file] , 
 DSC analysis of whole tardigrade (Figure 7) only shows unusually narrow temperature range for DSC analysis (shown only between 97~99u02daC, with extremely minute heat flow change (-0.39 ~ -0.41W g). We have asked Dr. Boothby and looking at the provided raw data, the thermal shift claimed by the author should be considered a noise by DSC analysis by standards of the field. ![file] In [Boothby (2021) Mol Cell rebuttal letter] 30901-1), the author replies that 1.tOur data is not replicated u2013 But this is not accurate, since we provide data using two species R. varieornatus and H. exemplaris, whereas Boothby et al. (2017) only showed a partial area of H. exemplaris. 2.tDifferent methodology of drying or that we did not take out of the survival of animals, but the latter is clearly a false claim (we surely tested the anhydrobiotic survival) These two are about our replication, and does not directly counter-argue the arbitrary close up of the data. Boothby (2021) further states: u201cAdditionally, Arakawa and Numata do not explain why their tardigrade DSC data did not replicate an independent study on tardigrade vitrification, which identified vitrification in 6 different tardigrade samples withu00a0Tgs closely mirroring the 98u00b0Cu00a0Tgu00a0Boothby etu00a0al. found (e.g., 92.9u00b0C u00b1 5.3u00b0C and 91.8u00b0C u00b1 9.4u00b0C).u00a0u201c This sentence is problematic in three points: 1.tThis sentence does not cite the source of data (which is [Hengherr et al. 2009, Physiological and Biochemical Zoology] ) 2.tThe two temperatures listed are arbitrary selected: the values Boothby (2021) uses are the two maximum values. Median values would be 86.8 u00b1 6.7 and 88.5 u00b1 3.5. 3.tWe clearly state in Arakawa and Numata (2021) Mol Cell that u201cHengherr etu00a0al. (2009)u00a0used 33% relative humidity condition for drying and could also have been affected by the remaining water content.u201c ![file] ,