One question in the origin of life is the time at which membrane compartments came into the picture as hosts for the first forms of metabolism. If we assume the proteins and nucleic acids came first, then it is difficult to conceive how all the macromolecular components could have been entrapped at a later time in a single compartment. On the other hand, the hypothesis that metabolism originated from inside the compartment means that we would then have to conceive semipermeable, sophisticated membranes in prebiotic times, which does not appear plausible. With this study, we believe that we can offer a partial solution to this riddle, at the same time opening a new vista on the principles of the entrapment of solute in vesicles. We used cryo-TEM to study the entrapment of the protein ferritin in liposomes. The novel, surprising principle that appears is that when lipid surfaces close up in a proteincontaining solution to form vesicles, the entrapment frequency does not follow the expected Poisson distribution, but tends to assume a power-law behaviour, characterized by many "empty" vesicles (no or very little entrapped solute), and a long decreasing tail with extremely crowded vesicles. Cryo-TEM analysis shows indeed some extremely crowded liposomes adjacent to empty ones. The conclusion is that membrane closure can accumulate a remarkable number of solutes inside some compartments. The possible mechanism and relevance of this extreme local super-concentration effect for the origin of life are discussed.The spontaneous formation of lipid vesicles (liposomes) in an aqueous phase containing one or more solutes produces a heterogeneous population of liposomes in terms of solute content. Such entrapments have generally been studied by averaging techniques, such as batch absorbance or fluorescence, whereas little attention has been devoted to studying individual encapsulation.[1] This is partly due to the technical difficulty of directly counting molecules inside liposomes. The encapsulation of biomacromolecules inside liposomes, on the other hand, is an important issue in origins of life research (protocell models) as well as in recent studies on synthetic cells. [2] A series of recent experiments within our project on the construction of minimal living cells [3] revealed possible deviations from the number of macromolecules expected to be entrapped inside liposomes of diameter d < 200 nm. In particular, with the aim of producing green fluorescent protein (GFP) inside liposomes, we prepared liposomes in the presence of the transcription-translation macromolecular machinery, namely E. coli extracts as well as PURESYSTEM [4] (a cell-free protein synthesis kit containing 36 purified components, t-RNAs, ribosomes, for a total of about 80 different macromolecules). We showed that GFP was synthesised inside liposomes, despite of the fact that the Poisson probability of liposome co-entrapment of about 80 different macromolecules (each at a concentration of 0.1-1 mm) is vanishingly small (~10 À26). In order to explain the obs...
The question of the minimal size of a cell that is still capable of endorsing life has been discussed extensively in the literature, but it has not been tackled experimentally by a synthetic-biology approach. This is the aim of the present work; in particular, we examined the question of the minimal physical size of cells using liposomes that entrapped the complete ribosomal machinery for expression of enhanced green fluorescence protein, and we made the assumption that this size would also correspond to a full fledged cell. We found that liposomes with a radius of about 100 nm, which is the smallest size ever considered in the literature for protein expression, are still capable of protein expression, and surprisingly, the average yield of fluorescent protein in the liposomes was 6.1-times higher than in bulk water. This factor would become even larger if one would refer only to the fraction of liposomes that are fully viable, which are those that contain all the molecular components (about 80). The observation of viable liposomes, which must contain all macromolecular components, indeed represents a conundrum. In fact, classic statistical analysis would give zero or negligible probability for the simultaneous entrapment of so many different molecular components in one single 100 nm radius spherical compartment at the given bulk concentration. The agreement between theoretical statistical predictions and experimental data is possible with the assumption that the concentration of solutes in the liposomes becomes larger by at least a factor twenty. Further investigation is required to understand the over-concentration mechanism, and to identify the several biophysical factors that could play a role in the observed activity enhancement. We conclude by suggesting that these entrapment effects in small-sized compartments, once validated, might be very relevant in the origin-of-life scenario.
One of the open questions in the origin of life is the spontaneous formation of primitive cell-like compartments from free molecules in solution and membranes. "Metabolism-first" and "replicator-first" theories claim that early catalytic cycles first evolved in solution, and became encapsulated inside lipid vesicles later on. "Compartment-first" theories suggest that metabolism progressively occurred inside compartments. Both views have some weaknesses: the low probability of co-entrapment of several compounds inside the same compartment, and the need to control nutrient uptake and waste release, respectively. By using lipid vesicles as early-cell models, we show that ribosomes, proteins and lipids spontaneously self-organise into cell-like compartments to achieve high internal concentrations, even when starting from dilute solutions. These findings suggest that the assembly of cell-like compartments, despite its low probability of occurrence, is indeed a physically realistic process. The spontaneous achievement of high local concentration might provide a rational account for the origin of primitive cellular metabolism.
Primitive cell models help to understand the role that compartmentalization plays in origin of life scenarios. Here we present a combined experimental and modeling approach towards the construction of simple model systems for primitive cellular assemblies. Charged lipid vesicles aggregate in the presence of oppositely charged biopolymers, such as nucleic acids or polypeptides. Based on zeta potential measurements, dynamic light scattering and cryo-transmission electron-microscopy, we have characterized the behavior of empty and ferritin-filled large unilamellar POPC vesicles, doped with different amounts of cationic (DDAB, CTAB) and anionic (sodium oleate) surfactants, and their aggregation upon the addition of anionic (tRNA, poly-l-glutamic acid) and cationic (poly-l-arginine) biopolymers, respectively. The experimental results are rationalized by a phenomenological modeling approach that predicts the average size of the vesicle aggregates as function of the amount of added biopolymers. In addition, we discuss the mechanism of vesicle aggregation induced by oppositely charged biopolymers. Our study complements previous reports about the formation of giant vesicle clusters and thus provides a general vista on primitive cell systems, based on the association of vesicles into compartmentalized aggregates.
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