Details of a Researcher - Fujiwara, Kei

Papers - Fujiwara, Kei

Division display 1 - 20 of about 56 ／ All the affair displays >>

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution

Sato G., Kinoshita S., Yamada T.G., Arai S., Kitaguchi T., Funahashi A., Doi N., Fujiwara K.

ACS Synthetic Biology (ACS Synthetic Biology) 13 ( 5 ) 1572 - 1581 2024.05

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

Access to Document (DOI)

Multimolecular Competition Effect as a Modulator of Protein Localization and Biochemical Networks in Cell-Size Space

Nishikawa S., Sato G., Takada S., Kohyama S., Honda G., Yanagisawa M., Hori Y., Doi N., Yoshinaga N., Fujiwara K.

Advanced Science (Advanced Science) 11 ( 6 ) 2024.02

Cells are small, closed spaces filled with various types of macromolecules. Although it is shown that the characteristics of biochemical reactions in vitro are quite different from those in living cells, the role of the co-existence of various macromolecules in cell-size space remains still elusive. Here, using a constructive approach, it is demonstrated that the co-existence of various macromolecules themselves has the ability to tune protein localization for spatiotemporal regulation and a biochemical reaction system in a cell-size space. Both experimental and theoretical analyses reveal that enhancement of interfacial effects by a large surface-area-to-volume ratio facilitates membrane localization of molecules in the cell-size space, and the interfacial effects are alleviated by competitive binding to lipid membranes among multiple proteins even if their membrane affinities are weak. These results indicate that competition for membrane binding among various macromolecules in the cell-size space plays a role in regulating the spatiotemporal molecular organization and biochemical reaction networks. These findings shed light on the importance of surrounding molecules for biochemical reactions using purified elements in small spaces.

Access to Document (DOI)

Phase-Separated Giant Liposomes for Stable Elevation of α-Hemolysin Concentration in Lipid Membranes

Kobayashi M., Noguchi H., Sato G., Watanabe C., Fujiwara K., Yanagisawa M.

Langmuir (Langmuir) 39 ( 32 ) 11481 - 11489 2023.08

ISSN 07437463

Staphylococcus aureus α-hemolysin (αHL) is one of the most popular proteins in nanopore experiments within lipid membranes. Higher concentrations of αHL within the lipid membrane are desirable to enhance the mass transport capacity through nanopores. However, the reconstitution of αHL at high concentrations is associated with the problem of membrane lytic disruption. In this study, we present a method that effectively increases αHL concentration while maintaining membrane stability. This method is achieved by using phase-separated giant liposomes, where coexisting liquid-disordered (Ld) and liquid-ordered phases (Lo) are enriched in unsaturated lipids and saturated lipids with cholesterol (Chol), respectively. Fluorescence observation of αHL in liposomes revealed that the presence of Chol facilitates αHL insertion into the membrane. Despite the preferential localization of αHL in the Ld phase rather than the Lo phase, the coexistence of both Lo and Ld phases prevents membrane disruption in the presence of concentrated αHL. We have explained this stabilization mechanism considering the lower membrane tension exhibited by phase-separated liposomes compared to homogeneous liposomes. Under hypertonic conditions, we have successfully increased the local concentration of αHL by invagination of the lipid-only region in the Ld phase, leaving αHL behind. This method exhibits potential for the reconstitution of various nanochannels and membrane proteins that prefer the Ld phase over the Lo phase, thus enabling the production of giant liposomes at high concentrations and the replication of the membrane-crowding condition observed in cells.

Access to Document (DOI)

Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide

Nakamura M., Fujiwara K., Doi N.

Journal of Nanobiotechnology (Journal of Nanobiotechnology) 20 ( 1 ) 2022.12

Background: Although protein-based methods using cell-penetrating peptides such as TAT have been expected to provide an alternative approach to siRNA delivery, the low efficiency of endosomal escape of siRNA/protein complexes taken up into cells by endocytosis remains a problem. Here, to overcome this problem, we adopted the membrane penetration-enhancing peptide S19 from human syncytin 1 previously identified in our laboratory. Results: We prepared fusion proteins in which the S19 and TAT peptides were fused to the viral RNA-binding domains (RBDs) as carrier proteins, added the RBD-S19-TAT/siRNA complex to human cultured cells, and investigated the cytoplasmic delivery of the complex and the knockdown efficiency of target genes. We found that the intracellular uptake of the RBD-S19-TAT/siRNA complex was increased compared to that of the RBD-TAT/siRNA complex, and the expression level of the target mRNA was decreased. Because siRNA must dissociate from RBD and bind to Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) after the protein/siRNA complex is delivered into the cytoplasm, a dilemma arises: stronger binding between RBD and siRNA increases intracellular uptake but makes RISC formation more difficult. Thus, we next prepared fusion proteins in which the S19 and TAT peptides were fused with Ago2 instead of RBD and found that the efficiencies of siRNA delivery and knockdown obtained using TAT-S19-Ago2 were higher than those using TAT-Ago2. In addition, we found that the smallest RISC delivery induced faster knockdown than traditional siRNA lipofection, probably due to the decreased time required for RISC formation in the cytoplasm. Conclusion: These results indicated that S19 and TAT-fused siRNA-binding proteins, especially Ago2, should be useful for the rapid and efficient delivery of siRNA without the addition of any endosome-disrupting agent.

Access to Document (DOI)

Controlling the Periodicity of a Reaction-Diffusion Wave in Artificial Cells by a Two-Way Energy Supplier

Takada S., Yoshinaga N., Doi N., Fujiwara K.

ACS Nano (ACS Nano) 16 ( 10 ) 16853 - 16861 2022.10

ISSN 19360851

Reaction-diffusion (RD) waves, which are dynamic self-organization structures generated by nanosize molecules, are a fundamental mechanism from patterning in nano- and micromaterials to spatiotemporal regulations in living cells, such as cell division and motility. Although the periods of RD waves are the critical element for these functions, the development of a system to control their period is challenging because RD waves result from nonlinear physical dynamics under far-from-equilibrium conditions. Here, we developed an artificial cell system with tunable period of an RD-driven wave (Min protein wave), which determines a cell division site plane in living bacterial cells. The developed system is based on our finding that Min waves are generated by energy consumption of either ATP or dATP, and the period of the wave is different between these two energy suppliers. We showed that the Min-wave period was modulated linearly by the mixing ratio of ATP and dATP and that it was also possible to estimate the mixing ratio of ATP and dATP from the period. Our findings illuminated a previously unidentified principle to control the dissipative dynamics of biomolecules and, simultaneously, built an important framework to construct molecular robots with spatiotemporal units.

Access to Document (DOI)

Cell-Size Space Regulates the Behavior of Confined Polymers: From Nano- and Micromaterials Science to Biology

Yanagisawa M., Watanabe C., Yoshinaga N., Fujiwara K.

Langmuir (Langmuir) 38 ( 39 ) 11811 - 11827 2022.10

ISSN 07437463

Polymer micromaterials in a liquid or gel phase covered with a surfactant membrane are widely used materials in pharmaceuticals, cosmetics, and foods. In particular, cell-sized micromaterials of biopolymer solutions covered with a lipid membrane have been studied as artificial cells to understand cells from a physicochemical perspective. The characteristics and phase transitions of polymers confined to a microscopic space often differ from those in bulk systems. The effect that causes this difference is referred to as the cell-size space effect (CSE), but the specific physicochemical factors remain unclear. This study introduces the analysis of CSE on molecular diffusion, nanostructure transition, and phase separation and presents their main factors, i.e., short- and long-range interactions with the membrane surface and small volume (finite element nature). This serves as a guide for determining the dominant factors of CSE. Furthermore, we also introduce other factors of CSE such as spatial closure and the relationships among space size, the characteristic length of periodicity, the structure size, and many others produced by biomolecular assemblies through the analysis of protein reaction-diffusion systems and biochemical reactions.

Access to Document (DOI)

Mode selection mechanism in traveling and standing waves revealed by Min wave reconstituted in artificial cells

Sakura Takada, Natsuhiko Yoshinaga, Nobuhide Doi, Kei Fujiwara

Science Advances (Science Advances) 8 ( 23 ) eabm8460 2022.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Reaction-diffusion coupling (RDc) generates spatiotemporal patterns, including two dynamic wave modes: traveling and standing waves. Although mode selection plays a substantial role in the spatiotemporal organization of living cell molecules, the mechanism for selecting each wave mode remains elusive. Here, we investigated a wave mode selection mechanism using Min waves reconstituted in artificial cells, emerged by the RDc of MinD and MinE. Our experiments and theoretical analysis revealed that the balance of membrane binding and dissociation from the membrane of MinD determines the mode selection of the Min wave. We successfully demonstrated that the transition of the wave modes can be regulated by controlling this balance and found hysteresis characteristics in the wave mode transition. These findings highlight a previously unidentified role of the balance between activators and inhibitors as a determinant of the mode selection of waves by RDc and depict an unexplored mechanism in intracellular spatiotemporal pattern formations.

Access to Document (DOI)

Characterization of the membrane penetration-enhancing peptide S19 derived from human syncytin-1 for the intracellular delivery of TAT-fused proteins

Suzuki M., Iwaki K., Kikuchi M., Fujiwara K., Doi N.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 586 63 - 67 2022.01

ISSN 0006291X

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high β-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the β-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a β-strand and that the subsequent parallel β-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

Access to Document (DOI)

Activation of a diluted E. coli cell-free transcription-translation system within liposomes by hypertonic concentration

Matsui Y., Akui T., Doi N., Fujiwara K.

STAR Protocols (STAR Protocols) 2 ( 4 ) 2021.12

We present a protocol for activating protein synthesis in liposomes encapsulating a diluted E. coli cell extract-based TX-TL (transcription-translation) system by hypertonic concentration. Protein expression is turned on in the liposome-encapsulated TX-TL system by simple treatment with a concentrated external solution. The expression of sfGFP is demonstrated here, but it can be applied to other proteins. This protocol can be applied to the development of artificial cells utilizing the switch-on mechanism to activate protein expression, responding to the outer environment. For complete details on the use and execution of this protocol, please refer to Akui et al. (2021).

Access to Document (DOI)

Transcription-translation of theEscherichia coligenome within artificial cells

Deyama T., Matsui Y., Chadani Y., Sekine Y., Doi N., Fujiwara K.

Chemical Communications (Chemical Communications) 57 ( 80 ) 10367 - 10370 2021.10

ISSN 13597345

Access to Document (DOI)

System concentration shift as a regulator of transcription-translation system within liposomes

Akui T., Fujiwara K., Sato G., Takinoue M., Nomura S.i.M., Doi N.

iScience (iScience) 24 ( 8 ) 2021.08

Biochemical systems in living cells have their optimum concentration ratio among each constituent element to maintain their functionality. However, in the case of the biochemical system with complex interactions and feedbacks among elements, their activity as a system greatly changes by the concentration shift of the entire system irrespective of the concentration ratio among elements. In this study, by using a transcription-translation (TX-TL) system as the subject, we illustrate the principle of the nonlinear relationship between the system concentration and the activity of the system. Our experiment and simulation showed that shifts of the system concentration of TX-TL by dilution and concentration works as a switch of activity and demonstrated its ability to induce a biochemical system to confer the permeability of small molecules to liposomes. These results contribute to the creation of artificial cells with the switch and provide an insight into the emergence of protocells.

Access to Document (DOI)

大学の枠を越えたオンライン生体分子デザインコンペティションの取り組み

7. 村田智,葛谷明紀,藤原慶,平順一,川又生吹,佐藤佑介,瀧ノ上正浩,野村M.慎一郎,角五彰,堀豊,安部桂太

工学教育 (日本工学教育協会) 69 ( 4 ) 31 - 39 2021.08

Switching ON of Transcription-Translation System Using GUV Fusion by Co-supplementation of Calcium with Long-Chain Polyethylene Glycol

Uwaguchi Y., Fujiwara K., Doi N.

ChemBioChem (ChemBioChem) 22 ( 13 ) 2319 - 2324 2021.07

ISSN 14394227

Giant unilamellar vesicles (GUVs) have been used as a material for bottom-up synthetic biology. However, due to the semi-permeability of the membrane, the need for methods to fuse GUVs has increased. To this aim, methods that are simple and show low leakage during fusion are important. In this study, we report a method of GUV fusion by a divalent cation (Ca2+) enhanced with a long chain polyethylene glycol (PEG20k). The methods showed significant GUV fusion without leakage of internal components of GUVs and maintained cell-free transcription-translation ability inside the GUVs without external supplementation of macromolecules. We demonstrate that the Ca-PEG method can be applied for switching ON of transcription-translation in GUVs in a fusion-dependent manner. The method developed here can be applied to extend bottom-up synthetic biology and molecular robotics that use GUVs as a chassis.

Access to Document (DOI)

A relationship between ntp and cell extract concentration for cell-free protein expression

Takahashi K., Sato G., Doi N., Fujiwara K.

Life (Life) 11 ( 3 ) 2021.03

The cell-free protein synthesis (CFPS) that synthesizes mRNA and protein from a template DNA has been featured as an important tool to emulate living systems in vitro. However, an obstacle to emulate living cells by CFPS is the loss of activity in the case of usage of high concentration cell extracts. In this study, we found that a high concentration of NTP which inhibits in the case of lower concentration cell extract restored the loss of CFPS activity using high concentration cell extracts. The NTP restoration was independent of the energy regeneration system used, and NTP derivatives also restored the levels of CFPS using a high concentration cell extract. Experiments using dialysis mode of CFPS showed that continuous exchange of small molecule reduced levels of NTP requirement and improved reaction speed of CFPS using the high concentration of cell extract. These findings contribute to the development of a method to understand the condition of living cells by in vitro emulation, and are expected to lead to the achievement of the reconstitution of living cells from biomolecule mixtures.

Access to Document (DOI)

Conformational equilibrium of MinE regulates the allowable concentration ranges of a protein wave for cell division

Kohyama S., Fujiwara K., Yoshinaga N., Doi N.

Nanoscale (Nanoscale) 12 ( 22 ) 11960 - 11970 2020.06

ISSN 20403364

The Min system for determining the cell division position at the center in bacteria has a unique character that uses a protein wave (Min wave) that emerges from its components (MinD and MinE). The Min wave emerges under the coupling of chemical reactions and molecular diffusions of MinDE and appears when the concentrations of MinD and MinE are similar. However, the nanoscale mechanism to determine their concentration ranges has remained elusive. In this study, by using artificial cells as a mimic of cells, we showed that the dominant MinE conformations determined the allowable concentration ranges for the emergence of the Min wave. Furthermore, the deletion of the membrane-binding region of MinE indicated that the region was essential for limiting the concentration ranges to be narrower. These findings illustrate a parameter tuning mechanism underlying complex molecular systems at the nanoscale for spatiotemporal regulation in living cells and show a possibility that the regulation of the equilibrium among molecular conformations can work as a switch for cell division.

Access to Document (DOI)

Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

Goto H., Kanai Y., Yotsui A., Shimokihara S., Shitara S., Oyobiki R., Fujiwara K., Watanabe T., Einaga Y., Matsumoto Y., Miki N., Doi N.

Lab on a Chip (Lab on a Chip) 20 ( 4 ) 852 - 861 2020.02

ISSN 14730197

We report the development of a micro total analysis system (μTAS) based on electrochemical measurements and dielectrophoretic sorting for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzymes immobilized on microbeads, together with their encoding DNA, can be measured with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. The 30 nL droplets containing microbead-displayed genes of enzymes with higher activity can then be recovered by dielectrophoretic sorting. Previously, we developed the NAD(P)H-measuring device containing the BDD electrode for high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. In this study, we fabricated an encapsulating device and a droplet-sorting device for nanoliter-size droplets, for the first time, and then combined these three devices to construct a μTAS for directed evolution of NAD(P)-dependent oxidoreductases. We confirmed that this system works by proof-of-principle experiments and successfully applied this system for screening of randomized libraries of NAD-dependent dehydrogenases.

Access to Document (DOI)

Dielectrophoretic sorting system for screening of NAD(P)-dependent oxidoreductases.

Kanai Y., Shimokihara S., Shitara S., Oyobiki R., Watanabe T., Einaga Y., Wakamatsu K., Matsumoto Y., Fujiwara K., Doi N.

21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 (21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017) 1383 - 1384 2020

Here we report the development of a flow injection analysis system based on electrochemical measurements and dielectrophoresis for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzyme which is immobilized on microbeads together with its encoding DNA can be measured with a boron-doped diamond (BDD) electrode in each compartment (~30 nL) of the microfluidic system, and the large droplets (~30 nL) containing genes of enzymes with higher activity can be recovered by dielectrophoretic sorting.

Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells

Kohyama S., Yoshinaga N., Yanagisawa M., Fujiwara K., Doi N.

eLife (eLife) 8 2019.07

The Min system, a system that determines the bacterial cell division plane, uses changes in the localization of proteins (a Min wave) that emerges by reaction-diffusion coupling. Although previous studies have shown that space sizes and boundaries modulate the shape and speed of Min waves, their effects on wave emergence were still elusive. Here, by using a microsized fully confined space to mimic live cells, we revealed that confinement changes the conditions for the emergence of Min waves. In the microsized space, an increased surface-tovolume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary for the stable generation of Min waves. Furthermore, we showed that the cell-sized space strictly limits parameters for wave emergence because confinement inhibits both the instability and excitability of the system. These results show that confinement of reaction-diffusion systems has the potential to control spatiotemporal patterns in live cells.

Access to Document (DOI)

Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system

Yoshida A., Kohyama S., Fujiwara K., Nishikawa S., Doi N.

Chemical Science (Chemical Science) 10 ( 48 ) 11064 - 11072 2019

ISSN 20416520

Spatiotemporal patterning is a fundamental mechanism for developmental differentiation and homeostasis in living cells. Because spatiotemporal patterns are based on higher-order collective motions of elements synthesized from genes, their behavior dynamically changes according to the element amounts. Thus, to understand life and use this process for material application, creation of artificial cells with time development of spatiotemporal patterning by changes of element levels is necessary. However, realizing coupling between spatiotemporal patterning and synthesis of elements in artificial cells has been particularly challenging. In this study, we established a system that can synthesize a patterning mechanism of the bacterial cell division plane (the so-called Min system) in artificial cells by modifying a defined protein expression system and demonstrated that artificial cells can show time development of spatiotemporal patterning similar to living cells. This system also allows generation and disappearance of spatiotemporal patterning, is controllable by a small molecule in artificial cells, and has the ability for application in cargo transporters. The system developed here provides a new material and a technique for understanding life, development of drug delivery tools, and creation of molecular robots.

Access to Document (DOI)

A dual system using compartmentalized partnered replication for selection of arsenic-responsive transcriptional regulator

Seaim Lwin Aye, Kei Fujiwara, Nobuhide Doi

The Journal of Biochemistry (Journal of Biochemistry) 164 ( 5 ) mvy055 - 348 2018.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

Engineering and design of genetic circuit in living cell is critical in accessing the beneficial application of synthetic biology. Directed evolution can avoid the complicated rational design of such circuit by screening or selecting functional circuit from non-functional one. Here, we proposed a positive–negative selection system for selecting a transcription factor that activates gene expression in response to arsenic in solution. First, we developed a whole cell biosensor for sensing arsenite in liquid using a regulator (ArsR) and a reporter (GFP), and evaluated its performance. Second, we developed a positive selection system for active ArsR using compartmentalized partnered replication that uses thermostable DNA polymerase as the reporter of activity. Third, we developed a negative selection system using sucrose-induced suicide gene SacB as the reporter for exclusion of inactive ArsR variants.

Access to Document (DOI)

　 Previous page - Next page 　