Endocytosis-Like Vesicle Fission Mediated by a Membrane-Expanding Molecular Machine Enables Virus Encapsulation for In Vivo Delivery.

Biological membranes are functionalized by membrane-associated protein machinery. Membrane-associated transport processes, such as endocytosis, represent a fundamental and universal function mediated by membrane-deforming protein machines, by which small biomolecules and even micrometer-size substances can be transported via encapsulation into membrane vesicles. Although synthetic molecules that induce dynamic membrane deformation have been reported, a molecular approach enabling membrane transport in which membrane deformation is coupled with substance binding and transport remains critically lacking. Here, we developed an amphiphilic molecular machine containing a photoresponsive diazocine core (AzoMEx) that localizes in a phospholipid membrane. Upon photoirradiation, AzoMEx expands the liposomal membrane to bias vesicles toward outside-in fission in the membrane deformation process. Cargo components, including micrometer-size M13 bacteriophages that interact with AzoMEx, are efficiently incorporated into the vesicles through the outside-in fission. Encapsulated M13 bacteriophages are transiently protected from the external environment and therefore retain biological activity during distribution throughout the body via the blood following administration. This research developed a molecular approach using synthetic molecular machinery for membrane functionalization to transport micrometer-size substances and objects via vesicle encapsulation. The molecular design demonstrated in this study to expand the membrane for deformation and binding to a cargo component can lead to the development of drug delivery materials and chemical tools for controlling cellular activities.

Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort.

Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip "fluorescent nucleic acid probe in droplets for bacterial sorting" (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.

Membrane Shape Dynamics-Based Analysis of the Physical Properties of Giant Unilamellar Vesicles Prepared by Inverted Emulsion and Hydration Techniques.

The giant unilamellar vesicle (GUV) is a basic model of the cell membrane that allows for the modulation and control of membrane shape dynamics, which play essential roles in the functions of living cell membranes. However, to properly use these artificial cell-like model systems, we need to understand their physical properties. GUV generation techniques are key technologies in the synthesis of artificial cell-like model systems. However, it is unclear whether GUVs produced by different techniques have the same physical properties. Here, we have investigated the physical properties of GUVs prepared by inverted emulsion and hydration techniques by examining the membrane shape deformation induced by external stimulation with a nonionic surfactant. We reveal differences in the spontaneous curvature of the membrane, the preferred differential area between the inner and outer leaflets of the membrane, and the edge tension of membrane pores between the GUVs prepared using the two distinct techniques.

DNA Origami Nanoplate-Based Emulsion with Nanopore Function.

Bio-inspired functional microcapsules have attracted increasing attention in many fields from physical/chemical science to artificial-cell engineering. Although particle-stabilised microcapsules are advantageous for their stability and functionalisation potential, versatile methods for their functionalisation are desired to expand their possibilities. This study reports a water-in-oil microdroplet stabilised with amphiphilic DNA origami nanoplates. By utilising DNA nanotechnology, DNA nanoplates were designed as a nanopore device for ion transportation and to stabilise the oil-water interface. Microscopic examination revealed the microcapsule formed by the accumulation of amphiphilic DNA nanoplates at the oil-water interface. Ion current measurements revealed the nanoplate pores functioned as channel to transport ions. These findings provide a general strategy for the programmable design of microcapsules to engineer artificial cells and molecular robots.

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10-30 µm GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates.

We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times.

Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

Invited for this month's cover picture are Dr. Masamune Morita, Dr. Kaoru Katoh and Dr. Naohiro Noda from the Biomedical Research Institute at the National Institute of Advanced Industrial Science and Technology (AIST, Japan). The cover picture shows direct monitoring of real-time activity of bacterial growth at the single-cell level inside giant unilamellar vesicles (GUVs); entrapped single bacterial cells are actively increasing to a great number of cells inside GUVs. This study shows new applications for GUVs, and can offer a novel tool for culturing bacteria in bacterial studies. Read the full text of their Communication at https://doi.org/10.1002/open.201800126.

Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

Bacterial cultivation techniques are classic, basic, and common processes used to characterize the physiological activity of bacteria in their environment. Owing to recent advances in bacterial cultivation techniques, the physiological activity of bacteria can be elucidated at the single-cell culture level. Here, we report a novel method to monitor the real-time activity of bacterial growth at the single-cell level inside giant unilamellar vesicles (GUVs). This method consists of two steps: 1) encapsulation of single bacteria in 1-33 pL scale GUVs and 2) immobilization of the GUVs on a planar lipid bilayer membrane on a glass surface. We directly observed single E. coli cells actively growing to a great number of cells inside GUVs. GUVs also protected the bacteria from external antibiotic compounds during prolonged cultivation for more than 24 h. This approach can be applied widely in the fields of biochemistry, biotechnology, microbiology, and synthetic biology.

DNA cytoskeleton for stabilizing artificial cells.

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.

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 µl) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions.

We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2 µL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems.

Generation of monodisperse cell-sized microdroplets using a centrifuge-based axisymmetric co-flowing microfluidic device.

We report an easy-to-use generation method of biologically compatible monodisperse water-in-oil microdroplets using a glass-capillary-based microfluidic device in a tabletop mini-centrifuge. This device does not require complicated microfabrication; furthermore, only a small sample volume is required in experiments. Therefore, we believe that this method will assist biochemical and cell-biological experiments.

Structure-dependent interactions of polyphenols with a biomimetic membrane system.

Polyphenols are naturally-occurring compounds, reported to be biologically active, and through their interactions with cell membranes. Although association of the polyphenols with the bilayer has been reported, the detailed mechanism of interaction is not yet well elucidated. We report on spatio-temporal real-time membrane dynamics observed in the presence of polyphenols. Two distinct membrane dynamics, corresponding to the two classes of polyphenols used, were observed. Flavonoids (epi-gallocatechin-3-gallate, gallocatechin, theaflavin and theaflavin-3-gallate) caused lipid membrane aggregation and rigidification. As simple structural modification through opening of the aromatic C-ring into an olefin bond, present in trans-stilbenes (resveratrol and picead), completely changed the membrane properties, increasing fluidity and inducing fluctuation. There were differences in the membrane transformations within the same class of polyphenols. Structure-dependent classification of membrane dynamics may contribute to a better understanding of the physicochemical mechanism involved in the bioactivity of polyphenols. In general, an increase in the number of hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the reactivity of the polyphenols. Most notable was the difference observed through a simple addition of the gallate group. Unraveling the importance of these polyphenols, at a functional group level further opens the key to tailored design of bioactive compounds as potential drug candidates.

Micrometer-size vesicle formation triggered by UV light.

Vesicle formation is a fundamental kinetic process related to the vesicle budding and endocytosis in a cell. In the vesicle formation by artificial means, transformation of lamellar lipid aggregates into spherical architectures is a key process and known to be prompted by e.g. heat, infrared irradiation, and alternating electric field induction. Here we report UV-light-driven formation of vesicles from particles consisting of crumpled phospholipid multilayer membranes involving a photoactive amphiphilic compound composed of 1,4-bis(4-phenylethynyl)benzene (BPEB) units. The particles can readily be prepared from a mixture of these components, which is casted on the glass surface followed by addition of water under ultrasonic radiation. Interestingly, upon irradiation with UV light, micrometer-size vesicles were generated from the particles. Neither infrared light irradiation nor heating prompted the vesicle formation. Taking advantage of the benefits of light, we successfully demonstrated micrometer-scale spatiotemporal control of single vesicle formation. It is also revealed that the BPEB units in the amphiphile are essential for this phenomenon.

Endo- and exocytic budding transformation of slow-diffusing membrane domains induced by Alzheimer's amyloid beta.

Cell-sized liposomes are a powerful tool for clarifying physicochemical mechanisms that govern molecular interactions. Herein, budding transformation of membrane domains was induced by amyloid beta peptides. The peptides increased the membrane viscosity as demonstrated by the Brownian motion of membrane domains. These results could aid in understanding the physicochemical mechanism of Alzheimer's disease.

The effect of oxysterols on the interaction of Alzheimer's amyloid beta with model membranes.

The interaction of amyloid beta (Aß) peptide with cell membranes has been shown to be influenced by Aß conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aß-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aß in membranes and the peptide-induced membrane dynamics in the presence of cholesterol and 7-ketocholesterol (7keto) or 25-hydroxycholesterol (25OH). Our results have indicated that oxysterols rendered membranes more sensitive to Aß, in contrast to role of cholesterol in inhibiting Aß/membrane interaction. We have demonstrated that two oxysterols had different impacts owing to distinct positions of the additional oxygen group in their structures. 7keto-containing cell-sized liposomes exhibited a high propensity toward association with Aß, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aß (Aß-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aß-40). These findings suggest the enhancing effect of oxysterols on interaction of Aß with membranes and contribute to clarify the harmful impact of cholesterol on Aß-induced neurotoxicity by means of its oxidation.

Membrane fusion and vesicular transformation induced by Alzheimer's amyloid beta.

Amyloid beta (Aß) peptides, produced through endo-proteolytic cleavage of amyloid precursor protein, are thought to be involved in the death of neural cells in Alzheimer's disease (AD). Although the mechanisms are not fully known, it has been suggested that disruption of cellular activity due to Aß interactions with the cell membrane may be one of the underlying causes. Here in, we have investigated the interaction between Aß-42 and biomimetic lipid membranes and the resulting perturbations in the lipid vesicles. We have shown that Aß oligomeric species localized closer to the membrane surface. Localization of the fibrillar species of Aß-42, although varied, was not as closely associated with the membrane surface. We have demonstrated that the presence of Aß-42 leads to an increase in membrane surface area, inducing lipid temporal vesicular transformation. Furthermore, we have unequivocally shown that Aß-peptides mediate membrane fusion. Although membrane fusion induced by Aß has been hypothesized/proposed, this is the first time it has been visually captured. This fusion may be one of the mechanisms behind the membrane increase in surface area and the resulting vesicular transformation. We have shown that the longer 'amyloidogenic' isoform causes vesicular transformation more readily, and has a higher membrane fusogenic potential than Aß-40. Although not core to this study, it is hugely interesting to observe the high agreement between membrane dynamics and the reported amyloidogenicity of the peptides and aggregation species opening up the potential role of vesicular dynamics for profiling and biosensing of Aß-induced neuro-toxicity.

Dynamic Morphological Changes Induced By GM1 and Protein Interactions on the Surface of Cell-Sized Liposomes.

It is important to understand the physicochemical mechanisms that are responsible for the morphological changes in the cell membrane in the presence of various stimuli such as osmotic pressure. Lipid rafts are believed to play a crucial role in various cellular processes. It is well established that Ctb (Cholera toxin B subunit) recognizes and binds to GM1 (monosialotetrahexosylganglioside) on the cell surface with high specificity and affinity. Taking advantage of Ctb-GM1 interaction, we examined how Ctb and GM1 molecules affect the dynamic movement of liposomes. GM1 a natural ligand for cholera toxin, was incorporated into liposome and the interaction between fluorescent Ctb and the liposome was analyzed. The interaction plays an important role in determining the various surface interaction phenomena. Incorporation of GM1 into membrane leads to an increase of the line tension leading to either rupture of liposome membrane or change in the morphology of the membrane. This change in morphology was found to be GM1 concentration specific. The interaction between Ctb-GM1 leads to fast and easy rupture or to morphological changes of the liposome. The interactions of Ctb and the glycosyl chain are believed to affect the surface and the curvature of the membrane. Thus, the results are highly beneficial in the study of signal transduction processes.

Ion permeation by a folded multiblock amphiphilic oligomer achieved by hierarchical construction of self-assembled nanopores.

A multiblock amphiphilic molecule 1, with a tetrameric alternating sequence of hydrophilic and hydrophobic units, adopts a folded structure in a liposomal membrane like a multipass transmembrane protein, and is able to transport alkali metal cations through the membrane. Hill's analysis and conductance measurements, analyzed by the Hille equation, revealed that the tetrameric assembly of 1 forms a 0.53 nm channel allowing for permeation of cations. Since neither 3, bearing flexible hydrophobic units and forming no stacked structures in the membrane, nor 2, a monomeric version of 1, is able to transport cations, the folded conformation of 1 in the membrane is likely essential for realizing its function. Thus, function and hierarchically formed higher-order structures of 1, is strongly correlated with each other like proteins and other biological macromolecules.