Characterization and regulation of 2D-3D convertible lipid membrane transformation.

Micro-nanomaterials that can adopt different structures are powerful tools in the fields of biological and medical sciences. We previously developed a lipid membrane that can convert between 2D nanosheet and 3D vesicle forms using cationic copolymer polyallylamine-graft-polyethylene glycol and the anionic peptide E5. The properties of the membrane during conversion have been characterized only by confocal laser scan microscopy. Furthermore, due to the 2D symmetry of the lipid nanosheet, the random folding of the lipid bilayer into either the original or the reverse orientation occurs during sheet-to-vesicle conversion, compromising the structural consistency of the membrane. In this study, flow cytometry was applied to track the conversion of more than 5000 lipid membranes from 3D vesicles to 2D nanosheets and back to 3D vesicles, difficult with microscopies. The lipid nanosheets exhibited more side scattering intensity than 3D vesicles, presumably due to free fluctuation and spin of the sheets in the suspension. Furthermore, by immobilizing bovine serum albumin as one of the representative proteins on the outer leaflet of giant unilamellar vesicles at a relatively low coverage, complete restoration of lipid membranes to the original 3D orientation was obtained after sheet-to-vesicle conversion. This convertible membrane system should be applicable in a wide range of fields. Our findings also provide experimental evidence for future theoretical studies on membrane behavior.

Switchable and orthogonal gene expression control inside artificial cells by synthetic riboswitches.

Here we report two novel synthetic riboswitches that respond to ASP2905 and theophylline and function in reconstituted cell-free protein synthesis (CFPS) system. We encapsulated the CFPS system as well as DNA-templated encoding reporter genes regulated by these orthogonal riboswitches inside liposomes, and achieved switchable and orthogonal control over gene expression by external stimulation with the cognate ligands.

Lipid vesicle-based molecular robots.

A molecular robot, which is a system comprised of one or more molecular machines and computers, can execute sophisticated tasks in many fields that span from nanomedicine to green nanotechnology. The core parts of molecular robots are fairly consistent from system to system and always include (i) a body to encapsulate molecular machines, (ii) sensors to capture signals, (iii) computers to make decisions, and (iv) actuators to perform tasks. This review aims to provide an overview of approaches and considerations to develop molecular robots. We first introduce the basic technologies required for constructing the core parts of molecular robots, describe the recent progress towards achieving higher functionality, and subsequently discuss the current challenges and outlook. We also highlight the applications of molecular robots in sensing biomarkers, signal communications with living cells, and conversion of energy. Although molecular robots are still in their infancy, they will unquestionably initiate massive change in biomedical and environmental technology in the not too distant future.

Identifying Conditions for Protein Synthesis Inside Giant Vesicles Using Microfluidics toward Standardized Artificial Cell Production.

To expand the range of practical applications of artificial cells, it is important to standardize the production process of giant (cell-sized) vesicles that encapsulate reconstituted biochemical reaction systems. For this purpose, a rapidly developing microfluidics-based giant vesicle generation system is a promising approach, similar to the droplet assay systems that are already widespread in the market. In this study, we examined the composition of the solutions used to generate vesicles encapsulating the in vitro transcription-translation (IVTT) system. We show that tuning of the lipid composition and adding poly(vinyl alcohol) to the outer solution improved the stability of the transition process into the lipid membrane so that protein synthesis proceeded in vesicles. The direct integration of α-hemolysin nanopores synthesized in situ was also demonstrated. These protein-synthesizing monodisperse giant vesicles can be prepared by using a simple microfluidic fabrication/operation with a commercial IVTT system.

Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane.

Artificial cells are membrane vesicles mimicking cellular functions. To date, giant unilamellar vesicles made from a single lipid membrane with a diameter of 10 µm or more have been used to create artificial cells. However, the creation of artificial cells that mimic the membrane structure and size of bacteria has been limited due to technical restrictions of conventional liposome preparation methods. Here, we created bacteria-sized large unilamellar vesicles (LUVs) with proteins localized asymmetrically to the lipid bilayer. Liposomes containing benzylguanine-modified phospholipids were prepared by combining the conventional water-in-oil emulsion method and the extruder method, and green fluorescent protein fused with SNAP-tag was localized to the inner leaflet of the lipid bilayer. Biotinylated lipid molecules were then inserted externally, and the outer leaflet was modified with streptavidin. The resulting liposomes had a size distribution in the range of 500-2000 nm with a peak at 841 nm (the coefficient of variation was 10.3%), which was similar to that of spherical bacterial cells. Fluorescence microscopy, quantitative evaluation using flow cytometry, and western blotting proved the intended localization of different proteins on the lipid membrane. Cryogenic electron microscopy and quantitative evaluation by α-hemolysin insertion revealed that most of the created liposomes were unilamellar. Our simple method for the preparation of bacteria-sized LUVs with asymmetrically localized proteins will contribute to the creation of artificial bacterial cells for investigating functions and the significance of their surface structure and size.

ePURE_JSBML: A Tool for Constructing a Deterministic Model of a Reconstituted Escherichia coli Protein Translation System with a User-Specified Nucleic Acid Sequence.

A protein synthesis system is one of the most important and complex biological networks, which translates DNA-encoded information into specific functions. Here, ePURE_JSBML, a tool for constructing biologically relevant large-scale and detailed computational models based on a reconstituted cell-free protein synthesis system, is presented; the user can specify the mRNA sequence, initial component concentration, and decoding rule. Model construction is based on Systems Biology Markup Language (SBML) using JSBML, a pure Java programming library. The tool generates simulation files, executable with Matlab, that enable a variety of simulation experiments including the synthesis of proteins of a few hundred residues.

Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Chimeric mutants of staphylococcal hemolysin, which act as both one-component and two-component hemolysin, created by grafting the stem domain.

Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane ß-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component α-hemolysin (α-HL) and two-component γ-hemolysin (γ-HL). The α-HL forms a homo-heptamer, whereas γ-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a ß-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the ß-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT ß-barrels for use in further promising applications.

Identification of conditions for efficient cell-sized liposome preparation using commercially available reconstituted in vitro transcription-translation system.

Attempts to create complex molecular systems that mimic parts of cellular systems using a bottom-up approach have become important in the field of biology. Among various molecular systems, in vitro protein synthesis inside lipid vesicles (liposomes), which we refer to as the artificial cell, has become an attractive system because it possesses two fundamental features of living cells: central dogma, and compartmentalization. Here, we investigated the effect of altering the amount or concentration of four constituents of the artificial cell consisting of a commercially available reconstituted in vitro transcription-translation (IVTT) system. As this IVTT system is available worldwide, the results will be useful to the scientific community when shared, unlike those from a lab-made IVTT system. We succeeded in revealing the effect and trend of altering each parameter and identified a suitable condition for preparing liposomes that are unilamellar and can synthesize proteins equally as well as the original IVTT system. Because the commercially available reconstituted IVTT system is an important standardization tool and the constituents can be adjusted as desired, our results will be useful for the bottom-up creation of more complex molecular systems.

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression.

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.

DNMT3.1 controls trade-offs between growth, reproduction, and life span under starved conditions in Daphnia magna.

The cladoceran crustacean Daphnia has long been a model of energy allocation studies due to its important position in the trophic cascade of freshwater ecosystems. However, the loci for controlling energy allocation between life history traits still remain unknown. Here, we report CRISPR/Cas-mediated target mutagenesis of DNA methyltransferase 3.1 (DNMT3.1) that is upregulated in response to caloric restriction in Daphnia magna. The resulting biallelic mutant is viable and did not show any change in growth rate, reproduction, and longevity under nutrient rich conditions. In contrast, under starved conditions, the growth rate of this DNMT3.1 mutant was increased but its reproduction was reciprocally reduced compared to the wild type when the growth and reproduction activities competed during a period from instar 4 to 8. The life span of this mutant was significantly shorter than that of the wild type. We also compared transcriptomes between DNMT3.1 mutant and wild type under nutrient-rich and starved conditions. Consistent with the DNMT3.1 mutant phenotypes, the starved condition led to changes in the transcriptomes of the mutant including differential expression of vitellogenin genes. In addition, we found upregulation of the I am not dead yet (INDY) ortholog, which has been known to shorten the life span in Drosophila, explaining the shorter life span of the DNMT3.1 mutant. These results establish DNMT3.1 as a key regulator for life span and energy allocation between growth and reproduction during caloric restriction. Our findings reveal how energy allocation is implemented by selective expression of a DNMT3 ortholog that is widely distributed among animals. We also infer a previously unidentified adaptation of Daphnia that invests more energy for reproduction than growth under starved conditions.

Mutation of the Cytochrome P450 CYP360A8 Gene Increases Sensitivity to Paraquat in Daphnia magna.

The freshwater crustacean Daphnia magna has traditionally been a model for ecotoxicological studies owing to its sensitivity to many xenobiotics. Because it is used in many toxicity assessments, its detoxification mechanism for xenobiotics is important and requires further study. However, studies related to detoxification genes are limited to transcriptomic profiling, and there are no D. magna mutants for use in the understanding of xenobiotic metabolism in vivo. We report the generation of a D. magna CYP360A8 mutant-the gene is a cytochrome P450 (CYP) clan 3 gene. Based on RNA sequencing of adult D. magna, we found that CYP360A8 has the highest expression level among all CYP genes. At ovarian maturation, its expression level is up-regulated 6-fold compared to the juvenile stages and is maintained thereafter. Using the CRISPR/CRISPR-associated 9 (Cas9) system, we disrupted CYP360A8 by coinjecting CYP360A8-targeting guide RNA and Cas9 proteins into D. magna eggs and established one monoallelic CYP360A8 mutant line. This CYP360A8 mutant had a higher sensitivity to the herbicide paraquat compared to the wild type. We confirmed the up-regulation of CYP360A8 by paraquat. The results demonstrate the role of CYP360A8 in paraquat detoxification. The present study establishes a CYP mutant of D. magna, and this strategy can be a basic platform to document a range of CYP gene-xenobiotic relationships in this species. Environ Toxicol Chem 2021;40:1279-1288. © 2020 SETAC.

Reduction of histamine and enhanced spinning behavior of Daphnia magna caused by scarlet mutant.

The ABC transporter, Scarlet, and its binding partner, White are involved in pigment synthesis in the insect eye and mutations in these genes are used as genetic markers. Recent studies have suggested that these transporters also have additional functions in the neuronal system. In our previous study, we generated scarlet mutant in the small crustacean, Daphnia magna and showed that the mutant lacked the eye pigment in the mutant. Here, we show that the scarlet mutant exhibits spinning behavior. This phenotype is partly associated with the presence of light. Metabolomic analysis of a juvenile mutant revealed that the scarlet mutant has approximately one-tenth of the histamine content of the wild type. Application of histamine to the scarlet mutant rescued the spinning behavior in juveniles, suggesting that the spinning behavior of the mutant is caused by the reduction of histamine. However, the altered behavior was not rescued in the adult mutant by the addition of histamine, suggesting that Scarlet plays an irreversible role in the development of histaminergic neurons. These results suggest that Scarlet plays an important role in histaminergic signaling, which might be related to control the spinning behavior, in addition to its role in eye pigmentation.

Production of genome-edited Daphnia for heavy metal detection by fluorescence.

Aquatic heavy metal pollution is a growing concern. To facilitate heavy metal monitoring in water, we developed transgenic Daphnia that are highly sensitive to heavy metals and respond to them rapidly. Metallothionein A, which was a metal response gene, and its promoter region was obtained from Daphnia magna. A chimeric gene fusing the promoter region with a green fluorescent protein (GFP) gene was integrated into D. magna using the TALEN technique and transgenic Daphnia named D. magna MetalloG were produced. When D. magna MetalloG was exposed to heavy metal solutions for 1 h, GFP expression was induced only in their midgut and hepatopancreas. The lowest concentrations of heavy metals that activated GFP expression were 1.2 µM Zn2+, 130 nM Cu2+, and 70 nM Cd2+. Heavy metal exposure for 24 h could lower the thresholds even further. D. magna MetalloG facilitates aqueous heavy metal detection and might enhance water quality monitoring.

Caloric restriction upregulates the expression of DNMT3.1, lacking the conserved catalytic domain, in Daphnia magna.

DNA methylation plays an important role in many aspects of biology, including development, disease, and phenotypic plasticity. In the branchiopod crustacean, Daphnia, de novo DNA methylation has been detected in specific environmental contexts. However, fundamental information on de novo DNA methyltransferase DNMT3 orthologs, including domain organization, developmental expression, and response to environmental stimuli, is lacking. In this study, we examined two DNMT3 orthologs in Daphnia magna, DapmaDNMT3.1 and DapmaDNMT3.2. Amino acid sequence alignment revealed that DapmaDNMT3.1 and DapmaDNMT3.2 lack the conserved methyltransferase motifs of the catalytic domain and the PWWP domain, respectively. We profiled the expression of the two orthologs during embryogenesis and under various feeding levels. During embryogenesis, in contrast to the low DapmaDNMT3.1 expression, DapmaDNTM3.2 was highly expressed at specific stages, that is, in the one cell-stage and at 48 hr post ovulation. In nutrient-rich condition, both genes were lowly expressed, whereas DapmaDNMT3.1 was upregulated at the lower food levels, suggesting a potential role of DapmaDNMT3.1 in gene regulation in response to caloric restriction. These findings provide a basis for understanding the developmental stage- and stress-dependent function of DNMT3 orthologs in D. magna.

Roles of and cross-talk between ecdysteroid and sesquiterpenoid pathways in embryogenesis of branchiopod crustacean Daphnia magna.

The ecdysteroid and sesquiterpenoid pathways control growth, developmental transition, and embryogenesis in insects. However, the function of orthologous genes and the cross-talk between both pathways remain largely uncharacterized in non-insect arthropods. Spook (Spo) and Juvenile hormone acid o-methyltransferase (Jhamt) have been suggested to function as rate-limiting factors in ecdysteroid and sesquiterpenoid biosynthesis, respectively, in insects. In this study, we report on the functions of Spo and Jhamt and the cross-talk between them in embryos of the branchiopod crustacean Daphnia magna. Spo expression was activated at the onset of gastrulation, with the depletion of Spo transcript by RNAi resulting in developmental arrest at this stage. This phenotype could be partially rescued by supplementation with 20-hydroxyecdysone, indicating that Spo may play the same role in ecdysteroid biosynthesis in early embryos, as reported in insects. After hatching, Spo expression was repressed, while Jhamt expression was activated transiently, despite its silencing during other embryonic stages. Jhamt RNAi showed little effect on survival, but shortened the embryonic period. Exposure to the sesquiterpenoid analog Fenoxycarb extended the embryonic period and rescued the Jhamt RNAi phenotype, demonstrating a previously unidentified role of sesquiterpenoid in the repression of precocious embryogenesis. Interestingly, the knockdown of Jhamt resulted in the derepression of ecdysteroid biosynthesis genes, including Spo, similar to regulation during insect hormonal biosynthesis. Sesquiterpenoid signaling via the Methoprene-tolerant gene was found to be responsible for the repression of ecdysteroid biosynthesis genes. It upregulated an ortholog of CYP18a1 that degrades ecdysteroid in insects. These results illuminate the conserved and specific functions of the ecdysteroid and sesquiterpenoid pathways in Daphnia embryos. We also infer that the common ancestor of branchiopod crustaceans and insects exhibited antagonism between the two endocrine hormones before their divergence 400 million years ago.

Two Doublesex1 mutants revealed a tunable gene network underlying intersexuality in Daphnia magna.

In recent years, the binary definition of sex is being challenged by repetitive reports about individuals with ambiguous sexual identity from various animal groups. This has created an urge to decode the molecular mechanism underlying sexual development. However, sexual ambiguities are extremely uncommon in nature, limiting their experimental value. Here, we report the establishment of a genetically modified clone of Daphnia magna from which intersex daphniids can be readily generated. By mutating the conserved central sex determining factor Doublesex1, body-wide feminization of male daphniid could be achieved. Comparative transcriptomic analysis also revealed a genetic network correlated with Doublesex1 activity which may account for the establishment of sexual identity in D. magna. We found that Dsx1 repressed genes related to growth and promoted genes related to signaling. We infer that different intersex phenotypes are the results of fluctuation in activity of these Dsx1 downstream factors. Our results demonstrated that the D. magna genome is capable of expressing sex in a continuous array, supporting the idea that sex is actually a spectrum.

Computational design of transmembrane pores.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA.

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.