Published papers

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.

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).

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.

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.

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.

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.

Here we report an improved μTAS based on electrochemical measurements and microbeads sorting for screening of NAD(P)-dependent oxidoreductases. By using this system, we measured the activity of model enzymes immobilized on microbeads by detection of NADH with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. Then, we recovered the magnetic microbeads or the droplets containing genes of enzymes with higher activity by magnetophoretic or dielectrophoretic sorting, respectively. Our μTAS should be useful for directed evolution of various NAD(P)-dependent oxidoreductases with high activity and stability in the future.

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.

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.

Here we created artificial cells in which information of the genome of living cells is expressed by the elements encoded in the genome. We confirmed that the system works normally within artificial cells, which paves the way for reconstructing living cells from biomolecules.

Physiological processes in cells are performed efficiently without getting jammed although cytoplasm is highly crowded with various macromolecules. Elucidating the physical machinery is challenging because the interior of a cell is so complex and driven far from equilibrium by metabolic activities. Here, we studied the mechanics of in vitro and living cytoplasm using the particle-tracking and manipulation technique. The molecular crowding effect on cytoplasmic mechanics was selectively studied by preparing simple in vitro models of cytoplasm from which both the metabolism and cytoskeletons were removed. We obtained direct evidence of the cytoplasmic glass transition; a dramatic increase in viscosity upon crowding quantitatively conformed to the super-Arrhenius formula, which is typical for fragile colloidal suspensions close to jamming. Furthermore, the glass-forming behaviors were found to be universally conserved in all the cytoplasm samples that originated from different species and developmental stages; they showed the same tendency for diverging at the macromolecule concentrations relevant for living cells. Notably, such fragile behavior disappeared in metabolically active living cells whose viscosity showed a genuine Arrhenius increase as in typical strong glass formers. Being actively driven by metabolism, the living cytoplasm forms glass that is fundamentally different from that of its non-living counterpart.

Artificial lipid membranes have been utilized to understand the physical mechanisms of the deformation patterns of live cells. However, typical artificial membrane systems contain only dilute components compared to those in the cytoplasm of live cells. By using giant unilamellar liposomes containing dense protein solutions similar to those in live cells, we here reveal that viscosity derived from internal crowding affects the deformation patterns of lipid membranes. After hypertonic treatment, liposome deformation patterns transitioned from budding to tubing when the initial internal macromolecular concentrations were increased. Remarkably, instead of observing different transition concentrations between two species of macromolecules, the viscosity at the transition concentration was found to be similar. Further analyses clearly demonstrated that the internal viscosity affects the deformation patterns of lipid membranes induced by hypertonic treatment. These results indicate that the viscosity of the cytoplasm is a key factor in determining cell deformation, and suggest the association of a process involving dynamic instability, such as a viscous fingering phenomenon, during the determination of deformation patterns by hypertonic treatment.

In vitro transcription-translation systems (TX-TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX-TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription-translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC-MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX-TL using genome as a template.

Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

The cytosolic delivery of therapeutic proteins (e.g., antibodies or enzymes) by cell-penetrating peptides (CPPs), such as a human immunodeficiency virus-derived TAT peptide, is facilitated by fusogenic peptides (FPs). For instance, we recently demonstrated that an FP, B18, which is derived from a sea urchin gamete fusion protein, promotes endosomal escape of an enhanced green fluorescent protein (eGFP)-TAT fusion protein directly conjugated to it. However, the potential clinical use of FPs raises concerns because all conventional FPs are non-human-derived. To solve this problem, we have attempted to identify novel human-derived FPs from two human proteins, including a human sperm protein, IZUMO1, which is involved in gamete recognition and fusion, and a human endogenous retroviral envelope protein, Syncytin1, which is involved in placental morphogenesis. Partial peptides from the core domains of the abovementioned proteins were chosen as candidates to generate human-derived FPs. We prepared fusion proteins of these peptides with eGFP and TAT in Escherichia coli and observed the localization of these fusion proteins in HeLa cells using confocal microscopy. Our results suggested that a 19-residues peptide of Syncytin1 (positions 322–340), named S19, possessed strong intracellular uptake activities with no detectable cytotoxicity. In addition, we estimated the number of molecules that escaped from endosomes using a nuclear localization signal, suggesting that the S19 peptide stimulated the intracellular delivery of TAT-fused eGFP by ~90-fold. Furthermore, we confirmed that S19 promoted the intracellular delivery of eGFP to various human cell lines, including HeLa, A431, HepG2, and SK-N-SH. In addition, we demonstrated that not only eGFP but also SNAP-tag and β-galactosidase were delivered efficiently and retained their activities.

Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activity is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we developed a microfluidic total analysis system for high-throughput screening of NAD(P)-dependent oxidoreductases. This system comprises three microfluidic devices: an encapsulating device, a NADH measuring device, and a magnetic beads sorting device. We demonstrated the utility of each part of the system.