α,ß-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances.

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,ß-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,ß-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,ß-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Distribution of soil testate amoeba assemblages along an elevation gradient on Mount Fuji (Japan).

Elevation gradients have been useful to study distributional patterns of soil organisms since the time of Humboldt but only recently these patterns have been studied for soil microorganisms. Here we report the results on species diversity and composition of soil- and moss-dwelling testate amoeba assemblages along a 1400 m elevation gradient (904-2377 m a.s.l.) on Mount Fuji (Japan) from temperate forest to alpine vegetation. In total, 95 testate amoeba taxa belonging to 29 genera were identified. The core of testate amoeba assemblages was formed by ubiquitous species such as Trinema lineare, Euglypha laevis, Cryptodifflugia oviformis, and Trinema complanatum. However, several taxa with limited geographic distribution were also observed (e.g., Centropyxis latideflandriana, C. stenodeflandriana, Plagiopyxis cf. barrosi, Heleopera rectangularis, and Distomatopyxis couillardi). Species diversity indices (species richness and Shannon's index) were characterised by bell-shaped patterns peaking at âˆ¼ 1700 m in the subalpine mixed conifer-deciduous forest. The species composition of testate amoeba assemblages was best explained by the vegetation types which accounted for 12.3% of the total variation. Overall, these findings indicate that elevation effects on species composition of testate amoeba assemblages are strongly mediated by vegetation.

An elevation transect study of testate amoeba communities up to 4000 m a.s.l. on Mount Kinabalu, Borneo.

Testate amoebae are a polyphyletic assemblage of unicellular eukaryotes with a specific shell. They occur frequently in soil and freshwater habitats all over the world, important for ecosystem functioning and useful for bio-indication. Terrestrial amoeba data from Borneo or Malaysia are rarely available. In this study, data on species composition and relative abundance were presented along a transect from 1730 m up to 4000 m above sea level from 10 elevational levels on Mt. Kinabalu, Malaysian Borneo. In total 78 morphotypes of testate amoebae were recorded. Most were small (under 100 µm) and supposedly cosmopolitan. However, Certesella certesi with limited geographical distribution and some rare taxa (e.g., Padaungiella lageniformis cordiformis, Placocista jurassica, Trinema chardezi) and two undescribed morphospecies were also observed. Presence/absence and relative abundance data of testate amoebae were associated to elevation with a pronounced change at the tree line, as revealed by non-metric multidimensional scaling ordination analysis. Our data from Southeast Asia contribute to the actual discussion on distributional patterns considering elevation gradients and biogeography of eukaryotic microbes.

Fabrication of a sugar-immobilized fluorescent PMMA shell on a Ni core particle via soap-free emulsion polymerization.

Sugar chain immobilized polymer particles having both magnetic and fluorescent properties can be expected to be useful in a wide variety of biomedical applications such as the detection, separation, and purification of proteins, viruses, or bacteria, because sugar chains specifically adsorb them. Since high magnetic responsiveness is required for such applications, we attempted to fabricate core-shell particles consisting of a submicron-sized magnetic core and a thin polymer shell (nano- to dozens of nanometers thick) that incorporates a fluorescent dye, with sugar molecules immobilized on the surface. Soap-free emulsion polymerization using methyl methacrylate (MMA) monomer and potassium persulfate (KPS) initiator in the presence of aminopropyltrimethoxysilane-treated Ni particles, octyl-ß-D-glucopyranoside (octyl-glc), and rhodamine B (RhB) produced a glucose-immobilized fluorescent PMMA thin shell on a Ni particle (Ni/PMMA/RhB/octyl-glc). Electrostatic interaction was used both to incorporate RhB into the PMMA shell and to coat the Ni core with the PMMA-RhB shell. Glucose was immobilized on the PMMA shell by embedding a hydrophobic octyl group derived from octyl-glc in the PMMA matrix, and the resulting sugar-immobilized PMMA shell was able to adsorb protein (concanavalin A; a protein that specifically adsorbs glucose). The resulting Ni/PMMA/RhB/octyl-glc particles were well-dispersed in water, detected by highly sensitive fluorescence techniques, and could be collected by a magnet within 10 sec. They are expected to be applied to detect biological substances such as various proteins and viruses by changing the glucose moiety of the particle surface to other functional glycans. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00396-022-04945-7.

Identification of a novel strong promoter from the anhydrobiotic midge, Polypedilum vanderplanki, with conserved function in various insect cell lines.

Larvae of the African midge Polypedilum vanderplanki (Diptera: Chironomidae) show a form of extreme desiccation tolerance known as anhydrobiosis. The cell line Pv11 was recently established from the species, and these cells can also survive under desiccated conditions, and proliferate normally after rehydration. Here we report the identification of a new promoter, 121, which has strong constitutive transcriptional activity in Pv11 cells and promotes effective expression of exogenous genes. Using a luciferase reporter assay, this strong transcriptional activity was shown to be conserved in cell lines from various insect species, including S2 (Drosophila melanogaster, Diptera), SaPe-4 (Sarcophaga peregrina, Diptera), Sf9 (Spodoptera frugiperda, Lepidoptera) and Tc81 (Tribolium castaneum, Coleoptera) cells. In conjunction with an appropriate selection maker gene, the 121 promoter was able to confer zeocin resistance on SaPe-4 cells and allowed the establishment of stable SaPe-4 cell lines expressing the fluorescent protein AcGFP1; this is the first report of heterologous gene expression in this cell line. These results show the 121 promoter to be a versatile tool for exogenous gene expression in a wide range of insect cell lines, particularly useful to those from non-model insect species.

Cooption of heat shock regulatory system for anhydrobiosis in the sleeping chironomid Polypedilum vanderplanki.

Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration-rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.

Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells.

Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic midge embryo endure complete desiccation in an ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells could preserve an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase (Luc), preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including preincubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pv11-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pv11-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation tolerance-related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

Current findings on the molecular mechanisms underlying anhydrobiosis in Polypedilum vanderplanki.

Water is an essential molecule for living organisms. However, some organisms can survive in environments which receive no rainfall for months and in which ordinary life cannot survive. How do they endure the extended dry season? The sleeping chironomid Polypedilum vanderplanki, which inhabits sub-Saharan Africa, exhibits extreme tolerance to complete desiccation, a process termed anhydrobiosis. During anhydrobiosis these organisms dry up and entirely shut down their metabolism. However, when the dried larvae are immersed in water, their metabolism is resumed. Interestingly, anhydrobiosis allows these organisms to tolerate not only desiccation but also high and low temperatures, the absence of oxygen, radiation, and chemical stresses. Here, we describe the mechanisms by which P. vanderplanki achieves anhydrobiosis revealed in our recent research.

Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line, Pv11.

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5-34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

Morphogenetic and molecular analyses of cyst wall components in the ciliated protozoan Colpoda cucullus Nag-1.

The cyst wall of the resting cyst of the ciliated protozoan Colpoda cucullus (Nag-1 strain) is composed of several layers of endocyst, a single layer of ectocyst associated with a mucous layer and lepidosomes composed of a fibrous or crystal-like structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the ectocyst associated with lepidosomes and mucous materials contained proteins corresponding to 27, 31, 45 kDa and smear bands ranging from 50 to 60 kDa. Liquid chromatography-tandem mass spectrometry of these proteins revealed that the 45-kDa protein (p45) was elongation factor Tu (EF-Tu). Immunofluorescence microscopy with an anti-EF-Tu polyclonal antibody showed that Colpoda EF-Tu (p45) was localized in the lepidosomes. The lepidosomes were stained vividly with Congo red, which is bound to the stacked ß-sheets of amyloid protofibrils. In the presence of puromycin, no cyst wall components including lepidosomes were formed, indicating that cyst wall formation requires synthesis of proteins including EF-Tu. Electron microscopy of encysting cells implied that vesicles which were presumably budded from endoplasmic reticula possibly fuse with a lepidosome-precursor vacuole containing electron-dense fine particles or fibrous structures, and followed by the subsequent fusion with other electron-lucent granules.

Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis.

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; ß-tubulin) was enhanced.

Excystment-dependent alteration of protein expression in terrestrial ciliate Colpoda cucullus.

Protein expression during the excystment of Colpoda cucullus was studied by SDS-PAGE. The expression levels of 60-, 50- and 49-kDa proteins were markedly changed from the early to later stage of excystment. The 60-kDa protein (p60) was temporarily expressed first, and its expression was inhibited by actinomycin D. LC-MS/MS analysis showed that the amino acid sequences of p60 partially coincided with those of the Paramecium tetraurelia unnamed protein homologous to DEAD-box RNA helicase. These results suggest that p60 expression is enhanced by transcriptional regulation and may be involved in initiating the molecular events leading to cellular morphogenesis.

EF-1α and mitochondrial ATP synthase ß chain: alteration of their expression in encystment-induced Colpoda cucullus.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase ß chains, and that p49 is elongation factor 1α.

Protein phosphorylation in encystment-induced Colpoda cucullus: localization and identification of phosphoproteins.

In Colpoda cucullus, the morphogenetic transformation was preceded by an enhancement of the in vivo protein phosphorylation level. Immunofluorescence microscopy using antiphosphoserine antibody showed that these phosphorylated proteins were localized in the macronucleus and other cytoplasmic regions. Biotinylated Phos-tag/ECL assays of isolated macronuclei showed that a 33-kDa protein (p33) was localized within them. The p33 obtained from isolated macronuclei was tentatively identified as ribosomal P0 protein by LC-MS/MS analysis. In addition, among the encystment-specific phosphoproteins obtained by phosphate-affinity chromatography, the 29-, 31-, and 33-kDa proteins (p29, p31, and p33) were tentatively identified as ribosomal P0 protein, whereas the 24-kDa phosphoprotein (p24) was tentatively identified as ribosomal S5 protein.

Culture Age, Intracellular Ca(2+) Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus.

In Colpoda cucullus, intracellular Ca(2+) mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca(2+) concentration (F340/F380 ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca(2+)-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus.

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca(2+) and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca(2+) or without the addition of Ca(2+). Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca(2+)/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca(2+) inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca(2+)-triggered signaling pathways, may be involved in encystment induction.