RESUMO
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.
Assuntos
Lipossomas Unilamelares/química , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Ferritinas/química , Ferritinas/metabolismo , Ácidos Nucleicos/química , Fosfatidilcolinas/química , Ácido Poliglutâmico/química , RNA de Transferência/química , RNA de Transferência/metabolismoRESUMO
The spontaneous formation of lipid vesicles, in particular fatty acid vesicles, is considered an important physical process at the roots of cellular life. It has been demonstrated previously that the addition of fatty acid micelles to preformed vesicles induces vesicle self-reproduction by a growth-division mechanism. Despite multiple experimental efforts, it remains unresolved how vesicles rearrange upon the addition of fresh membrane-forming compounds, and whether solutes that are initially encapsulated inside the mother vesicles are evenly redistributed among the daughter ones. Here we investigate the growth-division of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles, which, following the addition of oleate micelles, form mixed oleate/POPC vesicles. Our approach is based on free-flow electrophoresis (FFE) and cryogenic transmission electronmicroscopy (cryo-TEM). Two new features emerge from this study. FFE analysis unexpectedly reveals that the uptake of oleate micelles by POPC vesicles follows two different pathways depending on the micelles/vesicles ratio. At low oleate molar fractions (<0.35), plain incorporation of oleate into pre-existing POPC vesicles is our dominant observation. In contrast, oleate-rich and oleate-poor daughter vesicles are generated from parent POPC vesicles when the oleate molar fraction exceeds 0.35. Cryo-TEM reveals that when ferritin-filled vesicles grow and divide, some vesicles contain ferritin at increased concentrations, others are empty. Intriguingly, in some cases, ferritin appears to be highly concentrated inside the vesicles. These observations imply a specific redistribution (partitioning) of encapsulated solutes among nascent vesicles during the growth-division steps. We have interpreted our observations by assuming that freshly added oleate molecules are taken-up preferentially (cooperatively) by oleate-rich membrane regions that form spontaneously in POPC/oleate vesicles when a certain threshold (oleate molar fraction ca. 0.35) is surpassed. The proposed cooperative mechanism could be based on differential microscopic constants for oleate/oleic acid dynamics in oleate-rich and oleate-poor membrane regions, which eventually generate populations of oleate-rich and oleate-poor vesicles.
Assuntos
Simulação por Computador , Lipossomos/química , Modelos Químicos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Água/química , Microscopia Crioeletrônica , Fluidez de Membrana , MicelasRESUMO
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 (5-50â µm) to support protein synthesis efficiently; however data about smaller w/o compartments (<1â µm) are lacking. This work focuses on the biosynthesis of the enhanced green fluorescent protein (EGFP) inside sub-micrometric lecithin-based w/o droplets (0.8-1â 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.
Assuntos
Sistema Livre de Células/metabolismo , Emulsões/química , Proteínas de Fluorescência Verde/metabolismo , Óleos/química , Biossíntese de Proteínas , Água/química , Colesterol/metabolismo , Escherichia coli/metabolismo , Hexanóis/química , Lecitinas/química , Tensoativos/químicaRESUMO
How did primitive living cells originate? The formation of early cells, which were probably solute-filled vesicles capable of performing a rudimentary metabolism (and possibly self-reproduction), is still one of the big unsolved questions in origin of life. We have recently used lipid vesicles (liposomes) as primitive cell models, aiming at the study of the physical mechanisms for macromolecules encapsulation. We have reported that proteins and ribosomes can be encapsulated very efficiently, against statistical expectations, inside a small number of liposomes. Moreover the transcription-translation mixture, which realistically mimics a sort of minimal metabolic network, can be functionally reconstituted in liposomes owing to a self-concentration mechanism. Here we firstly summarize the recent advancements in this research line, highlighting how these results open a new vista on the phenomena that could have been important for the formation of functional primitive cells. Then, we present new evidences on the non-random entrapment of macromolecules (proteins, dextrans) in phospholipid vesicle, and in particular we show how enzymatic reactions can be accelerated because of the enhancement of their concentration inside liposomes.
RESUMO
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.
Assuntos
Evolução Biológica , Membrana Celular/química , Lipossomos/química , Origem da Vida , Células Artificiais/química , Células Artificiais/metabolismo , Engenharia Celular , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Composição de Medicamentos , Ferritinas/química , Ferritinas/metabolismo , Cinética , Peptídeos/química , Peptídeos/metabolismo , Ribossomos/química , Ribossomos/metabolismo , TermodinâmicaRESUMO
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.
Assuntos
Células Artificiais/metabolismo , Evolução Molecular Direcionada/métodos , Modelos Biológicos , Biologia Sintética , Aminoácidos/química , Aminoácidos/metabolismo , Células Artificiais/citologia , Evolução Molecular Direcionada/ética , Humanos , Imino Piranoses/química , Imino Piranoses/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Proteínas/química , Proteínas/metabolismo , Ribose/química , Ribose/metabolismo , Relação Estrutura-AtividadeRESUMO
In this mini-review we present some experimental approaches to the important issue in the origin of life, namely the origin of nucleic acids and proteins with specific and functional sequences. The formation of macromolecules on prebiotic Earth faces practical and conceptual difficulties. From the chemical viewpoint, macromolecules are formed by chemical pathways leading to the condensation of building blocks (amino acids, or nucleotides) in long-chain copolymers (proteins and nucleic acids, respectively). The second difficulty deals with a conceptual problem, namely with the emergence of specific sequences among a vast array of possible ones, the huge "sequence space", leading to the question "why these macromolecules, and not the others?" We have recently addressed these questions by using a chemical synthetic biology approach. In particular, we have tested the catalytic activity of small peptides, like Ser-His, with respect to peptide- and nucleotides-condensation, as a realistic model of primitive organocatalysis. We have also set up a strategy for exploring the sequence space of random proteins and RNAs (the so-called "never born biopolymer" project) with respect to the production of folded structures. Being still far from solved, the main aspects of these "open questions" are discussed here, by commenting on recent results obtained in our groups and by providing a unifying view on the problem and possible solutions. In particular, we propose a general scenario for macromolecule formation via fragment-condensation, as a scheme for the emergence of specific sequences based on molecular growth and selection.
RESUMO
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.
Assuntos
Evolução Biológica , Glicólise , Origem da Vida , Via de Pentose Fosfato/genéticaRESUMO
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.
Assuntos
Células Artificiais/metabolismo , Ferritinas/química , Lipossomos/química , Origem da Vida , Compartimento Celular , Ferritinas/metabolismo , RNA/química , RNA/metabolismo , Distribuições EstatísticasRESUMO
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.
Assuntos
Células Artificiais/química , Modelos Biológicos , Modelos Químicos , Origem da Vida , Anidridos/química , Células Artificiais/citologia , Células Artificiais/metabolismo , DNA/química , DNA/metabolismo , Enzimas/química , Enzimas/metabolismo , Código Genético , Homeostase , Hidrólise , Cinética , Ácidos Oleicos/química , OxirreduçãoRESUMO
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.
Assuntos
Aminoácidos/biossíntese , Modelos Biológicos , Origem da Vida , Peptídeo Hidrolases/química , Biossíntese de Proteínas , Sequência de Aminoácidos , Aminoácidos/química , Biocatálise , Evolução Biológica , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Conformação Proteica , Dobramento de Proteína , Solubilidade , ÁguaAssuntos
Sistema Livre de Células , Proteínas de Fluorescência Verde/biossíntese , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Biossíntese de Proteínas , Proteínas de Fluorescência Verde/química , Lipossomos/síntese química , Lipossomos/química , Fosfatidilcolinas/síntese química , Fosfatidilcolinas/química , Transcrição GênicaRESUMO
Chemical synthetic biology (CSB) is a branch of synthetic biology (SB) oriented toward the synthesis of chemical structures alternative to those present in nature. Whereas SB combines biology and engineering with the aim of synthesizing biological structures or life forms that do not exist in nature - often based on genome manipulation, CSB uses and assembles biological parts, synthetic or not, to create new and alternative structures. A short epistemological note will introduce the theoretical concepts related to these fields, whereas the text will be largely devoted to introduce and comment two main projects of CSB, carried out in our laboratory in the recent years. The "Never Born Biopolymers" project deals with the construction and the screening of RNA and peptide sequences that are not present in nature, whereas the "Minimal Cell" project focuses on the construction of semi-synthetic compartments (usually liposomes) containing the minimal and sufficient number of components to perform the basic function of a biological cell. These two topics are extremely important for both the general understanding of biology in terms of function, organization, and development, and for applied biotechnology.
RESUMO
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.
Assuntos
Sistema Livre de Células , Simulação por Computador , Processos Estocásticos , Biologia Sintética/métodos , Algoritmos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Cinética , Lipossomos/metabolismo , Biossíntese de Proteínas , Software , Transcrição GênicaRESUMO
The notion of minimal cells refers to cellular structures that contain the minimal and sufficient complexity to still be defined as living, or at least capable to display the most important features of biological cells. Here we briefly describe the laboratory construction of minimal cells, a project within the broader field of synthetic biology. In particular we discuss the advancements in the preparation of semi-synthetic cells based on the encapsulation of biochemicals inside liposomes, illustrating from the one hand the origin of this research and the most recent developments; and from the other the difficulties and limits of the approach. The role of physicochemical understandings is greatly emphasized.
Assuntos
Células Artificiais , Bactérias/citologia , Biologia Sintética , Vesículas Citoplasmáticas/química , Lipossomos/químicaRESUMO
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.