Entrapping desired amounts of actin filaments and molecular motor proteins in giant liposomes

We have successfully prepared cell-sized giant liposomes encapsulating desired amounts of actoHMM, a mixture of actin filament (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), in the presence of 5 mM MgCl 2 and 50 mM KCl. We employed a spontaneous transfer method to prepare those liposomes. In the absence of HMM, F-actin was distributed homogeneously inside the liposomes. In contrast, when F-actin was encapsulated in liposomes together with HMM, network structures were generated. Such network structures are attributable to the cross-linking of F-actin by HMM.     

Morphogenesis of liposomes caused by polycation-induced actin assembly formation

We investigated the effects of polycation-mediated actin assembly on the morphological transformation of the lipid vesicle membrane by spatiotemporally controlling actin assembly. By triggering the radical polymerization of the cationic monomer using UV irradiation, we achieved a varied photoinduced assembly of actin in bulk solution. Furthermore, we designed liposomes containing actin and cationic monomers. In these actin-encapsulated liposomes, various actin assemblies were formed by UV irradiation similar to that observed in bulk solution. Moreover, morphogenesis of actin-encapsulated liposomes was observed in liposomes encapsulated with G-actin but not with F-actin. This result indicates that a dynamic polymerization of G-actin is important for vesicle protrusion.     

Encapsulation of active cytoskeletal protein networks in cell-sized liposomes

We demonstrate that cytoskeletal actin-myosin networks can be encapsulated with high efficiency in giant liposomes by hydration of lipids in an agarose hydrogel. The liposomes have cell-sized diameters of 10-20 μm and a uniform actin content. We show by measurements of membrane fluorescence intensity and bending rigidity that the majority of liposomes are unilamellar. We further demonstrate that the actin network can be specifically anchored to the membrane by biotin-streptavidin linkages. These protein-filled liposomes are useful model systems for quantitative studies of the physical mechanisms by which the cytoskeleton actively controls cell shape and mechanics. In a broader context, this new preparation method should be widely applicable to encapsulation of proteins and polymers, for instance, to create polymer-reinforced liposomes for drug delivery.     

Structural transition of actin filament in a cell-sized water droplet with a phospholipid membrane

Actin filament, F-actin, is a semiflexible polymer with a negative charge, and is one of the main constituents of cell membranes. To clarify the effect of cross talk between a phospholipid membrane and actin filaments in cells, we conducted microscopic observations on the structural changes in actin filaments in a cell-sized (several tens of micrometers in diameter) water droplet coated with a phospholipid membrane such as phosphatidylserine (PS; negatively charged head group) or phosphatidylethanolamine (PE; neutral head group) as a simple model of a living cell membrane. With PS, actin filaments are distributed uniformly in the water phase without adsorption onto the membrane surface between 2 and 6 mM Mg2+, while between 6 and 12 mM Mg2+, actin filaments are adsorbed onto the inner membrane surface. With PE, the actin filaments are uniformly adsorbed onto the inner membrane surface between 2 and 12 mM Mg2+. With both PS and PE membranes, at Mg2+ concentrations higher than 12 mM, thick bundles are formed in the bulk water droplet accompanied by the dissolution of actin filaments from the membrane surface. The attraction between actin filaments and membrane is attributable to an increase in the translational entropy of counterions accompanied by the adsorption of actin filaments onto the membrane surface. These results suggest that a microscopic water droplet coated with phospholipid can serve as an easy-to-handle model of cell membranes.     

Giant vesicles with membranous microcompartments

Incubation of a cell-sized lipid membrane vesicle (giant vesicle, GV) in a diluted aqueous solution of neutral phosphate buffer salts or glucose transformed the GV to an oligovesicular vesicle (OVV) that encapsulates one or more smaller GVs. During the incubation, the membrane of flaccid vesicle invaginated and closed to form the inner vesicle of an OVV engulfing a part of the bulk aqueous phase. Using the GV-to-OVV transformation, an OVV that has different aqueous contents in each membranous microcompartment was constructed.     

Selective spatial localization of actomyosin motor function by chemical surface patterning

We have previously described the efficient guidance and unidirectional sliding of actin filaments along nanosized tracks with adsorbed heavy meromyosin (HMM; myosin II motor fragment). In those experiments, the tracks were functionalized with trimethylchlorosilane (TMCS) by chemical vapor deposition (CVD) and surrounded by hydrophilic areas. Here we first show, using in vitro motility assays on nonpatterned and micropatterned surfaces, that the quality of HMM function on CVD-TMCS is equivalent to that on standard nitrocellulose substrates. We further examine the influences of physical properties of different surfaces (glass, SiO(2), and TMCS) and chemical properties of the buffer solution on motility. With the presence of methylcellulose in the assay solution, there was HMM-induced actin filament sliding on both glass/SiO(2) and on TMCS, but the velocity was higher on TMCS. This difference in velocity increased with decreasing contact angles of the glass and SiO(2) surfaces in the range of 20-67 degrees (advancing contact angles for water droplets). The corresponding contact angle of CVD-TMCS was 81 degrees. In the absence of methylcellulose, there was high-quality motility on TMCS but no motility on glass/SiO(2). This observation was independent of the contact angle of the glass/SiO(2) surfaces and of HMM incubation concentrations (30-150 microg mL(-)(1)) and ionic strengths of the assay solution (20-50 mM). Complete motility selectivity between TMCS and SiO(2) was observed for both nonpatterned and for micro- and nanopatterned surfaces. Spectrophotometric analysis of HMM depletion during incubation, K/EDTA ATPase measurements, and total internal reflection fluorescence spectroscopy of HMM binding showed only minor differences in HMM surface densities between TMCS and SiO(2)/glass. Thus, the motility contrast between the two surface chemistries seems to be attributable to different modes of HMM binding with the hindrance of actin binding on SiO(2)/glass.     

Nano-biomachine from actin and myosin gels

We introduce here an ATP (adenosine triphosphate)-fueled nano-biomachine constructed from actin and myosin gels. Various types of chemically cross-linked actin gel, which are tens of times larger in size than native actin filaments (F-actin), were formed by complexing with cation-polymers and placed on a chemically cross-linked myosin gel. By adding dilute solution of ATP, they moved along the myosin gel with a velocity as high as that of native F-actin by coupling to ATP hydrolysis. Formation mechanism and structure of actin complexes as well as those of myosin gels were studied in detail and elucidated with the specific characteristics of the motility. These results demonstrate that one can construct nano-biomachines fueled by chemical energy of ATP with controlled motility. The text was submitted by the authors in English.     

Transmembrane Transport of Adenosine 5′-Triphosphate Using a Lipophilic Cholesteryl Derivative

The uptake of ATP in liposomes was achieved by using the lipophilic derivative cholesteryloxycarbonyl-ATP ( 1 ). Its hydrolysis leading to the release of ATP inside the vesicules (see scheme) was observed with the help of a pH gradient and monitored by ³¹P NMR spectroscopy. This is the first successful transfer of a nucleoside 5′-triphosphate across a membrane.     

Location of magnetic and fluorescent nanoparticles encapsulated inside giant liposomes

In this paper, we demonstrate the production of highly magnetic and fluorescent giant vesicles by encapsulating gamma-Fe2O3-rhodamine B nanoparticles. The liposomes containing the nanoparticles were made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We found that the ionic strength of the initial magnetic fluid is a crucial parameter in controlling the physicochemical properties of the bilayer. At high ionic strength, we obtained very important deformations of liposomes with high magnetic susceptibilities induced by an applied magnetic field. The encapsulation rate was studied using magnetophoresis and photobleaching tests, and the membrane properties were studied using confocal microscopy and elastic measurements.     

Hierarchical self-assembly of actin bundle networks: gels with surface protein skin layers

The networklike structure of actin bundles formed with the cross-linking protein alpha-actinin has been investigated via x-ray scattering and confocal fluorescence microscopy over a wide range of alpha-actinin/F-actin ratios. We describe the hierarchical structure of bundle gels formed at high ratios. Isotropic actin bundle gels form via cluster-cluster aggregation in the diffusion-limited aggregation regime at high alpha-actinin/actin ratios. This process is clearly observed by confocal fluorescence microscopy. Polylysine is investigated as an alternative bundling agent in the high-ratio regime and the effects of F-actin length are also discussed. One particularly fascinating aspect of this system is the presence of a structured skin layer at the gel/water interface. Confocal microscopy has elucidated the full three-dimensional structure of this layer and revealed several interesting morphologies. The protein skin layer is a micron-scale structure composed of a directed network of bundles and exhibits flat, crumpled, and tubelike shapes. We show that crumpling of the skin layer results from stresses due to the underlying gel. These biologically based geometric structures may detach from the gel, demonstrating potential for the generation of biological scaffolds with defined shapes for applications in cell encapsulation and tissue engineering. We demonstrate manipulation of the skin layer, producing hemispherical structures in solution.     

Structure of small actin-containing liposomes probed by atomic force microscopy: effect of actin concentration & liposome size

Actin-containing liposomes were prepared via extrusion through 400 and 600 nm pore diameter membranes at different monomeric actin concentrations in low ionic strength buffer (G-buffer). After subjecting the liposome dispersions to high ionic strength polymerization buffer (F-buffer), topological changes in liposome structure were studied using atomic force microscopy (AFM). Paired dumbbell, horseshoelike, and disklike assemblies were observed for actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes. The topology of actin-containing liposomes was found to be highly dependent on both liposome size and actin concentration. At 1 mg/mL actin, the actin-containing liposomes transformed into a disklike shape, whereas, at 5 mg/mL actin, the actin-containing liposomes retained a spherical shape. On the basis of these observations, we hypothesize that actin could either polymerize on the surface of the inner leaflet of the liposome membrane or polymerize in the aqueous core of the liposome. We explain the associated shape changes induced in actin-containing liposomes on the basis of the hypothesized mechanism of actin polymerization inside the liposomes. At higher actin concentrations (5 mg/mL), we observed membrane-induced actin self-assembly in G-buffer, which implies that G-actin is able to interact directly with lipid bilayers at sufficiently high concentrations.     

The bound nucleotide of actin.

The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G-actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F-actin and the interaction of F-actin - AMPPNP with myosin was studied. F-actin - AMPPNP activated the ATPase of myosin to the same extent as did F-actin - ADP. However, the rate of superprecipitation was slower in the case of F-actin - AMPPNP than in the control.     

An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles

A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 micro m in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5'-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length.An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.     

Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar

Giant liposomes, or giant vesicles, are cell-size (approximately 5-100 microm) compartments enclosed with phospholipid bilayers, and have often been used in biological research. They are usually generated using hydration methods, "electroformation" and "gentle hydration (or natural swelling)", in which dry lamellar films of phospholipids are hydrated with aqueous solutions. In gentle hydration, however, giant liposomes are difficult to prepare from an electrostatically neutral phospholipid because lipid lamellae cannot repel each other. In this study, we demonstrate the efficient formation of giant liposomes using the gentle hydration of neutral phospholipid (dioleoyl phosphatidylcholine, DOPC) dry films doped with nonelectrolytic monosaccharides (glucose, mannose, and fructose). A mixture of DOPC and such a sugar in an organic solvent (chloroform/methanol) was evaporated to form the films, which were then hydrated with distilled water or Tris buffers containing sodium chloride. Under these conditions, giant liposomes spontaneously formed rapidly and assumed a swollen cell-sized spherical shape with low lamellarity, whereas giant liposomes from pure DOPC films had multilamellar lipid layers, miscellaneous shapes and smaller sizes. This observation indicates that giant unilamellar vesicles (GUVs) of DOPC can be obtained efficiently through the gentle hydration of sugar-containing lipid dry films because repulsion between lipid lamellae is enhanced by the osmosis induced by dissolved sugar.     

Mode of heavy meromyosin adsorption and motor function correlated with surface hydrophobicity and charge

The in vitro motility assay is valuable for fundamental studies of actomyosin function and has recently been combined with nanostructuring techniques for the development of nanotechnological applications. However, the limited understanding of the interaction mechanisms between myosin motor fragments (heavy meromyosin, HMM) and artificial surfaces hampers the development as well as the interpretation of fundamental studies. Here we elucidate the HMM-surface interaction mechanisms for a range of negatively charged surfaces (silanized glass and SiO2), which is relevant both to nanotechnology and fundamental studies. The results show that the HMM-propelled actin filament sliding speed (after a single injection of HMM, 120 microg/mL) increased with the contact angle of the surfaces (in the range of 20-80 degrees). However, quartz crystal microbalance (QCM) studies suggested a reduction in the adsorption of HMM (with coupled water) under these conditions. This result and actin filament binding data, together with previous measurements of the HMM density (Sundberg, M.; Balaz, M.; Bunk, R.; Rosengren-Holmberg, J. P.; Montelius, L.; Nicholls, I. A.; Omling, P.; T?gerud, S.; M?nsson, A. Langmuir 2006, 22, 7302-7312. Balaz, M.; Sundberg, M.; Persson, M.; Kvassman, J.; M?nsson, A. Biochemistry 2007, 46, 7233-7251), are consistent with (1) an HMM monolayer and (2) different HMM configurations at different contact angles of the surface. More specifically, the QCM and in vitro motility assay data are consistent with a model where the molecules are adsorbed either via their flexible C-terminal tail part (HMMC) or via their positively charged N-terminal motor domain (HMMN) without other surface contact points. Measurements of zeta potentials suggest that an increased contact angle is correlated with a reduced negative charge of the surfaces. As a consequence, the HMMC configuration would be the dominant configuration at high contact angles but would be supplemented with electrostatically adsorbed HMM molecules (HMMN configuration) at low contact angles. This would explain the higher initial HMM adsorption (from probability arguments) under the latter conditions. Furthermore, because the HMMN mode would have no actin binding it would also account for the lower sliding velocity at low contact angles. The results are compared to previous studies of the microtubule-kinesin system and are also discussed in relation to fundamental studies of actomyosin and nanotechnological developments and applications.     

Enhanced Anticancer Efficacy by ATP‐Mediated Liposomal Drug Delivery

A liposome‐based co‐delivery system composed of a fusogenic liposome encapsulating ATP‐responsive elements with chemotherapeutics and a liposome containing ATP was developed for ATP‐mediated drug release triggered by liposomal fusion. The fusogenic liposome had a protein–DNA complex core containing an ATP‐responsive DNA scaffold with doxorubicin (DOX) and could release DOX through a conformational change from the duplex to the aptamer/ATP complex in the presence of ATP. A cell‐penetrating peptide‐modified fusogenic liposomal membrane was coated on the core, which had an acid‐triggered fusogenic potential with the ATP‐loaded liposomes or endosomes/lysosomes. Directly delivering extrinsic liposomal ATP promoted the drug release from the fusogenic liposome in the acidic intracellular compartments upon a pH‐sensitive membrane fusion and anticancer efficacy was enhanced both in vitro and in vivo.     

Endocytosis-like uptake of surface-modified drug nanocarriers into giant unilamellar vesicles

We had previously developed surface-modified poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for use as a cellular drug delivery system. The cellular uptake of PLGA-NPs was mediated predominantly by endocytosis, and this uptake was increased by surface modifications with polymers, such as chitosan (CS) and polysorbate 80 (P80). In the present study, we prepared a cell-sized giant unilamellar vesicle (GUV) that mimics a cell membrane to investigate the interaction between cell membranes and NPs. Endocytosis-like uptake of NPs into a GUV was observed when the NPs were modified with nonionic surfactant P80 probably due to change in viscoelasticity and enhanced fusion activity of the membrane induced by P80. In contrast, unmodified NPs and those modified with CS were not internalized into a GUV. These results suggest that surface properties of PLGA-NPs are an important formulation parameter for their interaction with lipid membranes.     

Inter-monomer cross-linking affects the thermal transitions in F-actin

Chemical cross-links which covalently connected the Cys-374 and Glu-41 residues of adjacent monomers in the same strand of F-actin were used to follow the consequences of the modification for the motional and structural properties of the actin filaments. DSC measurements reported that the inter-monomer cross-links shifted the thermal transition temperature and affected strongly the cooperativity of the transition in comparison with uncross-linked F-actin. Addition of HMM to F-actin induced significant decrease of the transition temperature to lower value from 69.4 to 67. 5 °C.