Thermally Driven Membrane Phase Transitions Enable Content Reshuffling in Primitive Cells

Abstract

Self-assembling single-chain amphiphiles available in the prebiotic environment likely played a fundamental role in the advent of primitive cell cycles. However, the instability of prebiotic fatty acid-based membranes to temperature and pH seems to suggest that primitive cells could only host prebiotically relevant processes in a narrow range of nonfluctuating environmental conditions. Here we propose that membrane phase transitions, driven by environmental fluctuations, enabled the generation of daughter protocells with reshuffled content. A reversible membrane-to-oil phase transition accounts for the dissolution of fatty acid-based vesicles at high temperatures and the concomitant release of protocellular content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled material in a new cohort of protocells. Notably, we find that our disassembly/reassembly cycle drives the emergence of functional RNA-containing primitive cells from parent nonfunctional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally driven tunable prebiotic process, which supports the release and reshuffling of oligonucleotides and membrane components, potentially leading to a new generation of protocells with superior traits. In the absence of protocellular transport machinery, the environmentally driven disassembly/assembly cycle proposed herein would have plausibly supported protocellular content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early life forms.

Figure 1

Thermal cycling drives reversible and tunable fatty acid phase transitions. (a) Overlapped profiles of turbidity and fluorescence intensity as a function of temperature for 100-nm-radius vesicles made of 50 mM myristoleic acid (in 200 mM Tris-HCl, pH 7, 25 °C → 95 °C). For fluorescence experiments, Laurdan (20 μM) was embedded in the lipid bilayer. Absorbance (gray) is monitored at 420 nm, whereas Laurdan fluorescence (purple) is monitored at λ_exc = 350 nm, λ_em = 426 and 496 nm. (b) Schematic representation of thermal cycles explored in this study. Microscopy images after the heating ramp (95 °C, hot-stage epifluorescence microscopy images) and after the cooling ramp (25 °C, confocal microscopy images) are shown for myristoleic acid vesicles. (c) Membrane budding observed during the cooling ramp on the surface of myristoleic acid droplets by hot-stage epifluorescence microscopy. n = 3 for data in (a).

Figure 2

Thermal cycling drives disassembly and reassembly of fatty acid membranes. (a) Electron cryo-microscopy (top) and confocal microscopy (bottom) images collected before and after thermal cycles for myristoleic acid vesicles. (b) Analyses of myristoleic acid samples showing how the size (left) and number (right) of the next-generation population of vesicles are affected by lipid concentration. (c) Analyses of independently prepared myristoleic acid samples showing how the size (left) and number (right) of regenerated vesicles are affected by heating time. Analyses in (b) and (c) were performed on collected confocal microscopy images for 100-nm-radius vesicles made of 50 mM myristoleic acid vesicles in 200 mM Tris-HCl, pH 8, after thermal cycling. Upon cooling the samples to 25 °C, vesicles were allowed to re-equilibrate for 1 h. Minimal diameter cutoff for image processing = 500 nm. Statistical significance was assessed using the one-way ANOVA test, n = 10 independent experiments. Statistical values obtained for *P = < 0.05, ****P = < 0.0001. Center dashed line represents median; dotted lines represent upper and lower quartiles. n = 3 for data in (a).

Figure 3

Thermal cycling drives release and re-encapsulation of protocellular content. (a) Size-exclusion chromatograms showing partial re-encapsulation (3.2%) of FITC-dextran in 100-nm-radius vesicles made of 50 mM myristoleic acid in 200 mM Tris-HCl, pH 8, upon heat exposure. Content uptake was monitored by fluorescence (λ_exc = 495 nm). (b) Size-exclusion chromatograms showing partial encapsulation (4.1%) of FITC-dextran in 100-nm-radius vesicles made of 50 mM myristoleic acid in 200 mM Tris-HCl, pH 8, upon heat exposure. Content uptake was monitored by fluorescence (λ_exc = 495 nm). (c) Microscopy images corresponding to experiments reported in (a) (top row) and (b) (bottom row) before the thermal cycles (25 °C, confocal microscopy images), after the heating ramps (95 °C, hot-stage epifluorescence microscopy images) and after the cooling ramps (25 °C, confocal microscopy images) are shown for 50 mM myristoleic acid vesicles in 200 mM Tris-HCl, pH 8. (d) Hot-stage epifluorescence microscopy images corresponding to the experiment reported in (a) at high temperature values (90 °C, left; 95 °C, right) show the formation of faceted myristoleic acid structures (90 °C, left), which transiently trap the aqueous content. Such multilayer structures then convert to lipid droplets (95 °C, right), completely releasing the encapsulated material. Samples for confocal microscopy analyses were diluted after thermal cycling to reduce the background noise of the unencapsulated fluorescent material. Data are mean and SEM, n = 3 independent experiments.

Figure 4

Thermal cycling drives reassembly of protocells with reshuffled membrane material and encapsulated content. (a) Schematic representation and confocal microscopy images for experiments with populations of vesicles with different fluorescent content. 100-nm-radius vesicles, made of 50 mM myristoleic acid in 200 mM Tris-HCl, pH 8, and containing either FITC-10nt or TYE665-10nt oligonucleotides, were mixed before undergoing thermal cycling and, after 1 h re-equilibration at room temperature, were visualized by confocal microscopy. (b) Schematic representation and confocal microscopy images for experiments with populations of vesicles, labeled with different fluorescent lipids. 100-nm-radius vesicles, made of 50 mM myristoleic acid in 200 mM Tris-HCl, pH 8, and either NBD-PE or Rh-DHPE, were mixed before undergoing thermal cycling and, after 1 h re-equilibration at room temperature, were visualized by confocal microscopy. (c) Schematic representation of the experiment to reconstitute a split Broccoli aptamer inside myristoleic acid vesicles. (d) Size-exclusion chromatograms showing no reconstitution of the split Broccoli aptamer when vesicle mixing occurs after thermal cycling. (e) Size-exclusion chromatograms showing reconstitution of the split Broccoli aptamer (20.3%) when vesicle mixing occurs before thermal cycling. Experiments for Broccoli aptamer reconstitution were performed with 100-nm-radius vesicles made of 50 mM myristoleic acid in 200 mM Tris-HCl, pH 8. Broccoli aptamer reconstitution was monitored by fluorescence of DFHBI (λ_exc = 505 nm). Confocal images were collected prior to purification. Data are mean and SEM, n = 3 independent experiments.