Entrapping Desired Amounts of Actin Filaments and Molecular Motor Proteins in Giant Liposomes

Takiguchi, K.; Yamada, Ayako; Negishi, Michiharu; Tanaka-Takiguchi, Yohko; Yoshikawa, Kenichi

doi:10.1021/la802031n

Cited by 59 publications

(52 citation statements)

References 24 publications

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“…In cases where it is necessary to experiment with repeated trials at high concentrations, a restriction is imposed even on proteins obtained with a high yield such as actin. It is important to ensure efficiency and reproducibility in the method for encapsulating high concentrations of proteins in membrane vesicles 16 . The amount-reduced water-in-oil (W/O) emulsion centrifugation method employed here meets those requirements 8 .…”

Section: Resultsmentioning

confidence: 99%

“…It is noted that approximately 200 μM actin is comparable to the actin concentration in living cells 19,20 . Also, this is the upper limit concentration of F-actin that can be handled, such as by pipetting, due to its very high viscosity 16 . Incidentally, rare liposomal morphologies, such as triangle or cruciform shapes, could be observed when higher concentrations of actin were encapsulated.…”

Section: Resultsmentioning

confidence: 99%

“…Labeling of actin with fluorescent dyes (Alexa 488 or 546 maleimide) was performed as described previously 34,40 . Otherwise, in order to visualize actin, rhodamine-phalloidin was added to F-actin prior to the preparation of liposomes encapsulating actin 16 . G-actin was stored in salt-free buffer (0.2 mM ATP, 1 mM NaHCO 3 , 0.2 mM CaCl 2 with or without 2-5 mM Tris-HCl (pH 8.0)), and F-actin was polymerized in salt-containing buffer (5 mM Tris-HCl (pH 8.0), 30 mM KCl, 0.2 mM ATP, 5 mM DTT, 0.9 mM NaHCO 3 , 0.18 mM CaCl 2 ) in general.…”

Section: Methodsmentioning

confidence: 99%

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Repetitive stretching of giant liposomes utilizing the nematic alignment of confined actin

2018

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Giant liposomes encapsulating cytoskeletons have been constructed to further understand the mechanisms of cell movement and develop cell-sized chemical machineries. Innovative studies demonstrating liposomal movements using microtubules and the molecular motors kinesin/dynein have been reported. However, no one has succeeded in generating repetitive motions controlled by external stimuli. Here we show that if the actin concentration in liposomes is comparable to that of cytoplasm of living cells, the liposomes can be deformed into spindle shapes by encapsulating only actin filaments, even without the molecular motor myosin. Furthermore, their shapes can be changed reversibly between spindle and sphere shapes by adjusting osmotic pressure or by light irradiation of fluorescent-labeled actin. In the latter case, the repetitive shape changes are accompanied with stretching and shrinking of filopodia-or acrosome projection-like extensions. Our results indicate that filamentous polymer of variable length like actin filament is a potential material for the reproduction of cell-like movement.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Repetitive stretching of giant liposomes utilizing the nematic alignment of confined actin

2018

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“…We prepared GUVs with the membrane consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and cholesterol at a weight ratio of 18∶2∶1 using the water-in-oil (W∕O) emulsion transfer method (38,39), which can produce unilamellar vesicles with defined inner and outer aqueous compositions (38,40,41). A small portion of POPG, a negatively charged lipid, was added to obtain dispersed GUVs by electrostatic repulsion (Fig.…”

Section: Resultsmentioning

confidence: 99%

Coupling of the fusion and budding of giant phospholipid vesicles containing macromolecules

Terasawa

Nishimura

Suzuki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

160

166

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Mechanisms that enabled primitive cell membranes to self-reproduce have been discussed based on the physicochemical properties of fatty acids; however, there must be a transition to modern cell membranes composed of phospholipids [Budin I, Szostak JW (2011) Proc Natl Acad Sci USA 108:5249–5254]. Thus, a growth-division mechanism of membranes that does not depend on the chemical nature of amphiphilic molecules must have existed. Here, we show that giant unilamellar vesicles composed of phospholipids can undergo the coupled process of fusion and budding transformation, which mimics cell growth and division. After gaining excess membrane by electrofusion, giant vesicles spontaneously transform into the budded shape only when they contain macromolecules (polymers) inside their aqueous core. This process is a result of the vesicle maximizing the translational entropy of the encapsulated polymers (depletion volume effect). Because the cell is a lipid membrane bag containing highly concentrated biopolymers, this coupling process that is induced by physical and nonspecific interactions may have a general importance in the self-reproduction of the early cellular compartments.

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“…Actin gels show high elasticity (6), which ensures the stability of cell membranes against various forces. For liposomes, the use of actin filaments as a cytoskeleton is not an optimal strategy for the following three reasons: First, although actin bundles and actomyosin rings have been reconstituted in artificial cells (7,8), formation of an actin cortex underneath artificial membranes has been still challenging. Second, actin is hard to modify by chemical and genetic means because of its essentiality for cell growth.…”

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confidence: 99%

DNA cytoskeleton for stabilizing artificial cells

Kurokawa

Fujiwara

Morita

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

130

123

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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.iposomes have been used as artificial cell models to understand cell shape, membrane protein function, and lipid− protein interaction, among other biological functions (1-3). In addition, liposomes have been used as a platform for biosensing and as drug delivery systems (DDS) because of their excellent biocompatibility and biodegradability (4). However, liposomes collapse easily against environmental shifts and mechanical forces because of their low bending modulus. The fragility of liposomes causes uncontrolled leakage of the entrapped compounds and thus inhibits their use in biomedical applications and artificial cells experiments.In contrast, cell membranes are tolerant against environmental shifts and mechanical forces. The stability of cell membrane arises from the cytoskeleton underneath the membrane. The major component of cytoskeletons is actin (5). Actin gels show high elasticity (6), which ensures the stability of cell membranes against various forces. For liposomes, the use of actin filaments as a cytoskeleton is not an optimal strategy for the following three reasons: First, although actin bundles and actomyosin rings have been reconstituted in artificial cells (7,8), formation of an actin cortex underneath artificial membranes has been still challenging. Second, actin is hard to modify by chemical and genetic means because of its essentiality for cell growth. Third, the physicochemical properties of actin gels are still unclear (9, 10). Hence, the cytoskeleton of liposomes should be constructed with defined and designable materials. To accomplish this aim, DNA nanotechnology, which uses limited components with high designability in a nanometer scale (11), is a feasible candidate to construct cytoskeleton structures in artificial cells.DNA nanostructure...

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Entrapping Desired Amounts of Actin Filaments and Molecular Motor Proteins in Giant Liposomes

Cited by 59 publications

References 24 publications

Repetitive stretching of giant liposomes utilizing the nematic alignment of confined actin

Repetitive stretching of giant liposomes utilizing the nematic alignment of confined actin

Coupling of the fusion and budding of giant phospholipid vesicles containing macromolecules

DNA cytoskeleton for stabilizing artificial cells

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