Shin-ichiro M. Nomura

Gene Expression within Cell-Sized Lipid Vesicles

Functional protein synthesis was observed in cell‐sized lipid vesicles following encapsulation of a gene‐expression system. Expression of rsGFP (red‐shifted green fluorescent protein) within individual vesicles was observed by fluorescence microscopy. Interestingly, at the early stage of the reaction, the expression efficiency inside the vesicle was remarkably higher than that in the solution outside. The synthesized rsGFP in individual vesicles is safe from attack by proteinase K added to the external aqueous solution. Studies on cell‐sized vesicles expressing protein should contribute to a fundamental understanding of certain aspects of living systems and will be useful for practical applications, such as the construction of microreactors.

Direct formation of proteo-liposomes by in vitro synthesis and cellular cytosolic delivery with connexin-expressing liposomes

Liposomes are widely utilized in molecular biology and medicine as drug carriers. Here we report a new liposome-cell interaction through connexins. Connexin 43 (Cx43)-containing liposomes were prepared by using cell-free transcription/translation systems with plasmids encoding Cx43 in the presence of liposome. The expressed membrane protein, Cx43, was directly constituted to the liposome membrane upon in vitro synthesis, leading to pure membrane protein-containing liposomes. The hydrophilic dye calcein was efficiently transferred from Cx43-expressing liposomes to cultured cells (Cx43 expressing). The transfer is significantly blocked in the presence of gap junction inhibitor (18beta-glycyrrhetinic acid) and in the case of the other type of connexin (Cx32)-expressing cell. The results show that calcein entered the cell through connexin-mediated pathway. Cx43 liposomes containing a soluble NEMO-binding domain peptide suppressed the intracellular signaling cascade IL-1beta-induced NF-kappaB activation and cyclooxygenase-2 expression in Cx43-expressing cells, confirming effective peptide transfer into the cell. This is a new method for direct cytosolic delivery of hydrophilic molecules.

Direct integration of cell‐free‐synthesized connexin‐43 into liposomes and hemichannel formation

Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated from Escherichia coli (the PURE system) in the presence of liposomes. Connexin‐43 (Cx43), which is a water‐insoluble integral membrane protein that forms a hexameric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. The addition of liposomes following protein expression (post‐translational presence of liposomes) did not lead to the integration of Cx43 into the liposome membranes. The amount of integrated Cx43 increased as the liposome concentration increased. The presence of liposomes did not influence the total amount of synthesized Cx43. The Cx43 integrated into the liposome membranes formed open membrane pores. These results indicate that the liposomes act in a chaperone‐like manner by preventing Cx43 from aggregating in solution, because of integration into the bilayer, and also by functionalization of the integrated Cx43 in the membrane. This is the first report that cell‐free‐synthesized water‐insoluble membrane protein is directly integrated with a uniform orientation as a functional oligomer into liposome membranes. This simple proteoliposome preparation procedure should be a valuable approach for structural and functional studies of membrane proteins.

Condensation of an additive-free cell extract to mimic the conditions of live cells.

The cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living cells, we developed a method to prepare additive-free, highly concentrated cell extracts. First, we verified the requirement for specific salts and buffers for functional cell-free translation extracts. The S30 fraction of Escherichia coli cell extracts without additives exhibited sufficient cell-free protein production. Next, we established a method to accumulate biological components by gradual evaporation by using a vacuum desiccator. Bovine serum albumin, green fluorescent protein, alkaline phosphatase, and a diluted reconstituted protein expression system were successfully condensed in their active forms using this method. The protein concentration of the prepared cell extract was elevated to 180 mg/mL, which was expected to contain approximately 260 mg/mL macromolecules, without the loss of cell-free protein expression activity. Such a condensed cell extract may be useful for investigating the differences between cells and reconstituted materials and may contribute to the development of methods to synthesize cells from cell extracts in the future.

Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds.

Micrometer-sized molecular robot changes its shape in response to signal molecules

An amoeba-like molecular robot changes its shape in response to sequence-designed DNA signal molecules. Rapid progress in nanoscale bioengineering has allowed for the design of biomolecular devices that act as sensors, actuators, and even logic circuits. Realization of micrometer-sized robots assembled from these components is one of the ultimate goals of bioinspired robotics. We constructed an amoeba-like molecular robot that can express continuous shape change in response to specific signal molecules. The robot is composed of a body, an actuator, and an actuator-controlling device (clutch). The body is a vesicle made from a lipid bilayer, and the actuator consists of proteins, kinesin, and microtubules. We made the clutch using designed DNA molecules. It transmits the force generated by the motor to the membrane, in response to a signal molecule composed of another sequence-designed DNA with chemical modifications. When the clutch was engaged, the robot exhibited continuous shape change. After the robot was illuminated with light to trigger the release of the signal molecule, the clutch was disengaged, and consequently, the shape-changing behavior was successfully terminated. In addition, the reverse process—that is, initiation of shape change by input of a signal—was also demonstrated. These results show that the components of the robot were consistently integrated into a functional system. We expect that this study can provide a platform to build increasingly complex and functional molecular systems with controllable motility.

A Communication interface using vesicles embedded with channel forming proteins in molecular communication

This paper describes design and empirically study of a communication interface in molecular communication. The communication interface hides the characteristics of the molecules during the propagation from the sender to a receiver to allow a generic transport of molecules independent of the characteristics. The authors of this paper propose a communication interface that uses a vesicle embedded with channel forming proteins. The channel forming proteins embedded with the vesicle form communication channels between the vesicle itself and a sender/receiver. The proposed molecular communication interface uses vesicle to hide the characteristics of the molecules and the communication channels to encapsulate/decapsulate the molecules into/from the vesicle. The vesicle receives the molecules from the sender through the communication channels formed between the vesicle itself and the sender. The molecules are encapsulated in the vesicle, and their characteristics are hidden by the vesicle structure during the propagation to a receiver. At the receiver, the molecules are transferred into the receiver through the communication channels formed between the vesicle itself and the receiver. The authors of this paper constructed the vesicles embedded with channel forming proteins and the molecules were successfully encapsulated into the vesicle. It was also demonstrated that molecules were transferred through the communication channels.

A molecular communication interface using liposomes with gap junction proteins

Molecular communication is an emerging communication paradigm that uses molecules as a communication medium. Molecular communication allows biological and artificially created nano- or cell-scale devices to communicate with each other. This paper proposes a molecular communication system that uses liposomes with gap junction proteins as an interface. A liposome acts as a container of information molecules, and information molecules propagate through gap junctions from a sender to a liposome and also from a liposome to a receiver

DNA cytoskeleton for stabilizing artificial cells

Significance Although liposomes and lipid droplets have been used for numerous applications, the fragility of the lipid membrane causes an unintentional collapse, which is problematic for advanced applications. To solve this problem, we constructed an artificial cytoskeleton with DNA nanotechnology (a DNA cytoskeleton). The DNA cytoskeleton is a DNA network formed underneath the membrane of positively charged lipids through electrostatic interactions without the need for special handling. The DNA cytoskeleton significantly improves mechanical stability and, therefore, confers tolerance against osmotic shock to liposomes like the cytoskeleton in live cells. Because of its biocompatibility and the easiness of implementing design changes, the DNA cytoskeleton could become a tool for great stabilizer of liposomes and lipid droplets. 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.

Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell–GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami) into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines) in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.