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

Abstract

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.

Fig. 1.

Electrofusion and budding transformation of GUVs. (A) Schematic representation of the electrofusion experimental setup. (B) Sequential epifluorescence images of the electrofusion of GUVs containing GFP (green) and R-PE (orange) without polymer. White and black-filled arrows at time zero indicate vesicles to fuse together. (C–F) Sequential images of budding transformations of vesicles containing 3 mM PEG 6000. White-filled arrows at time zero indicate vesicles to fuse together. Gray-filled arrows indicate the neck formation before budding. (C) Bright-field images. (D) Epifluorescence images of the membrane marked with fluorescence lipids. (E) Confocal fluorescence images of the membrane marked with fluorescence lipids. (F) Confocal fluorescence images of the vesicles encapsulating FITC-BSA. (Scale bars: 10 μm.)

Fig. 2.

Illustration of the depletion volume effect (not to scale). (A) Classical representation of the depletion volume effect. Larger particles in the polymer solution aggregate to reduce V_dep, in turn increasing V_free. (B) In the system consisting of the GUV containing the polymer, V_free increases when the curved area of the membrane increases, under the constraint of constant volume and surface area. Thickness of the polymer depletion volume is exaggerated to clarify the relative difference of V_free.

Fig. 3.

Probability of budding transformation of vesicles encapsulating polymers after electrofusion. (A) PEG 6000 at various concentrations. (B) PEG with various molecular weight and concentrations. (C) Dextran with various molecular weight and concentrations.

Fig. 4.

Estimation of the free energy. (A) Confocal images and 3D representation of the typical shape transformation at the late phase of budding. Constriction of the elongated shape develops towards budding. (Scale bar: 5 μm.) (B) ΔE_dep calculated for the hypothetical transformation from a spherocylinder into two spheres (5-μm diameter was assumed) in various experimental conditions. Red and blue bars, respectively, represent the conditions in which the budding transformation occurred (P_bud > 0.1) and did not occur (P_bud < 0.1). The green horizontal line shows the estimated increase of bending energy of the membrane.

Fig. 5.

Repetitive cycles of fusion-to-budding transformation. After the first fusion (t = 12 s), an ac signal was applied after each budding event (t = 94 and 132 s). The vesicles contain 3 mM PEG 6000 (5%wt/wt). White arrows indicate the vesicles to be fused. Gray arrows show the neck formation. (Scale bar: 10 μm.)