Vesicle aggregates as a model for primitive cellular assemblies.

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.

Protein Synthesis in Sub-Micrometer Water-in-Oil Droplets.

Water-in-oil (w/o) emulsions are used as a cellular model because of their unique cell-like architecture. Previous works showed the capability of eukaryotic-cell-sized w/o droplets (5-50 µm) to support protein synthesis efficiently; however data about smaller w/o compartments (<1 µm) are lacking. This work focuses on the biosynthesis of the enhanced green fluorescent protein (EGFP) inside sub-micrometric lecithin-based w/o droplets (0.8-1 µm) and on its dependence on the compartments' dynamic properties in terms of solute exchange mechanisms. We demonstrated that protein synthesis is strongly affected by the nature of the lipid interface. These findings could be of value and interest for both basic and applied research.

Prebiotic metabolic networks?

A prebiotic origin of metabolism has been proposed as one of several scenarios for the origin of life. In their recent work, Ralser and colleagues (Keller et al, 2014) observe an enzyme-free, metabolism-like reaction network under conditions reproducing a possible prebiotic environment.

Spontaneous encapsulation and concentration of biological macromolecules in liposomes: an intriguing phenomenon and its relevance in origins of life.

One of the main open questions in origin of life research focuses on the formation, by self-organization, of primitive cells composed by macromolecular compounds enclosed within a semi-permeable membrane. A successful experimental strategy for studying the emergence and the properties of primitive cells relies on a synthetic biology approach, consisting in the laboratory assembly of cell models of minimal complexity (semi-synthetic minimal cells). Despite the recent advancements in the construction and characterization of synthetic cells, an important physical aspect related to their formation is still not well known, namely, the mechanism of solute entrapment inside liposomes (in particular, the entrapment of macromolecules). In the past years, we have investigated this phenomenon and here we shortly review our experimental results. We show how the detailed cryo-transmission electron microscopy analyses of liposome populations created in the presence of ferritin (taken as model protein) or ribosomes have revealed that a small fraction of liposomes contains a high number of solutes, against statistical expectations. The local (intra-liposomal) macromolecule concentration in these liposomes largely exceeds the bulk concentration. A similar behaviour is observed when multi-molecular reaction mixtures are used, whereby the reactions occur effectively only inside those liposomes that have entrapped high number of molecules. If similar mechanisms operated in early times, these intriguing results support a scenario whereby the formation of lipid compartments plays an important role in concentrating the components of proto-metabolic systems-in addition to their well-known functions of confinement and protection.

Chemical synthetic biology.

Although both the most popular form of synthetic biology (SB) and chemical synthetic biology (CSB) share the biotechnologically useful aim of making new forms of life, SB does so by using genetic manipulation of extant microorganism, while CSB utilises classic chemical procedures in order to obtain biological structures which are non-existent in nature. The main query concerning CSB is the philosophical question: why did nature do this, and not that? The idea then is to synthesise alternative structures in order to understand why nature operated in such a particular way. We briefly present here some various examples of CSB, including those cases of nucleic acids synthesised with pyranose instead of ribose, and proteins with a reduced alphabet of amino acids; also we report the developing research on the "never born proteins" (NBP) and "never born RNA" (NBRNA), up to the minimal cell project, where the issue is the preparation of semi-synthetic cells that can perform the basic functions of biological cells.

The minimal autopoietic unit.

It is argued that closed, cell-like compartments, may have existed in prebiotic time, showing a simplified metabolism which was bringing about a primitive form of stationary state- a kind of homeostasis. The autopoietic primitive cell can be taken as an example and there are preliminary experimental data supporting the possible existence of this primitive form of cell activity. The genetic code permits, among other things, the continuous self-reproduction of proteins; enzymic proteins permit the synthesis of nucleic acids, and in this way there is a perfect recycling between the two most important classes of biopolymers in our life. On the other hand, the genetic code is a complex machinery, which cannot be posed at the very early time of the origin of life. And the question then arises, whether some form of alternative beginning, prior to the genetic code, would have been possible: and this is the core of the question asked. Is something with the flavor of early life conceivable, prior to the genetic code? My answer is positive, although I am too well aware that the term "conceivable" does not mean that this something is easily to be performed experimentally. To illustrate my answer, I would first go back to the operational description of cellular life as given by the theory of autopoiesis. Accordingly, a living cell is an open system capable of self-maintenance, due to a process of internal self-regeneration of the components, all within a boundary which is itself product from within. This is a universal code, valid not only for a cell, but for any living macroscopic entity, as no living system exists on Earth which does not obey this principle. In this definition (or better operational description) there is no mention of DNA or genetic code. I added in that definition the term "open system"-which is not present in the primary literature (Varela, et al., 1974) to make clear that every living system is indeed an open system-without this addition, it may seem that with autopoiesis we are dealing with a perpetuum mobile, against the second principle of thermodynamics. Now consider the following figure (Fig. 1). It represents in a very schematic form a cell, as an open system, with a semipermeable membrane constituted by the chemical S, which permits the entrance of the nutrient A and the elimination of the decay product P. A is transformed inside the cell into S by a chemical reaction characterized by kgen, and S can be transformed into P by the reaction kdec. The two reactions actually may represent two entire families of reaction, in the sense that one can envisage several A and several S and several P.

A new start from ground zero?

It is pointed out that one of the main reasons of lack of real conceptual progress in the field may lie in the fact that questions concerning the biogenesis of macromolecules have never been asked or addressed in a proper way. We should start again research on the origin of life starting from "ground zero" and focusing on the prebiotic synthesis of ordered sequences of proteins and nucleic acids.

Spontaneous overcrowding in liposomes as possible origin of metabolism.

We emphasize here that, in considering the initial prebiotic reactions, it is fundamental to take into consideration the critical threshold concentration, in particular when talking about self-replication and initial metabolism. It is also shown that the in situ formation of vesicles in a solution containing macromolecular solutes, permits to obtain filled vesicles which are overcrowded of those solutes and therefore viable for complex biochemical reactions.

Quasi-cellular systems: stochastic simulation analysis at nanoscale range.

BACKGROUND: The wet-lab synthesis of the simplest forms of life (minimal cells) is a challenging aspect in modern synthetic biology. Quasi-cellular systems able to produce proteins directly from DNA can be obtained by encapsulating the cell-free transcription/translation system PURESYSTEM(PS) in liposomes. It is possible to detect the intra-vesicle protein production using DNA encoding for GFP and monitoring the fluorescence emission over time. The entrapment of solutes in small-volume liposomes is a fundamental open problem. Stochastic simulation is a valuable tool in the study of biochemical reaction at nanoscale range. QDC (Quick Direct-Method Controlled), a stochastic simulation software based on the well-known Gillespie's SSA algorithm, was used. A suitable model formally describing the PS reactions network was developed, to predict, from inner species concentrations (very difficult to measure in small-volumes), the resulting fluorescence signal (experimentally observable). RESULTS: Thanks to suitable features specific of QDC, we successfully formalized the dynamical coupling between the transcription and translation processes that occurs in the real PS, thus bypassing the concurrent-only environment of Gillespie's algorithm. Simulations were firstly performed for large liposomes (2.67µm of diameter) entrapping the PS to synthetize GFP. By varying the initial concentrations of the three main classes of molecules involved in the PS (DNA, enzymes, consumables), we were able to stochastically simulate the time-course of GFP-production. The sigmoid fit of the GFP-production curves allowed us to extract three quantitative parameters which are significantly dependent on the various initial states. Then we extended this study for small-volume liposomes (575 nm of diameter), where it is more complex to infer the intra-vesicle composition, due to the expected anomalous entrapment phenomena. We identified almost two extreme states that are forecasted to give rise to significantly different experimental observables. CONCLUSIONS: The present work is the first one describing in the detail the stochastic behavior of the PS. Thanks to our results, an experimental approach is now possible, aimed at recording the GFP production kinetics in very small micro-emulsion droplets or liposomes, and inferring, by using the simulation as a reverse-engineering procedure, the internal solutes distribution, and shed light on the still unknown forces driving the entrapment phenomenon.

Formation of RNA phosphodiester bond by histidine-containing dipeptides.

A new scenario for prebiotic formation of nucleic acid oligomers is presented. Peptide catalysis is applied to achieve condensation of activated RNA monomers into short RNA chains. As catalysts, L-dipeptides containing a histidine residue, primarily Ser-His, were used. Reactions were carried out in self-organised environment, a water-ice eutectic phase, with low concentrations of reactants. Incubation periods up to 30 days resulted in the formation of short oligomers of RNA. During the oligomerisation, an active intermediate (dipeptide-mononucleotide) is produced, which is the reactive species. Details of the mechanism and kinetics, which were elucidated with a set of control experiments, further establish that the imidazole side chain of a histidine at the carboxyl end of the dipeptide plays a crucial role in the catalysis. These results suggest that this oligomerisation catalysis occurs by a transamination mechanism. Because peptides are much more likely products of spontaneous condensation than nucleotide chains, their potential as catalysts for the formation of RNA is interesting from the origin-of-life perspective. Finally, the formation of the dipeptide-mononucleotide intermediate and its significance for catalysis might also be viewed as the tell-tale signs of a new example of organocatalysis.

Giant vesicles "colonies": a model for primitive cell communities.

Current research on the origin of life typically focuses on the self-organisation of molecular components in individual cell-like compartments, thereby bringing about the emergence of self-sustaining minimal cells. This is justified by the fact that single cells are the minimal forms of life. No attempts have been made to investigate the cooperative mechanisms that could derive from the assembly of individual compartments. Here we present a novel experimental approach based on vesicles "colonies" as a model of primitive cell communities. Experiments show that several advantages could have favoured primitive cell colonies when compared with isolated primitive cells. In fact there are two novel unexpected features typical of vesicle colonies, namely solute capture and vesicle fusion, which can be seen as the basic physicochemical mechanisms at the origin of life.

Encapsulation of ferritin, ribosomes, and ribo-peptidic complexes inside liposomes: insights into the origin of metabolism.

Here we summarize the main results of our latest investigation on the spontaneous encapsulation of proteins (ferritin) and ribosomes inside lipid vesicles. We show that when vesicles form in a solution containing some macromolecules (even at low concentration), in contrast to the expectations, a few but measurable number of vesicles is able to capture a very high number of solutes, up to 60 times the external concentration. We also show preliminary evidences on the encapsulation of additional solutes (ribo-peptidic complexes, fluorescent proteins and enzymes), and shortly present our current approach aimed at exploiting this phenomenon. In particular, we would like to reveal how the formation of compartments can trigger effective intra-vesicle reactions starting from diluted solutions. Although the mechanistic details for this phenomenon are still missing, we claim that these new evidences are highly relevant for the origin of the first functional cells in primitive times.

An open question on the origin of life: the first forms of metabolism.

The general framework of the origin of life on Earth is outlined, emphasizing that the so-called prebiotic 'RNA world' is as yet on shaky scientific ground, and that one should any way ask the question of the structure of the first protocellular compartments capable of the initial forms of metabolism. This question is the basis of the research project on the minimal cells, containing the minimal and sufficient complexity capable of leading to life. Such research is briefly summarized, highlighting experiments with liposome-based semisynthetic cells which are capable of ribosomal protein synthesis with a very minimal number of enzymes. The most recent finding in this area of research is the unexpected observation that the formation and closure of liposomes in situ acts as an attractor for the solute molecules in solution, bringing about a very high local concentration in some of the liposomes. It is argued that this spontaneous overcrowding, which permits reactions which are not possible in the original dilute solution, might be the origin of cellular metabolism for the origin of life on Earth.

Spontaneous crowding of ribosomes and proteins inside vesicles: a possible mechanism for the origin of cell metabolism.

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.

A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells.

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity--inside liposomes--of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within - which corresponds to the autopoietic growth mechanism.

Towards an autopoietic redefinition of life.

In this paper we develop the autopoietic approach to the definition of the living developed by Maturana and Varela in the Seventies. Starting from very simple observations concerning the phenomenology of life, we propose a reformulation of the autopoietic original definition of life which integrates some of the contemporary criticism to it. Our definitional proposal, aiming to stimulate the further development of the autopoietic approach, expresses what remains implicit in the definition of the living originally given by Maturana and Varela: life, as self-production, is a process of cognitive coupling with the environment.

Massive non-natural proteins structure prediction using grid technologies.

BACKGROUND: The number of natural proteins represents a small fraction of all the possible protein sequences and there is an enormous number of proteins never sampled by nature, the so called "never born proteins" (NBPs). A fundamental question in this regard is if the ensemble of natural proteins possesses peculiar chemical and physical properties or if it is just the product of contingency coupled to functional selection. A key feature of natural proteins is their ability to form a well defined three-dimensional structure. Thus, the structural study of NBPs can help to understand if natural protein sequences were selected for their peculiar properties or if they are just one of the possible stable and functional ensembles. METHODS: The structural characterization of a huge number of random proteins cannot be approached experimentally, thus the problem has been tackled using a computational approach. A large random protein sequences library (2 x 10(4) sequences) was generated, discarding amino acid sequences with significant similarity to natural proteins, and the corresponding structures were predicted using Rosetta. Given the highly computational demanding problem, Rosetta was ported in grid and a user friendly job submission environment was developed within the GENIUS Grid Portal. Protein structures generated were analysed in terms of net charge, secondary structure content, surface/volume ratio, hydrophobic core composition, etc. RESULTS: The vast majority of NBPs, according to the Rosetta model, are characterized by a compact three-dimensional structure with a high secondary structure content. Structure compactness and surface polarity are comparable to those of natural proteins, suggesting similar stability and solubility. Deviations are observed in alpha helix-beta strands relative content and in hydrophobic core composition, as NBPs appear to be richer in helical structure and aromatic amino acids with respect to natural proteins. CONCLUSION: The results obtained suggest that the ability to form a compact, ordered and water-soluble structure is an intrinsic property of polypeptides. The tendency of random sequences to adopt alpha helical folds indicate that all-alpha proteins may have emerged early in pre-biotic evolution. Further, the lower percentage of aromatic residues observed in natural proteins has important evolutionary implications as far as tolerance to mutations is concerned.

The minimal size of liposome-based model cells brings about a remarkably enhanced entrapment and protein synthesis.

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.

Vesicle behavior: in search of explanations.

In this paper we will present a series of experiments in the general field of surfactant aggregates and, in particular, in vesicle chemistry, which have found no definitive explanation until now. These experiments concern vesicle self-reproduction (in particular, the so-called matrix effect); the interaction between vesicle and RNA, where RNA appears capable of discriminating between vesicles differing slightly in size; the fusion of oppositely charged vesicles, which brings about unexpected behavior of size distribution; and some aspects of local concentration inside vesicles, which still lack clarification in terms of local versus overall concentration. The theoretical and experimental implications of this not yet understood behavior will be discussed, emphasizing that progress in the field must face the difficulty of applying thermodynamics to these kinetically trapped systems, and the general difficulty of understanding how kinetic and thermodynamic factors interplay with each other.