Membranous and Membraneless Interfaces-Origins of Artificial Cellular Complexity.

Living cell architecture is based on the concept of micro-compartmentation at different hierarchical levels [...].

Microscopic Observation of Membrane Fusion between Giant Liposomes and Baculovirus Budded Viruses Activated by the Release of a Caged Proton.

Baculovirus (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) is an envelope virus possessing a fusogenic protein, GP64, which can be activated under weak acidic conditions close to those in endosomes. When the budded viruses (BVs) are bathed at pH 4.0 to 5.5, they can bind to liposome membranes with acidic phospholipids, and this results in membrane fusion. In the present study, using the caged-proton reagent 1-(2-nitrophenyl)ethyl sulfate, sodium salt (NPE-caged-proton), which can be uncaged by irradiation with ultraviolet light, we triggered the activation of GP64 by lowering the pH and observed membrane fusion on giant liposomes (giant unilamellar vesicles, GUVs) by visualizing the lateral diffusion of fluorescence emitted from a lipophilic fluorochrome (octadecyl rhodamine B chloride, R18) that stained viral envelopes of BVs. In this fusion, entrapped calcein did not leak from the target GUVs. The behavior of BVs prior to the triggering of membrane fusion by the uncaging reaction was closely monitored. BVs appeared to accumulate around a GUV with DOPS, implying that BVs preferred phosphatidylserine. The monitoring of viral fusion triggered by the uncaging reaction could be a valuable tool for revealing the delicate behavior of viruses affected by various chemical and biochemical environments.

Self-emergent vortex flow of microtubule and kinesin in cell-sized droplets under water/water phase separation.

By facilitating a water/water phase separation (w/wPS), crowded biopolymers in cells form droplets that contribute to the spatial localization of biological components and their biochemical reactions. However, their influence on mechanical processes driven by protein motors has not been well studied. Here, we show that the w/wPS droplet spontaneously entraps kinesins as well as microtubules (MTs) and generates a micrometre-scale vortex flow inside the droplet. Active droplets with a size of 10-100 µm are generated through w/wPS of dextran and polyethylene glycol mixed with MTs, molecular-engineered chimeric four-headed kinesins and ATP after mechanical mixing. MTs and kinesin rapidly created contractile network accumulated at the interface of the droplet and gradually generated vortical flow, which can drive translational motion of a droplet. Our work reveals that the interface of w/wPS contributes not only to chemical processes but also produces mechanical motion by assembling species of protein motors in a functioning manner.

Advanced hybridoma technology for selective production of high-affinity monoclonal antibodies through B-cell receptors.

In general, it is difficult to raise novel monoclonal antibodies against relatively low-molecular weight antigen, and particularly those with high homology for the mouse protein. The optimized B-cell targeting (BCT) technique can overcome this limitation. The point of this advanced technology is the selection of sensitized B lymphocytes by the antigen through B-cell receptors (BCRs). This strict selection by specific and strong interaction between antigen and antibody enables the efficient production of monoclonal antibodies with high specificity and affinity. It also offers the condensation of sensitized target B lymphocytes to selectively generate hybridoma cells secreting desired monoclonal antibodies. In this study, several kinds of biotinylated human myoglobin (hMyo) were prepared to select sensitized B lymphocytes via BCRs. Biotinylated hMyo prepared by a 3.75- and 7.5-fold molar excess of N-hydroxysuccinimide (NHS)-biotin provided high antigenicity of 68-88%. B lymphocytes selected by these biotinylated antigens had an ELISA-positive rate >17 times higher than that with usual biotinylated antigen. Monoclonal antibodies generated by the optimized BCT technology by preselecting sensitized B lymphocytes with the target antigen were identified to specifically recognize lower antigenic epitopes in hMyo with high affinity, while this would be impossible by the polyethylene glycol (PEG) method. Furthermore, combination of these high-affinity monoclonal antibodies gave the best binding rate in an epitope binning assay. These outcomes could be attributed to the unique characteristic that BCRs on sensitized B lymphocytes themselves can select the target epitopes in the antigen. The BCRs may act as a strict sensor of B lymphocytes to precisely select the target epitopes, even though the number of immunized B lymphocytes is low.

Water-in-Water Droplets Selectively Uptake Self-Assembled DNA Nano/Microstructures: a Versatile Method for Purification in DNA Nanotechnology.

DNA is an excellent material for constructing self-assembled nano/microstructures. Owing to the widespread use of DNA as a building block in laboratories and industry, it is desirable to increase the efficiency of all steps involved in producing self-assembled DNA structures. One of the bottlenecks is the purification required to separate the excess components from the target structures. This paper describes a purification method based on the fractionation by water-in-water (W/W) droplets composed of phase-separated dextran-rich droplets in a polyethylene glycol (PEG)-rich continuous phase. The dextran-rich droplets facilitate the selective uptake of self-assembled DNA nano/microstructures and allow the separation of the target structure. This study investigates the ability to purify DNA origami, DNA hydrogels, and DNA microtubes. The W/W-droplet fractionation allows the purification of structures of a broad size spectrum without changes to the protocol. By quantifying the activity of deoxyribozyme-modified DNA origami after W/W-droplet purification, this study demonstrates that this method sufficiently preserves the accessibility to the surface of a functional DNA nanostructure. It is considered that the W/W-droplet fractionation could become one of the standard methods for the purification of self-assembled DNA nano/microstructures for biomedical and nanotechnology applications owing to its low cost and simplicity.

Mechanism of Budded Virus Envelope Fusion into a Planar Bilayer Lipid Membrane on a SiO2 Substrate.

Artificial planar bilayer lipid membranes (BLMs) are simple models of cellular systems under physically and chemically controlled conditions, and they have been used to investigate membrane protein activity. Baculovirus-budded virus (BV) systems can express recombinant membrane proteins. In this study, aiming for membrane protein reconstitution, we examined the fusion of BVs containing recombinant membrane proteins into artificial planar BLMs on a Si microwell substrate. BV fusion with the BLMs depended on the pH of the solution, and it was enhanced at lower pH. Based on fluorescence recovery after photobleaching (FRAP) measurement, the fusion state of BVs was evaluated, and full fusion at low pH was confirmed. The fluorescent labeling the membrane proteins was also observed in the freestanding part of the BLMs as well as in the supported part. These results demonstrate the effectiveness of BLMs as a platform to examine detailed fusion dynamics of BVs. Furthermore, this study revealed that the fusion of BVs is a promising method for reconstituting membrane proteins to artificial freestanding BLMs for the development of biodevices with which we can examine membrane protein activity.

Electrofusion of cells with different diameters by generating asymmetrical electric field in the microwell array.

This paper reports a superiority of the asymmetric electric field formed in the rectangle microwell array for the electrofusion of splenocytes and myeloma cells with different diameters. The upper substrate with microband electrodes was mounted on the lower substrate with the microwell array. Two electrodes were arranged at the both sides of the microwells on the bottom surface. An attractive force of positive dielectrophoresis was employed to capture splenocytes with smaller diameter and myeloma cells with larger diameter at the right and left of microwells by applying AC electric field. The splenocytes and myeloma cells were fused by the asymmetric electric field that was generated in the microwells by applying DC electric pulse to the bottom electrode at the right side. The asymmetric field could allow to the formation of small openings on the membrane for the fusion of smaller splenocytes by experiencing higher field and the suppression for the disruption of larger myeloma cells by experiencing lower field.

Polymerization/depolymerization of actin cooperates with the morphology and stability of cell-sized droplets generated in a polymer solution under a depletion effect.

Intercellular fluids in living organisms contain high concentrations of macromolecules such as nucleic acid and protein. Over the past few decades, several studies have examined membraneless organelles in terms of liquid-liquid phase separation. These studies have investigated aggregation/attraction among a rich variety of biomolecules. Here, we studied the association between the polymerization/depolymerization of actin, interconversion between monomeric (G-actin) and filamentous states (F-actin), and water/water phase separation in a binary polymer solution using polyethylene glycol (PEG) and dextran (DEX). We found that actin, which is a representative cytoskeleton, changes its distribution in a PEG/DEX binary solution depending on its polymerization state: monomeric G-actin is distributed homogeneously throughout the solution, whereas polymerized F-actin is localized only within the DEX-rich phase. We extended our study by using fragmin, which is a representative actin-severing and -depolymerizing factor. It took hours to restore a homogeneous actin distribution from localization within the DEX-rich phase, even with the addition of fragmin in an amount that causes complete depolymerization. In contrast, when actin that had been depolymerized by fragmin in advance was added to a solution with microphase-separation, F-actin was found in DEX-rich phase droplets. The micro-droplets tended to deform into a non-spherical morphology under conditions where they contained F-actin. These findings suggest that microphase-separation is associated with the dynamics of polymerization and localization of the actin cytoskeleton. We discuss our observations by taking into consideration the polymer depletion effect.

Conformation-specific monoclonal antibodies recognizing the native structure of G protein-coupled receptor (GPCR).

It is quite difficult to generate monoclonal antibodies that recognize the three-dimensional structures of the antigens of interest. To address this limitation, we developed a new hybridoma technology termed "optimized stereospecific targeting (SST)". Here we aimed at generating stereospecific monoclonal antibodies against a G protein-coupled receptor (GPCR). The optimized SST technique enabled the efficient production of conformation-specific monoclonal antibodies against human corticotropin-releasing hormone receptor 1 (huCRHR1). Hybridoma cells secreting stereospecific monoclonal antibodies were selectively cloned by a limiting dilution method and the target monoclonal antibodies were purified by protein A column chromatography. They specifically cross-reacted with native huCRHR1 expressed on the surface of CHO cells, whereas they showed no affinity for MDA-MB-231 cancer cells, which abundantly express EphA2 on the cell surface. Furthermore, immunofluorescence analysis revealed that treatment of huCRHR1-expressing CHO cells with 4% paraformaldehyde led to a decrease in the affinity of purified monoclonal antibodies for intact huCRHR1 on the cell surface. In addition, purified monoclonal antibodies showed no cross-reactivity with huCRHR1 expressed on Sf9 insect cells. These results strongly suggest that monoclonal antibodies generated by the optimized SST technique feature specific binding to the intact form of the target GPCR on mammalian cells.

Self-Emergent Protocells Generated in an Aqueous Solution with Binary Macromolecules through Liquid-Liquid Phase Separation.

Recently, liquid-liquid phase separation (LLPS) has attracted considerable attention among researchers in the life sciences as a plausible mechanism for the generation of microstructures inside cells. LLPS occurs through multiple nonspecific interactions and does not always require a lock-and-key interaction with a binary macromolecular solution. The remarkable features of LLPS include the non-uniform localization and concentration of solutes, resulting in the ability to isolate certain chemical systems and thereby parallelize multiple chemical reactions within the limited space of a living cell. We report that, by using the macromolecules, poly(ethylene glycol) (PEG) and dextran, that exhibit LLPS in an aqueous solution, cell-sized liposomes are spontaneously formed therein in the presence of phospholipids. In this system, LLPS is generated through the depletion effect of macromolecules. The results showed that cell-like microdroplets entrapping DNA wrapped by a phospholipid layer emerge in a self-organized manner.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

Nonspecific characteristics of macromolecules create specific effects in living cells.

Recently, the important role of microphase separation in living cells has been attracting considerable interest in relation to cell organization and function. For example, many studies have focused on liquid-liquid phase separation (LLPS) as a very plausible mechanism for the presence of membraneless organelles. To confirm the role of phase separation in living cells, experimental studies on models and/or reconstructed systems are needed. In this short review, we discuss current paradigms of LLPS and provide some example "review data" to demonstrate particular points relating to the specific localization of biological macromolecules like DNAs and actin proteins with spontaneous domain formation in microdroplets emerging in an aqueous two-phase system (ATPS) (we use polyethylene glycol (PEG)/dextran (DEX)-a binary polymer solution). We also suggest that phase separation and transition may play basic roles in regulation of the biochemical reactivity of individual long genomic DNAs.

Membrane fusion and infection abilities of baculovirus virions are preserved during freezing and thawing in the presence of trehalose.

Budded viruses (BVs) of baculovirus such as Autographa californica nucleopolyhedrovirus (AcNPV) have recently been studied as biological nanomaterials, and methods for their longer-term storage without deterioration would be desirable. The cryopreservation of virions with a naturally occurring saccharide like trehalose as a cryoprotectant is known to be useful for maintaining the viral structure and function. In this study, we examined how useful trehalose is as protectant for BV cryopreservation during repeated freeze-thaw cycles: 1) membrane fusion between liposomes (multilamellar vesicles, MLVs) and BVs, 2) infection of insect culture cells (Sf9 cells) by RFP-expressing BVs, and 3) morphologies of these BVs were investigated by fluorescent dequenching assay, fluorescence microscopy, and transmission electron microscopy (TEM), respectively. The results suggest that the BVs deteriorate in quality with each freeze-thaw cycle, and this deterioration can be diminished with the use of trehalose to an extent similar to that seen with storage on ice.

B-cell receptor-based multitargeting method for simultaneous production of novel multiple monoclonal antibodies.

Until now, various kinds of monoclonal antibodies have been raised against many antigens. Nevertheless, the production of these monoclonal antibodies was usually limited to only one antigen. If simultaneous generation of monoclonal antibodies against multiple antigens were available at one time, we could reduce not only laborious work, but also experimental animals. Here, we developed a multitargeting (MT) method that enables simultaneous production of monoclonal antibodies against multiple antigens on the basis of strict selection of sensitized B lymphocytes by the target antigens via B-cell receptors. After immunization using multiple antigens, monoclonal antibodies against four different antigens containing lower antigenic one were successfully generated only in one experiment. At maximum, more than 90 % of ELISA-positive wells to hybridoma-positive ones was obtained by this advanced technology. This must be attributed to strict selection of sensitized B lymphocytes by different antigens. In the MT method, sensitized B lymphocytes were selected by means of each desired antigen regardless of their antigenic differences. Selective fusion of B cell-myeloma cell complexes by electrical pulses was also of critical importance for efficient generation of hybridoma cells secreting desired monoclonal antibodies. This study strongly suggests that simultaneous production of novel monoclonal antibodies directed against multiple antigens of interest by the MT method can be feasible.

Future perspectives of therapeutic monoclonal antibodies.

Attention to therapeutic monoclonal antibodies has been dramatically increasing year by year. Their highly specific targeting of antigens can provide very effective medical treatment, and the advent of molecular-targeting medicine is allowing development of a new generation of therapeutic agents. However, there is one critical obstacle to overcome. Most of the established therapeutic monoclonal antibodies have specificity for the primary structures of target antigens, although all proteins harbor original native intact structures for their own specific functions. Stereo-specific monoclonal antibodies recognizing conformational structures of target antigens may thus offer a markedly more versatile approach. Their application may change the very concepts underlying use of therapeutic antibodies.

Aqueous/Aqueous Micro Phase Separation: Construction of an Artificial Model of Cellular Assembly.

To artificially construct a three-dimensional cell assembly, we investigated the availability of long-duration microdroplets that emerged near a critical point in an aqueous two-phase system (ATPS) with the hydrophilic binary polymers, polyethylene glycol (PEG), and dextran (DEX), as host containers. We found that erythrocytes (horse red blood cells; RBCs) and NAMRU mouse mammary gland epithelial cells (NMuMG cells) were completely and spontaneously entrapped inside DEX-rich microdroplets. RBCs and NMuMG cells were located in the interior and at the periphery of the droplets at PEG/DEX = 5%:5%. In contrast, the cells exhibited opposite localizations at PEG/DEX = 10%:5%, where, interestingly, NMuMG cells apparently assembled to achieve cell adhesion. We simply interpreted such specific localizations by considering the alternative responses of these cells to the properties of the PEG/DEX interfaces with different gradients in polymer concentrations.

Specific Spatial Localization of Actin and DNA in a Water/Water Microdroplet: Self-Emergence of a Cell-Like Structure.

The effect of binary hydrophilic polymers on a pair of representative bio-macromolecules in a living cell has been examined. The results showed that these bio-macromolecules exhibited specific localization in cell-sized droplets that were spontaneously formed through water/water microphase segregation under crowding conditions with coexisting polymers. In these experiments, a simple binary polymer system with poly(ethylene glycol) (PEG) and dextran (DEX) was used. Under the conditions of microphase segregation, DNA was entrapped within cell-sized droplets rich in DEX. Similarly, F-actin, linearly polymerized actin, was entrapped specifically within microdroplets rich in DEX, whereas G-actin, a monomeric actin, was distributed evenly inside and outside these droplets. This study has been extended to a system with both F-actin and DNA, and it was found that DNA molecules were localized separately from aligned F-actin proteins to create microdomains inside microdroplets, reflecting the self-emergence of a cellular morphology similar to a stage of cell division.

Opposite effect of polyamines on In vitro gene expression: Enhancement at low concentrations but inhibition at high concentrations.

BACKGROUND: Polyamines have various biological functions including marked effects on the structure and function of genomic DNA molecules. Changes in the higher-order structure of DNA caused by polyamines are expected to be closely related to genetic activity. To clarify this issue, we examined the relationship between gene expression and the higher-order structure of DNA under different polyamine concentrations. PRINCIPAL FINDINGS: We studied the effects of polyamines, spermidine SPD(3+) and spermine SP(4+), on gene expression by a luciferase assay. The results showed that gene expression is increased by ca. 5-fold by the addition of SPD(3+) at 0.3 mM, whereas it is completely inhibited above 2 mM. Similarly, with SP(4+), gene expression is maximized at 0.08 mM and completely inhibited above 0.6 mM. We also performed atomic force microscopy (AFM) observations on DNA under different polyamine concentrations. AFM revealed that a flower-like conformation is generated at polyamine concentrations associated with maximum expression as measured by the luciferase assay. On the other hand, DNA molecules exhibit a folded compact conformation at polyamine concentrations associated with the complete inhibition of expression. Based on these results, we discuss the plausible mechanism of the opposite effect, i.e., enhancement and inhibition, of polyamines on gene expression. CONCLUSION AND SIGNIFICANCE: It was found that polyamines exert opposite effect, enhancement and inhibition, on gene expression depending on their concentrations. Such an opposite effect is argued in relation to the conformational change of DNA: enhancement is due to the parallel ordering of DNA segments that is accompanied by a decrease in the negative charge of double-stranded DNA, and inhibition is caused by the compaction of DNA into a tightly packed state with almost perfect charge-neutralization.

Huntingtin Polyglutamine-Dependent Protein Aggregation in Reconstituted Cells.

One of the aims of synthetic biology is bottom-up construction of reconstituted human cells for medical uses. To that end, we generated giant unilamellar vesicles (GUVs) that contained a HeLa cell extract, which comprises a cell-free protein synthesis (CFPS) system. Then we expressed Huntingtin protein fragments that contained polyglutamine (polyQ) sequences (Htt-polyQ), a hallmark of Huntington's disease. That system produced polyQ-dependent protein aggregates, as previously demonstrated in living cells. We next simplified the system by generating GUVs that contained purified human factors, which reconstituted a CFPS system. Htt-polyQ fragments expressed in these GUVs also formed protein aggregates. Moreover, an N-terminal deletion mutant, which had failed to form protein aggregates in living cells, also failed to form protein aggregates in the reconstituted GUVs. Thus, the GUV systems that encapsulated a human CFPS system could serve as reconstituted cells for studying neurological diseases.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases.