Suggested Involvement of PP1/PP2A Activity and De Novo Gene Expression in Anhydrobiotic Survival in a Tardigrade, Hypsibius dujardini, by Chemical Genetic Approach

Abstract

Upon desiccation, some tardigrades enter an ametabolic dehydrated state called anhydrobiosis and can survive a desiccated environment in this state. For successful transition to anhydrobiosis, some anhydrobiotic tardigrades require pre-incubation under high humidity conditions, a process called preconditioning, prior to exposure to severe desiccation. Although tardigrades are thought to prepare for transition to anhydrobiosis during preconditioning, the molecular mechanisms governing such processes remain unknown. In this study, we used chemical genetic approaches to elucidate the regulatory mechanisms of anhydrobiosis in the anhydrobiotic tardigrade, Hypsibius dujardini. We first demonstrated that inhibition of transcription or translation drastically impaired anhydrobiotic survival, suggesting that de novo gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades.

Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation

“…Specimens of the tardigrade H. dujardini that had been dried slowly could subsequently survive more rapid desiccation (Figure 1B), suggesting that a sufficient protectant(s) was made during slow drying. This finding, in addition to the fact that H. dujardini requires de novo transcription and translation to robustly survive desiccation (Kondo et al, 2015), makes H. dujardini attractive for differential gene expression studies.…”

“…This trend suggests that tardigrades need time to produce protectants, a theory supported by the recent evidence that de novo transcription and translation are required for the tardigrade Hypsibius dujardini to robustly survive desiccation (Kondo et al, 2015). …”

Structural insights into a secretory abundant heat‐soluble protein from an anhydrobiotic tardigrade,Ramazzottius varieornatus

“… and Tanaka et al . took molecular biological approaches using a tardigrade Ramazzottius varieornatus , which can enter anhydrobiosis in a shorter time than other tardigrades such as Hypsibius dujardini . Consequently, four new heat‐soluble protein families named secretory abundant heat‐soluble (SAHS) proteins, cytoplasmic abundant heat‐soluble (CAHS) proteins, mitochondrial abundant heat‐soluble (MAHS) proteins, and Rv LEAM were discovered.…”

AMPK activity is required for the induction of anhydrobiosis in a tardigrade Hypsibius exemplaris, and its potential up‐regulator is PP2A

“…The procedure was carried out as described previously with slight modifications (Figure a) (Kondo et al, ). Compound C (Merck Millipore), AMPK activator (Santa Cruz Biotechnologies) and cantharidic acid (CA; Enzo Life Sciences) were dissolved in dimethyl sulfoxide special grade (DMSO; FUJIFILM Wako Pure Chemical Corporation), AICAR (AdipoGen) was dissolved in fresh Milli‐Q (MQ) water as a stock solution, and all were stored at −20°C.…”

“…Genes to Cells KONDO et al have recently become a model system for the study of molecular basis of anhydrobiosis as several rearing systems of some different types of anhydrobiotic species and nonanhydrobiotic species have been established (Gabriel et al, 2007;Horikawa et al, 2008;Ito, Saigo, Abe, Kubo, & Kunieda, 2016;Suzuki, 2003), high-quality genome databases are available (Hashimoto et al, 2016;Yoshida et al, 2017), gene knockdown by RNA interference is functional (Tenlen, McCaskill, & Goldstein, 2013), and several candidate proteins possibly involved in anhydrobiosis (such as tardigrade unique heat-soluble proteins (Tanaka et al, 2015;Yamaguchi et al, 2012) and DNA-binding proteins (Hashimoto et al, 2016)) have been identified. Although studies on the molecular basis of anhydrobiosis in tardigrades have been mainly focused on the exploration of new protectants and functional analyses, studies on molecular regulatory mechanisms have also recently begun (Kondo, Kubo, & Kunieda, 2015). Anhydrobiotic style is categorized by the necessity of preconditioning, the pre-exposure of animals to high relative humidity (RH) condition in order to make animals tolerate subsequent exposure to low RH condition.…”