Miho Yanagisawa

Oriented Reconstitution of a Membrane Protein in a Giant Unilamellar Vesicle: Experimental Verification with the Potassium Channel KcsA

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.

UV-Induced Bursting of Cell-Sized Multicomponent Lipid Vesicles in a Photosensitive Surfactant Solution

We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (Lo) phase and a DOPC-rich liquid-disordered (Ld) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Yburst), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Yburst decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Yburst increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of Lo phase (with increasing [Chol] ≥ 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Yburst = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.

Cell-Sized confinement in microspheres accelerates the reaction of gene expression

Cell-sized water-in-oil droplet covered by a lipid layer was used to understand how lipid membranes affect biochemical systems in living cells. Here, we report a remarkable acceleration of gene expression in a cell-sized water-in-oil droplet entrapping a cell-free translation system to synthesize GFP (green fluorescent protein). The production rate of GFP (VGFP) in each droplet remained almost constant at least for on the order of a day, which implies 0th-order reaction kinetics. Interestingly, VGFP was inversely proportional to radius of droplets (R) when R is under 50 μm, and VGFP in droplets with R ∼ 10 μm was more than 10 times higher than that in the bulk. The acceleration rates of GFP production in cell-sized droplets strongly depended on the lipid types. These results demonstrate that the membrane surface has the significant effect to facilitate protein production, especially when the scale of confinement is on the order of cell-size.

Physicochemical analysis from real-time imaging of liposome tubulation reveals the characteristics of individual F-BAR domain proteins.

The Fer-CIP4 homology-BAR (F-BAR) domain, which was identified as a biological membrane-deforming module, has been reported to transform lipid bilayer membranes into tubules. However, details of the tubulation process, the mechanism, and the properties of the generated tubules remain unknown. Here, we successfully monitored the entire process of tubulation and the behavior of elongated tubules caused by four different F-BAR domain family proteins (FBP17, CIP4, PSTPIP1, and Pacsin2) using direct real-time imaging of giant unilamellar liposomes with dark-field optical microscopy. FBP17 and CIP4 develop many protrusions simultaneously over the entire surface of individual liposomes, whereas PSTPIP1 and Pacsin2 develop only a few protrusions from a narrow restricted part of the surface of individual liposomes. Tubules formed by FBP17 or CIP4 have higher bending rigidities than those formed by PSTPIP1 or Pacsin2. The results provide striking evidence that these four F-BAR domain family proteins should be classified into two groups: one group of FBP17 and CIP4 and another group of PSTPIP1 and Pacsin2. This classification is consistent with the phylogenetic proximity among these proteins and suggests that the nature of the respective tubulation is associated with biological function. These findings aid in the quantitative assessment with respect to manipulating the morphology of lipid bilayers using membrane-deforming proteins.

Generation of giant unilamellar liposomes containing biomacromolecules at physiological intracellular concentrations using hypertonic conditions

Artificial cells, particularly cell-sized liposomes, serve as tools to improve our understanding of the physiological conditions of living cells. However, such artificial cells typically contain a more dilute solution of biomacromolecules than that found in living cells (300 mg mL(-1)). Here, we reconstituted the intracellular biomacromolecular conditions in liposomes using hyperosmotic pressure. Liposomes encapsulating 80 mg mL(-1) of macromolecules of BSA or a protein mixture extracted from Escherichia coli were immersed in hypertonic sucrose. The concentration of macromolecules in BSA-containing liposomes was increased in proportion to the initial osmotic pressure ratio between internal and external media. On the other hand, the concentration of the protein mixture in liposomes could be saturated to reach the physiological concentration of macromolecules in cells. Furthermore, membrane transformation after the hypertonic treatment differed between BSA- and protein mixture-containing liposomes. These results strongly suggested that the crowded environment in cells is different from that found in typical single-component systems.

Adhesive force between paired microdroplets coated with lipid monolayers

We created pairs of adhering water-in-oil microdroplets coated with lipid monolayers as model cells and studied the effects of the physicochemical properties of the lipids on the adhesive force ΔF. Four species of liquid-phase lipids were used: dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC). The dependence of ΔF on the choice of lipid was evaluated by independently measuring the interfacial tension at the oil–water interface, γ, and the contact angle between the adhering droplets, θ. It was found that a difference in size between the hydrophilic head and hydrophobic tail of the lipids results in an increase in γ. Hence, cone-shaped DOPE had a larger γ than did cylinder-shaped PC (γ: DOPE ≫ DMPC ∼ DPPC > DOPC). On the other hand, DMPC with the shortest tail length had the smallest θ among the lipids (θ: DOPC > DPPC > DOPE ≫ DMPC). Finally, it was found that ΔF drastically decreases when the carbon number of the alkyl chain in the tails is smaller than 16 (ΔF: DOPE > DOPC ∼ DPPC ≫ DMPC). Furthermore, using polyethylene glycol (PEG)-conjugated DOPE, we demonstrated that the conjugation of shorter PEG molecules (<750) to the head part of the DOPE changes its molecular shape to cylindrical, and thus its γ and ΔF become similar to those of the DOPC system.

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.

Adhesion of binary giant vesicles containing negative spontaneous curvature lipids induced by phase separation

Abstract.We report the adhesion of binary giant vesicles composed of two types of phospholipids, one has negative spontaneous curvature which tends to bend toward the head group and the other has zero spontaneous curvature. In a homogeneous one-phase region, the giant vesicles do not adhere to each other, whereas in a coexisting two-phase region, the giant vesicles show adhesion. A fluorescence microscope observation reveals that the adhesion takes place through the domains rich in phospholipids having negative spontaneous curvature. We propose a phase separation induced hemifusion model where two apposed monolayers of adjacent vesicles are hemifused in order to reduce the bending energy of monolayers with negative spontaneous curvature and the boundary energy between the domains and matrix. We provide a strong evidence for the hemifusion model by lipid transfer experiments.

Multiple patterns of polymer gels in microspheres due to the interplay among phase separation, wetting, and gelation.

Significance We investigate how microdroplet confinement affects pattern formation of a polymer blend in the liquid-and-gel coexisting phase, wherein interactions between the droplet surface and the polymers regulate wettability of the gelation polymer. The complete and partial wetting of the polymers produces two stable states: hollow microspheres and hemisphere microgels. In addition, gelation during phase separation produces various shapes as trapped states. The relation between capsule thickness and droplet size is changed by the dynamical coupling. Furthermore, multiple patterns with spherical asymmetric shapes are produced by the partial wetting and shape deformation along the phase boundaries between the sol/gel phases. These findings reveal a complex pattern formation arising from the interplay among the interfacial tensions, gel elasticity, and wetting in microspheres. We report the spontaneous patterning of polymer microgels by confining a polymer blend within microspheres. A poly(ethylene glycol) (PEG) and gelatin solution was confined inside water-in-oil (W/O) microdroplets coated with a layer of zwitterionic lipids: dioleoylphosphatidylethanolamine (PE) and dioleoylphosphatidylcholine (PC). The droplet confinement affected the kinetics of the phase separation, wetting, and gelation after a temperature quench, which determined the final microgel pattern. The gelatin-rich phase completely wetted to the PE membrane and formed a hollow microcapsule as a stable state in the PE droplets. Gelation during phase separation varied the relation between the droplet size and thickness of the capsule wall. In the case of the PC droplets, phase separation was completed only for the smaller droplets, wherein the microgel partially wetted the PC membrane and had a hemisphere shape. In addition, the temperature decrease below the gelation point increased the interfacial tension between the PEG/gelatin phases and triggered a dewetting transition. Interestingly, the accompanying shape deformation to minimize the interfacial area was only observed for the smaller PC droplets. The critical size decreased as the gelatin concentration increased, indicating the role of the gel elasticity as an inhibitor of the deformation. Furthermore, variously patterned microgels with spherically asymmetric shapes, such as discs and stars, were produced as kinetically trapped states by regulating the incubation time, polymer composition, and droplet size. These findings demonstrate a way to regulate the complex shapes of microgels using the interplay among phase separation, wetting, and gelation of confined polymer blends in microdroplets.

Phase behaviors of agarose gel

We present evidence for the existence of phase separation in the gel state of agarose having the mixture of water and methanol as the gel solvent. Firstly, the sol-gel transition line and the cloud point line are determined independently as a function of the concentration of agarose as well as the concentration of methanol in the mixed solvent by the quasi-equilibrium cooling of the solutions. Then the spinodal line is determined by quenching the solutions below the sol-gel transition line. We find that the spinodal line appears below the cloud point line and both lines are entirely buried below the sol-gel transition line in the aqueous agarose system. The concentration fluctuations are, therefore, frozen into the polymer network of agarose gel that promotes the opacity of the resultant gel. The structure of agarose gel is observed by the confocal laser scanning microscope (CLSM) imaging technique that reveals that the density fluctuations are grown up to micrometer scale in space. The phase separation boundary is found to shift to the higher temperature region than the sol-gel transition line when the concentration of methanol in the mixed solvent is increased. The results indicate that the position of the phase separation boundary in relative to the sol-gel transition line varies with the quality of solvent. These results are in agreement with the theory of the sol-gel transition in which both the divergence of the connectivity and the thermodynamic instability are taken into account.