Coupled growth and division of model protocell membranes

Abstract

The generation of synthetic forms of cellular life requires solutions to the problem of how biological processes such as cyclic growth and division could emerge from purely physical and chemical systems. Small unilamellar fatty acid vesicles grow when fed with fatty acid micelles and can be forced to divide by extrusion, but this artificial division process results in significant loss of protocell contents during each division cycle. Here we describe a simple and efficient pathway for model protocell membrane growth and division. The growth of large multilamellar fatty acid vesicles fed with fatty acid micelles, in a solution where solute permeation across the membranes is slow, results in the transformation of initially spherical vesicles into long thread-like vesicles, a process driven by the transient imbalance between surface area and volume growth. Modest shear forces are then sufficient to cause the thread-like vesicles to divide into multiple daughter vesicles without loss of internal contents. In an environment of gentle shear, protocell growth and division are thus coupled processes. We show that model protocells can proceed through multiple cycles of reproduction. Encapsulated RNA molecules, representing a primitive genome, are distributed to the daughter vesicles. Our observations bring us closer to the laboratory synthesis of a complete protocell consisting of a self-replicating genome and a self-replicating membrane compartment. In addition, the robustness and simplicity of this pathway suggests that similar processes might have occurred under the prebiotic conditions of the early Earth.

Figure 1

Vesicle growth and division. (A, B) Epifluorescence micrographs of vesicle shape transformations during growth, 10 and 30 min after the addition of 5 equiv of oleate micelles to multilamellar oleate vesicles (in 0.2 M Na-bicine, pH 8.5, ∼1 mM initial oleic acid), respectively. All vesicles were labeled with 2 mM encapsulated HPTS, a water-soluble fluorescent dye, in their internal aqueous space. Scale bar, 50 μm. (C) Schematic diagram of cyclic multilamellar vesicle growth and division: vesicles remain multilamellar before and after division (shown as, but not limited to, two layers). (D−F) Growth of a single multilamellar oleate vesicle, 3 min, 10 min, and 25 min after the addition of 5 equiv of oleate micelles, respectively. (G, H) In response to mild fluid agitation, this thread-like vesicle divided into multiple smaller daughter vesicles (also shown in movie S1). Scale bar for D−H, 20 μm.

Figure 2

Cycles of vesicle growth and division. (A) Relative surface area after two cycles of addition of 5 equiv of oleate micelles (solid circles) or 5 equiv of NaOH (open circles) to oleate vesicles, each followed by agitation. Inset micrographs show vesicle shapes at indicated times. Scale bar, 10 μm. (B) Vesicle shapes during cycles of growth and division in a model prebiotic buffer (0.2 M Na-glycine, pH 8.5, ∼1 mM initial oleic acid, vesicles contain 10 mM HPTS for fluorescence imaging). Scale bar, 20 μm.

Figure 3

Growth of vesicles containing encapsulated RNA, and redistribution of RNA molecules into daughter vesicles. (A, B) Oleate vesicle (in 0.2 M Na-bicine, pH 8.5, ∼1 mM initial oleic acid) containing 5′-DY547-labeled RNA (DY547-A₁₀, 0.5 mM) at 10 and 30 min after the addition of 5 equiv of oleate micelles, respectively. Scale bar, 10 μm. (C) Number of RNA-containing oleate vesicles before and after division, n = 3. (D) Percentage RNA leakage after the division of thread-like oleate vesicles by agitation, versus the leakage from 4-μm diameter vesicles extruded through 2 μm pores, n = 3. (E) Number of RNA-containing decanoate:decanol (2:1) vesicles (in 0.2 M Na-bicine, pH 8.5, at room temperature, ∼20 mM initial amphiphile concentration) before and after division, n = 3. (F) Total amount of RNA leakage from decanoate:decanol (2:1) vesicles after division, versus the leakage from vesicles lysed by adding 1% Triton X-100, n = 3.

Figure 4

Vesicle growth and division in various buffers and with various lipid compositions (A) Oleate vesicle (containing 2 mM HPTS, in 60 mM Na-glycine, 30 mM Na-alanine, 10 mM Na-aspartate, and 10 mM Na-glutamate, pH 8.5, ∼1 mM initial oleic acid) at 30 min after the addition of 5 equiv of oleate micelles. (B) Myristoleate vesicle (containing 2 mM HPTS, in 0.2 M Na-bicine, pH 8.5, ∼4 mM initial myristoleic acid) at 30 min after the addition of 5 equiv of myristoleate micelles. (C) Decanoate vesicle (containing 2 mM HPTS, in 0.2 M Na-bicine, pH 7.4, at 50 °C, ∼60 mM initial decanoic acid) at 30 min after the addition of 1.7 equiv of decanoate micelles. (D) Decanoate:decanol (2:1) vesicle (containing 2 mM HPTS, in 0.2 M Na-bicine, pH 8.5, at room temperature, ∼20 mM initial amphiphile concentration) at 30 min after the addition of 2 equiv of decanoate micelles and 1 equiv of decanol emulsion. Scale bar for A−D, 10 μm. (E) Number of dye-containing oleate vesicles in a mixed amino acid solution (conditions as above) before and after growth and division, n = 3. (F) Number of dye-containing myristoleate vesicles (conditions as above) before and after growth and division, n = 3. (G) Number of dye-containing decanoate vesicles (conditions as above) before and after growth and division, n = 3.

Figure 5

Growth of multilamellar versus unilamellar vesicles (A) Schematic diagram of incorporation of micelles into a multilamellar vesicle: the outermost membrane grows faster than the inner membrane layers. (B, C) Confocal images of multilamellar oleate vesicle (0.2 mol % Rh-DHPE, in 0.2 M Na-bicine, pH 8.5, ∼1 mM initial oleic acid) before and 10 min after the addition of 1 equiv of oleate micelles, respectively. (D) Confocal image of multilamellar vesicle after division. (E) Schematic diagram of incorporation of micelles into a unilamellar vesicle. (F, G) Confocal images of unilamellar oleate vesicle (conditions as above) before and 10 min after the addition of 1 equiv of oleate micelles, respectively. (H) Confocal image of a multilamellar vesicle formed after the agitation of elongated unilamellar vesicles. Scale bar for B−D, F−H; 2 μm.

Figure 6

Vesicle growth in a highly permeable buffer (A) Schematic diagram of growth of a multilamellar vesicle in ammonium acetate: as the surface area of the outermost membrane increases, the solutes in their neutral forms (NH₃ and CH₃COOH) permeate the membrane, allowing the internal volume to increase. (B, C) Confocal images of multilamellar oleate vesicle (0.2 mol % Rh-DHPE, in 0.2 M ammonium acetate, pH 8.5, ∼1 mM initial oleic acid) before and 10 min after the addition of 1 equiv of oleate micelles, respectively. (D, E) Confocal images of multilamellar oleate vesicle (containing 2 mM HPTS, in 0.2 M ammonium acetate, pH 8.5, ∼1 mM initial oleic acid) before and 10 min after the addition of 1 equiv of oleate micelles, respectively. Scale bar for B−E, 2 μm.