Molecular Transportation Conversion of Membrane Tension Using a Mechanosensitive Channel in Asymmetric Lipid-Protein Vesicles.

Giant lipid vesicles composed of a lipid bilayer form complex membrane structures and enzyme network reactions that can be used to construct well-defined artificial cell models based on microfluidic technologies and synthetic biology. As a different approach to cell-mimicking systems, we formed an asymmetric lipid-amphiphilic protein (oleosin) vesicle containing a lipid and an oleosin monolayer in the outer and inner leaflets, respectively. These asymmetric vesicles enabled the reconstitution and function of ß-barrel types of membrane proteins (OmpG) and the fission of vesicles stimulated by lysophospholipids. These applications combine the advantages of the high stability of lipids and oleosin leaflets in asymmetric lipid-oleosin vesicles. In this study, to evaluate the versatility of this asymmetric lipid-oleosin vesicle, the molecular transport of the mechanosensitive channel of large conductance (MscL) with an α-helix was evaluated by changing the tension of the asymmetric vesicle membrane with lysophospholipid. A nanopore of MscL assembled as a pentamer of MscLs transports small molecules of less than 10 kDa by sensing physical stress at the lipid bilayer. The amount and maximum size of the small molecules transported via MscL in the asymmetric lipid-oleosin vesicles were compared to those in the lipid vesicles. We revealed the existence of the C- and N-terminal regions (cytoplasmic side) of MscL on the inner leaflet of the asymmetric lipid-oleosin vesicles using an insertion direction assay. Furthermore, the change in the tension of the lipid-oleosin membrane activated the proteins in these vesicles, inducing their transportation through MscL nanopores. Therefore, asymmetric lipid-oleosin vesicles containing MscL can be used as substrates to study the external environment response of complex artificial cell models.

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements.

To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle-vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5-20 µm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30 min, 1 h, 2 h, and 4 h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3'-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches.

Formation of Cell-Sized Liposomes Incorporating a ß-Barrel-Structured Porin through Rehydration of a Phospholipid-Membrane Protein Dried Film.

Giant unilamellar vesicles (GUVs) integrated with membrane proteins (proteo-GUVs) are attractive tools for visualizing membrane protein functions such as enzyme reactions and molecular transportation. In the dehydration-rehydration method, one of the methods used to form proteo-GUVs, they are formed by using a dried film containing phospholipids and membrane proteins through rehydration with an alternating current electric field and a supporting gel. However, these methods make it difficult to form proteo-GUVs under physiological salt concentration and charged phospholipid conditions or carry the risk of gel contamination of lipid membranes. Therefore, proteo-GUVs formed by these rehydration methods may be harmful to membrane proteins. Here, we propose a method for the formation of proteo-GUVs containing physiological salt concentrations and negatively charged phospholipids that do not require an electric field and a supporting gel. To investigate the molecular transport of modified outer membrane protein G (OmpG), OmpG-giant unilamellar vesicles (GUVs) and OmpG-large unilamellar vesicles (LUVs) were formed. The structure and function of different mutants reconstituted into LUVs were evaluated by using circular dichroism spectroscopy and electrophysiological measurements. In addition, the molecular transport of OmpG in GUVs was evaluated by monitoring the Ca2+ influx into GUVs and fluorescent molecule leakage from GUVs through OmpG nanopores. We found that the amount of Ca2+ influx into GUVs through the OmpG nanopores depended on the pore size of OmpG. Our method for forming proteo-GUVs can be applied for the functional evaluation of ß-barrel porin and in biological sensors using ß-barrel porin.

Control of Reversible Formation and Dispersion of the Three Enzyme Networks Integrating DNA Computing.

The majority of biological reactions in the cytoplasm of living cells occur via enzymatic cascade reactions. To achieve efficient enzyme cascade reactions mimicking the proximity conditions of enzymes in the cytoplasm, the proximity of each enzyme, creating a high local concentration of proteins, has been recently investigated using the conjugation of synthetic polymer molecules, proteins, and nucleic acids. Although there have been methodologies reported for the complex formation and enhanced activity of cascade reactions due to the proximity of each enzyme using DNA nanotechnology, one pair of the enzyme (GOx and HRP) complex is only assembled by the mutual independence of various shapes of the DNA structure. This study reports the network formation of three enzyme complexes assembled by a triple-branched DNA structure as a unit, thus enabling the reversible formation and dispersion of the three enzyme complex networks using single-stranded DNA, RNA, and enzymes. It was found that the activities of the three enzyme cascade reactions in the enzyme-DNA complex network were controlled by formation and dispersion of the three enzyme complex networks, due to the proximity of each enzyme with the enzyme-DNA complex network. Furthermore, three micro RNA sequences for breast cancer biomarkers were successfully detected using an enzyme-DNA complex network integrated with DNA computing. Overall, the reversible formation and dispersion of the enzyme-DNA complex network through the external stimulation of biomolecules and DNA computing provide a novel platform for controlling the production amount, diagnosis, theranostics, and biological or environmental sensing.

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes.

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

Cell-sized asymmetric phospholipid-amphiphilic protein vesicles with growth, fission, and molecule transportation.

Lipid vesicles, which mimic cell membranes in structure and components, have been used to study the origin of life and artificial cell construction. A different approach to developing cell-mimicking systems focuses on the formation of protein- or polypeptide-based vesicles. However, micro-sized protein vesicles that are similar in membrane dynamics to the cell and that reconstitute membrane proteins are difficult to form. In this study, we generated cell-sized asymmetric phospholipid-amphiphilic protein (oleosin) vesicles that allow the reconstitution of membrane proteins and the growth and fission of vesicles. These vesicles are composed of a lipid membrane on the outer leaflet and an oleosin membrane on the inner leaflet. Further, we elucidated a mechanism for the growth and fission of cell-sized asymmetric phospholipid-oleosin vesicles by feeding phospholipid micelles. Our asymmetric phospholipid-oleosin vesicles with the advantages of the lipid leaflet and the protein leaflet will potentially promote understanding of biochemistry and synthetic biology.

Cu(II)-Triggered Ion Channel Properties of a 2,2'-Bipyridine-Modified Amphotericin B.

The development of stimuli-responsive synthetic channels that open and close in response to physical and chemical changes in the surrounding environment has attracted attention because of their potential bioapplications such as sensing, drug release, antibiotics, and molecular manipulation tools to control membrane transport in cells. Metal coordination is ideal as a stimulus for stimuli-responsive channels because it allows for reversible gating behavior through the addition and removal of metal ions and fine-tuning of channel structure through coordination geometry defined by the type of the metal ion and ligand. We have previously reported on transition metal-ion dependent ion permeability control of Amphotericin B (AmB) modified with a metal coordination site, 2,2'-bipyridine ligand (bpy-AmB). AmB is one of the polyene macrolide antibiotics, and it is known that the interaction between AmB and ergosterol molecules is required for AmB channel formation. In contrast, the Cu2+ coordination to the bpy moiety of bpy-AmB induces formation of Ca2+ ion-permeable channels in the ergosterol-free POPC membrane. However, the details of bpy-AmB properties such as channel stability, ion selectivity, pore size, and the effect of ergosterol on channel formation remain unclear. Here, we investigate bpy-AmB channels triggered by transition metal coordination in POPC or ergosterol-containing POPC liposomes using an HPTS assay, electrophysiological measurements, and time-resolved UV-vis spectral measurements. These analyses reveal that bpy-AmB channels triggered by Cu2+ ions are more stable and have larger pore sizes than the original AmB channels and enable efficient permeation of various cations. We believe that our channel design will lead to the construction of metal coordination-triggered synthetic ion channels.

Control of Enzyme Reaction Initiation inside Giant Unilamellar Vesicles by the Cell-Penetrating Peptide-Mediated Translocation of Cargo Proteins.

Cell-penetrating peptides (CPPs) play important roles in directly delivering biomolecules, such as DNA, proteins, and peptides, into living cells. In artificial lipid membranes, such as planar lipid bilayers, the direct membrane translocation of ß-galactosidase via Pep-1 (one of the CPPs) is dependent upon a voltage gradient between the inner and outer leaflets of the lipid membranes. Giant unilamellar vesicles (GUVs) with asymmetric lipid distributions, which are recently generated using microfluidic technologies, can be observed by optical microscopy. Therefore, interactions between CPPs and asymmetric lipid bilayers in different kinds of lipids and the translocation mechanism of proteins via CPPs into GUVs can be investigated at the level of a single asymmetric GUV. This CPP-based system for transporting proteins into GUVs will be applied to control the start of enzyme reactions in GUVs. This study aimed to explore efficient protein translocation into GUVs via CPP and demonstrate that enzymatic reactions start in GUVs using a CPP-mediated direct translocation. The interactions and the enzyme reactions between the CPP (Pep-1 or penetratin)-DNase I complexes and the asymmetric or symmetric GUV membranes containing the negatively or neutrally charged lipids were observed by confocal laser-scanning microscopy. The asymmetric GUVs containing phosphatidylserine (PS) in the inner leaflet showed efficient DNase I translocation into GUVs via penetratin. Finally, the formation of a cross-linked actin network was observed in asymmetric PS GUVs incubated with Pep-1-streptavidin complexes. The CPP-mediated direct translocation can contribute to developing artificial cell models with the capacity to control the initiation of enzymatic reactions.

A case of cerebral paragonimiasis misdiagnosed as eosinophilic granulomatosis with polyangiitis.

Paragonimiasis is a parasitic disease caused by Paragonimus westermani infection, and migration to the brain results in cerebral paragonimiasis. Cerebral paragonimiasis is now extremely rare, but a few cases are still reported. A 48-year-old Japanese woman presented with right-hand convulsion, right-hand numbness, sputum, and fatigue. Chest computed tomography demonstrated multiple nodular lesions, and head computed tomography revealed a hemorrhagic lesion in the left motor cortex. Magnetic resonance imaging revealed multiple small ring-shaped lesions with surrounding edema. Laboratory evaluation demonstrated peripheral eosinophilia. We considered eosinophilic granulomatosis with polyangiitis and started steroid treatment as a diagnostic therapy since we wanted to avoid cerebral lesion biopsy if possible. However, the patient underwent craniotomy surgery after steroid treatment for four months because a new intracerebral mass lesion had appeared. Trematode eggs were detected in the sample, and the final diagnosis was cerebral paragonimiasis. The patient was successfully treated with praziquantel. Cerebral paragonimiasis is extremely rare but should be considered in the differential diagnosis if atypical intracranial hemorrhage and peripheral eosinophilia are observed.

Formation and function of OmpG or OmpA-incorporated liposomes using an in vitro translation system.

Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a ß-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from Escherichia coli (E. coli). Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes with various lipid compositions was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.

Formation of Giant Lipid Vesicle Containing Dual Functions Facilitates Outer Membrane Phospholipase.

Giant lipid vesicles are used to study artificial cell models, as well as the encapsulation of biomolecules, and the reconstitution of membrane proteins on these vesicles. Recently, complex reactions in giant vesicles have been controlled by reconstituting numerous kinds of biomolecules. However, it is challenging to generate giant lipid vesicles containing a diverse set of proteins at concentrations sufficient to ensure proper functioning. Here, we describe an artificial cell model showing dual functions of small molecule transportation and small vesicle budding, using a dual functional membrane protein (transportation and phosphatase activity) called the outer membrane phospholipase (OmpLA). To the best of our knowledge, we have revealed for the first time the transportation of ions or small molecules through OmpLA on the charged lipid bilayer. The lipid composition controlled the orientation of OmpLA through proteinase K digestion. Finally, OmpLA enzyme activity of phospholipid hydrolysis caused the budding of small vesicles.

Investigation of Fusion between Nanosized Lipid Vesicles and a Lipid Monolayer Toward Formation of Giant Lipid Vesicles with Various Kinds of Biomolecules.

We determined the properties of fusion between large unilamellar vesicles (LUVs) and the lipid monolayer by measuring the fluorescence intensity of rhodamine-conjugated phospholipids in cell-sized lipid vesicles. The charge of LUVs (containing cationic lipids) and lipid droplets (containing anionic lipids) promoted lipid membrane fusion. We also investigated the formation of cell-sized lipid vesicles with asymmetric lipid distribution using this fusion method. Moreover, cell-sized asymmetric ganglioside vesicles can be generated from the planar lipid bilayer formed at the interface between the lipid droplets with/without LUVs containing ganglioside. The flip-flop dynamics of ganglioside were observed on the asymmetric ganglioside vesicles. This fusion method can be used to form asymmetric lipid vesicles with poor solubility in n-decane or lipid vesicles containing various types of membrane proteins for the development of complex artificial cell models.

Highly sensitive VOC detectors using insect olfactory receptors reconstituted into lipid bilayers.

This paper reports a volatile organic compound (VOC) sensor based on olfactory receptors that were reconstituted into a lipid bilayer and used in a specifically designed gas flow system for rapid parts per billion (ppb)-level detection. This VOC sensor achieves both rapid detection and high detection probability because of its gas flow system and array design. Specifically, the gas flow system includes microchannels and hydrophobic microslits, which facilitate both the introduction of gas into the droplet and droplet mixing. We installed this system into a parallel lipid bilayer device and subsequently demonstrated parts per billion-level (0.5 ppb) detection of 1-octen-3-ol in human breath. Therefore, this system extends the various applications of biological odorant sensing, including breath diagnosis systems and environmental monitoring.

Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded Microperforated Sheet in Aqueous Solution.

This paper describes a method for a bilayer lipid membrane (BLM) formation using a perforated sheet along with an open chamber. Microscopic observation of the formed membrane showed a typical droplet interface bilayer. We proved that the formed membrane was a BLM based on electrical measurements of the membrane protein α-hemolysin, which produces nanopores in BLMs. Unlike the conventional approach for BLM formation based on the droplet contact method, this method provides aqueous surfaces with no organic solvent coating layer. Hence, this method is suitable for producing BLMs that facilitate the direct addition of chemicals into the aqueous phase.

A Lipid-Bilayer-On-A-Cup Device for Pumpless Sample Exchange.

Lipid-bilayer devices have been studied for on-site sensors in the fields of diagnosis, food and environmental monitoring, and safety/security inspection. In this paper, we propose a lipid-bilayer-on-a-cup device for serial sample measurements using a pumpless solution exchange procedure. The device consists of a millimeter-scale cylindrical cup with vertical slits which is designed to steadily hold an aqueous solution and exchange the sample by simply fusing and splitting the solution with an external solution. The slit design was experimentally determined by the capabilities of both the retention and exchange of the solution. Using the optimized slit, a planar lipid bilayer was reconstituted with a nanopore protein at a microaperture allocated to the bottom of the cup, and the device was connected to a portable amplifier. The solution exchangeability was demonstrated by observing the dilution process of a blocker molecule of the nanopore dissolved in the cup. The pumpless solution exchange by the proposed cup-like device presents potential as a lipid-bilayer system for portable sensing applications.

Rapid and Resilient Detection of Toxin Pore Formation Using a Lipid Bilayer Array.

An artificial cell membrane is applied to study the pore formation mechanisms of bacterial pore-forming toxins for therapeutic applications. Electrical monitoring of ionic current across the membrane provides information on the pore formation process of toxins at the single pore level, as well as the pore characteristics such as dimensions and ionic selectivity. However, the efficiency of pore formation detection largely depends on the encounter probability of toxin to the membrane and the fragility of the membrane. This study presents a bilayer lipid membrane array that parallelizes 4 or 16 sets of sensing elements composed of pairs of a membrane and a series electrical resistor. The series resistor prevents current overflow attributed to membrane rupture, and enables current monitoring of the parallelized membranes with a single detector. The array system shortens detection time of a pore-forming protein and improves temporal stability. The current signature represents the states of pore formation and rupture at respective membranes. The developed system will help in understanding the toxic activity of pore-forming toxins.

Development of Artificial Cell Models Using Microfluidic Technology and Synthetic Biology.

Giant lipid vesicles or liposomes are primarily composed of phospholipids and form a lipid bilayer structurally similar to that of the cell membrane. These vesicles, like living cells, are 5-100 µm in diameter and can be easily observed using an optical microscope. As their biophysical and biochemical properties are similar to those of the cell membrane, they serve as model cell membranes for the investigation of the biophysical or biochemical properties of the lipid bilayer, as well as its dynamics and structure. Investigation of membrane protein functions and enzyme reactions has revealed the presence of soluble or membrane proteins integrated in the giant lipid vesicles. Recent developments in microfluidic technologies and synthetic biology have enabled the development of well-defined artificial cell models with complex reactions based on the giant lipid vesicles. In this review, using microfluidics, the formations of giant lipid vesicles with asymmetric lipid membranes or complex structures have been described. Subsequently, the roles of these biomaterials in the creation of artificial cell models including nanopores, ion channels, and other membrane and soluble proteins have been discussed. Finally, the complex biological functions of giant lipid vesicles reconstituted with various types of biomolecules has been communicated. These complex artificial cell models contribute to the production of minimal cells or protocells for generating valuable or rare biomolecules and communicating between living cells and artificial cell models.

Molecular and Functional Analysis of Pore-Forming Toxin Monalysin From Entomopathogenic Bacterium Pseudomonas entomophila.

Pseudomonas entomophila is a highly pathogenic bacterium that infects insects. It is also used as a suitable model pathogen to analyze Drosophila's innate immunity. P. entomophila's virulence is largely derived from Monalysin, a ß-barrel pore-forming toxin that damages Drosophila tissues, inducing necrotic cell death. Here we report the first and efficient purification of endogenous Monalysin and its characterization. Monalysin is successfully purified as a pro-form, and trypsin treatment results in a cleaved mature form of purified Monalysin which kills Drosophila cell lines and adult flies. Electrophysiological measurement of Monalysin in a lipid membrane with an on-chip device confirms that Monalysin forms a pore, in a cleavage-dependent manner. This analysis also provides a pore-size estimate of Monalysin using current amplitude for a single pore and suggests lipid preferences for the insertion. Atomic Force Microscope (AFM) analysis displays its structure in a solution and shows that active-Monalysin is stable and composed of an 8-mer complex; this observation is consistent with mass spectrometry data. AFM analysis also shows the 8-mer structure of active-Monalysin in a lipid bilayer, and real-time imaging demonstrates the moment at which Monalysin is inserted into the lipid membrane. These results collectively suggest that endogenous Monalysin is indeed a pore-forming toxin composed of a rigid structure before pore formation in the lipid membrane. The endogenous Monalysin characterized in this study could be a desirable tool for analyzing host defense mechanisms against entomopathogenic bacteria producing damage-inducing toxins.

A pumpless solution exchange system for nanopore sensors.

This paper proposes a nanopore-based sensor exploiting the solution exchange of a droplet-based lipid bilayer driven by a superabsorbent polymer. Biological nanopores are candidates for use in portable sensors because of their potential to recognize and detect single molecules. One of the current challenges in the development of portable nanopore sensors is the inability to achieve continuous detection. To achieve continuous detection, we have exploited the suction force of a superabsorbent polymer to drive the continuous microfluidic flow required to wash the analyte out of the droplet. The superabsorbent polymer drives the microfluidic flow without electricity, and the developed solution exchange system remains compact. To demonstrate solution exchange in the droplet containing the lipid bilayer, the concentration of heptakis(6-O-sulfo)-ß-cyclodextrin was monitored in a time-dependent manner using α-hemolysin nanopores. A reduction in the concentration, attributable to solution exchange, was successfully observed. We believe that the proposed system will increase the portability and usability of nanopore sensors.

Construction of a Biohybrid Odorant Sensor Using Biological Olfactory Receptors Embedded into Bilayer Lipid Membrane on a Chip.

This paper describes an odorant sensor based on mosquito olfactory receptors (ORs) that is sensitive to the volatile organic compound octenol. The ORs and OR coreceptors were reconstructed in the lipid bilayer membrane in a chamber device equipped with electrodes. Using this odorant sensor, we obtained ion current changes caused by specific OR responses to octenol. We installed the odorant sensor into a mobile robot and succeeded in the demonstration of coupling octenol gas detection and robot actuation. We believe that this biohybrid odorant sensing system will be a key technology for future artificial olfaction.