FRET sensor-based quantification of intracellular trehalose in mammalian cells

Kikuta, Shingo; Hou, Bi-Huei; Satō, Ryōichi; Frommer, Wolf B.; Kikawada, Takahiro

doi:10.1080/09168451.2015.1069699

Cited by 14 publications

(8 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This enhanced approach, which we term DIP-seq, was used to assess the potential of TMBP as a biosensor scaffold for trehalose. Trehalose is a molecule of biological interest that is implicated in stress tolerance and metabolic regulation of yeast and plants but for which there is no available SFPB 23 24 25 . Independent transposon libraries, in triplicate, were used to create libraries of cpGFP insertions into the TMBP ligand-binding domain.…”

Section: Resultsmentioning

confidence: 99%

Rapid construction of metabolite biosensors using domain-insertion profiling

et al. 2016

View full text Add to dashboard Cite

Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery.

show abstract

Section: Resultsmentioning

confidence: 99%

Rapid construction of metabolite biosensors using domain-insertion profiling

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Desiccation-intolerant mammalian cultured cells do not endogenously express genes encoding anhydroprotectants such as late embryogenesis-abundant (LEA) proteins, enzymes for synthesis of trehalose and trehalose transporter 27, 34 . Even though the mammalian cells were pretreated with trehalose prior dehydration/rehydration as Pv11 cells, desiccation stress resulted in the loss of both cell viability and luciferase activity during desiccation (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Kikuta

Watanabe

Satō

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Desiccation-intolerant mammalian cultured cells do not endogenously express genes encoding anhydroprotectants such as late embryogenesis-abundant (LEA) proteins, enzymes for synthesis of trehalose and trehalose transporter 21,29 . Even though the mammalian cells were pretreated with trehalose prior dehydration/rehydration as Pv11 cells, desiccation stress resulted in the loss of both cell viability and luciferase activity during desiccation (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Kikuta

Watanabe

Satō

et al. 2017

Preprint

Self Cite

View full text Add to dashboard Cite

Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic Polypedilum vanderplanki embryo endure complete desiccation because of their 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 preserved an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase, preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including pre-incubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pvll-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pvll-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-tolerant related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

show abstract

FRET sensor-based quantification of intracellular trehalose in mammalian cells

Cited by 14 publications

References 22 publications

Rapid construction of metabolite biosensors using domain-insertion profiling

Rapid construction of metabolite biosensors using domain-insertion profiling

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

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

Contact Info

Product

Resources

About