Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells.

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5'-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5'-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s-1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).

Exploiting the photoactivity of bacterial reaction center to investigate liposome dynamics.

Charge recombination kinetics of bacterial photosynthetic protein Reaction Center displays an exquisite sensitivity to the actual occupancy of ubiquinone-10 in its QB-binding site. Here, we have exploited such phenomenon for assessing the growth and the aggregation/fusion of phosphocholine vesicles embedding RC in their membrane, when treated with sodium oleate.

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells.

Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min-1 Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

A Simple Protein Synthesis Model for the PURE System Operation.

The encapsulation of transcription-translation (TX-TL) cell-free machinery inside lipid vesicles (liposomes) is a key element in synthetic cell technology. The PURE system is a TX-TL kit composed of well-characterized parts, whose concentrations are fine tunable, which works according to a modular architecture. For these reasons, the PURE system perfectly fulfils the requirements of synthetic biology and is widely used for constructing synthetic cells. In this work, we present a simplified mathematical model to simulate the PURE system operations. Based on Michaelis-Menten kinetics and differential equations, the model describes protein synthesis dynamics by using 9 chemical species, 6 reactions and 16 kinetic parameters. The model correctly predicts the time course for messenger RNA and protein production and allows quantitative predictions. By means of this model, it is possible to foresee how the PURE system species affect the mechanism of proteins synthesis and therefore help in understanding scenarios where the concentration of the PURE system components has been modified purposely or as a result of stochastic fluctuations (for example after random encapsulation inside vesicles). The model also makes the determination of response coefficients for all species involved in the TX-TL mechanism possible and allows for scrutiny on how chemical energy is consumed by the three PURE system modules (transcription, translation and aminoacylation).

The binding of quinone to the photosynthetic reaction centers: kinetics and thermodynamics of reactions occurring at the QB-site in zwitterionic and anionic liposomes.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

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.

Structure and enzymatic properties of molecular dendronized polymer-enzyme conjugates and their entrapment inside giant vesicles.

Macromolecular hybrid structures were prepared in which two types of enzymes, horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD), were linked to a fluorescently labeled, polycationic, dendronized polymer (denpol). Two homologous denpols of first and second generation were used and compared, and the activities of HRP and SOD of the conjugates were measured in aqueous solution separately and in combination. In the latter case the efficiency of the two enzymes in catalyzing a two-step cascade reaction was evaluated. Both enzymes in the two types of conjugates were highly active and comparable to free enzymes, although the efficiency of the enzymes bound to the second-generation denpol was significantly lower (up to a factor of 2) than the efficiency of HRP and SOD linked to the first-generation denpol. Both conjugates were analyzed by atomic force microscopy (AFM), confirming the expected increase in object size compared to free denpols and demonstrating the presence of enzyme molecules localized along the denpol chains. Finally, giant phospholipid vesicles with diameters of up to about 20 µm containing in their aqueous interior pool a first-generation denpol-HRP conjugate were prepared. The HRP of the entrapped conjugate was shown to remain active toward externally added, membrane-permeable substrates, an important prerequisite for the development of vesicular multienzyme reaction systems.

Light energy transduction in liposome-based artificial cells.

In this work we review the latest strategies for the bottom-up assembly of energetically autonomous artificial cells capable of transducing light energy into chemical energy and support internalized metabolic pathways. Such entities are built by taking inspiration from the photosynthetic machineries found in nature which are purified and reconstituted directly in the membrane of artificial compartments or encapsulated in form of organelle-like structures. Specifically, we report and discuss recent examples based on liposome-technology and multi-compartment (nested) architectures pointing out the importance of this matter for the artificial cell synthesis research field and some limitations and perspectives of the bottom-up approach.

Stochastic simulations of minimal cells: the Ribocell model.

BACKGROUND: Over the last two decades, lipid compartments (liposomes, lipid-coated droplets) have been extensively used as in vitro "minimal" cell models. In particular, simple and complex biomolecular reactions have been carried out inside these self-assembled micro- and nano-sized compartments, leading to the synthesis of RNA and functional proteins inside liposomes. Despite this experimental progress, a detailed physical understanding of the underlying dynamics is missing. In particular, the combination of solute compartmentalization, reactivity and stochastic effects has not yet been clarified. A combination of experimental and computational approaches can reveal interesting mechanisms governing the behavior of micro compartmentalized systems, in particular by highlighting the intrinsic stochastic diversity within a population of "synthetic cells". METHODS: In this context, we have developed a computational platform called ENVIRONMENT suitable for studying the stochastic time evolution of reacting lipid compartments. This software - which implements a Gillespie Algorithm - is an improvement over a previous program that simulated the stochastic time evolution of homogeneous, fixed-volume, chemically reacting systems, extending it to more general conditions in which a collection of similar such systems interact and change over the course of time. In particular, our approach is focused on elucidating the role of randomness in the time behavior of chemically reacting lipid compartments, such as micelles, vesicles or micro emulsions, in regimes where random fluctuations due to the stochastic nature of reacting events can lead an open system towards unexpected time evolutions. RESULTS: This paper analyses the so-called Ribocell (RNA-based cell) model. It consists in a hypothetical minimal cell based on a self-replicating minimum RNA genome coupled with a self-reproducing lipid vesicle compartment. This model assumes the existence of two ribozymes, one able to catalyze the conversion of molecular precursors into lipids and the second able to replicate RNA strands. The aim of this contribution is to explore the feasibility of this hypothetical minimal cell. By deterministic kinetic analysis, the best external conditions to observe synchronization between genome self-replication and vesicle membrane reproduction are determined, while its robustness to random fluctuations is investigated using stochastic simulations, and then discussed.

Cell Hypertrophy: A "Biophysical Roadblock" to Reversing Kidney Injury.

In anamniotes cell loss can typically be compensated for through proliferation, but in amniotes, this capacity has been significantly diminished to accommodate tissue complexity. In order to cope with the increased workload that results from cell death, instead of proliferation highly specialised post-mitotic cells undergo polyploidisation and hypertrophy. Although compensatory hypertrophy is the main strategy of repair/regeneration in various parenchymal tissues, the long-term benefits and its capacity to sustain complete recovery of the kidney has not been addressed sufficiently. In this perspective article we integrate basic principles from biophysics and biology to examine whether renal cell hypertrophy is a sustainable adaptation that can efficiently regenerate tissue mass and restore organ function, or a maladaptive detrimental response.

Stochastic simulations of mixed-lipid compartments: from self-assembling vesicles to self-producing protocells.

The computational platform ENVIRONMENT, developed to simulate stochastically reaction systems in varying compartmentalized conditions [Mavelli and Ruiz-Mirazo: Philos Trans R Soc Lond B Biol Sci 362:1789-1802, 2007; Physical Biology 7(3): 036002, 2010], is here applied to study the dynamic properties and stability of model protocells that start producing their own lipid molecules (e.g., phospholipids), which get inserted in previously self-assembled vesicles, made of precursor amphiphiles (e.g., fatty acids). Attention is mainly focused on the changes that this may provoke in the permeability of the compartment, as well as in its eventual osmotic robustness.

ENVIRONMENT: a computational platform to stochastically simulate reacting and self-reproducing lipid compartments.

'ENVIRONMENT' is a computational platform that has been developed in the last few years with the aim to simulate stochastically the dynamics and stability of chemically reacting protocellular systems. Here we present and describe some of its main features, showing how the stochastic kinetics approach can be applied to study the time evolution of reaction networks in heterogeneous conditions, particularly when supramolecular lipid structures (micelles, vesicles, etc) coexist with aqueous domains. These conditions are of special relevance to understand the origins of cellular, self-reproducing compartments, in the context of prebiotic chemistry and evolution. We contrast our simulation results with real lab experiments, with the aim to bring together theoretical and experimental research on protocell and minimal artificial cell systems.

Kinetic models for autopoietic chemical systems: the role of fluctuations in a homeostatic regime.

Two kinetic models describing the emergence of autopoietic chemical units are presented and discussed: the single reagent autopoietic mechanism (SRAM) and a reduced version (rSRAM). The proposed schemes are inspired to the autopoietic vesicles studied by Zepik et al (2001 Angew. Chem., Int. Ed. Engl. 40 199-202). Deterministic and stochastic analyses are then performed in order to obtain conditions for growth, homeostasis and decay time behaviours of the overall amphiphiles concentration. Only the reduced SRAM is able to exhibit all the three regimes as experimentally observed and in order to obtain details on the time evolution of the aggregates' size distribution, stochastic simulations are carried out. What emerges from the rSRAM simulation outcomes is that random fluctuations can act as selection rules for the size of the autopoietic units in the homeostatic regime suggesting how, in a prebiotic scenario, stochastic fluctuations can select the more robust, in this case larger, as the fittest 'organisms'.

Tailoring density and optical and thermal behavior of gold surfaces and nanoparticles exploiting aromatic dithiols.

Self-assembled monolayers (SAMs) derived of 4-methoxy-terphenyl-3'',5''-dimethanethiol (TPDMT) and 4-methoxyterphenyl-4''-methanethiol (TPMT) have been prepared by chemisorption from solution onto gold thin films and nanoparticles. The SAMs have been characterized by spectroscopic ellipsometry, Raman spectroscopy and atomic force microscopy to determine their optical properties, namely the refractive index and extinction coefficient, in an extended spectral range of 0.75-6.5 eV. From the analysis of the optical data, information on SAMs structural organization has been inferred. Comparison of SAMs generated from the above aromatic thiols to well-known SAMs generated from the alkanethiol dodecanethiol revealed that the former aromatic SAMs are densely packed and highly vertically oriented, with a slightly higher packing density and a absence of molecular inclination in TPMT/Au. The thermal behavior of SAMs has also been monitored using ellipsometry in the temperature range 25-500 degrees C. Gold nanoparticles functionalized by the same aromatic thiols have also been discussed for surface enhanced Raman spectroscopy applications. This study represents a step forward tailoring the optical and thermal behavior of surfaces as well as nanoparticles.

Table-top combined scanning X-ray small angle scattering and transmission microscopies of lipid vesicles dispersed in free-standing gel.

A mm thick free-standing gel containing lipid vesicles made of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) was studied by scanning Small Angle X-ray Scattering (SAXS) and X-ray Transmission (XT) microscopies. Raster scanning relatively large volumes, besides reducing the risk of radiation damage, allows signal integration, improving the signal-to-noise ratio (SNR), as well as high statistical significance of the dataset. The persistence of lipid vesicles in gel was demonstrated, while mapping their spatial distribution and concentration gradients. Information about lipid aggregation and packing, as well as about gel density gradients, was obtained. A posteriori confirmation of lipid presence in well-defined sample areas was obtained by studying the dried sample, featuring clear Bragg peaks from stacked bilayers. The comparison between wet and dry samples allowed it to be proved that lipids do not significantly migrate within the gel even upon drying, whereas bilayer curvature is lost by removing water, resulting in lipids packed in ordered lamellae. Suitable algorithms were successfully employed for enhancing transmission microscopy sensitivity to low absorbing objects, and allowing full SAXS intensity normalization as a general approach. In particular, data reduction includes normalization of the SAXS intensity against the local sample thickness derived from absorption contrast maps. The proposed study was demonstrated by a room-sized instrumentation, although equipped with a high brilliance X-ray micro-source, and is expected to be applicable to a wide variety of organic, inorganic, and multicomponent systems, including biomaterials. The employed routines for data reduction and microscopy, including Gaussian filter for contrast enhancement of low absorbing objects and a region growing segmentation algorithm to exclude no-sample regions, have been implemented and made freely available through the updated in-house developed software SUNBIM.

Movement of giant lipid vesicles induced by millimeter wave radiation change when they contain magnetic nanoparticles.

Superparamagnetic iron oxide nanoparticles are used in a rapidly expanding number of research and practical applications in biotechnology and biomedicine. Recent developments in iron oxide nanoparticle design and understanding of nanoparticle membrane interactions have led to applications in magnetically triggered, liposome delivery vehicles with controlled structure. Here we study the effect of external physical stimuli-such as millimeter wave radiation-on the induced movement of giant lipid vesicles in suspension containing or not containing iron oxide maghemite (γ-Fe2O3) nanoparticles (MNPs). To increase our understanding of this phenomenon, we used a new microscope image-based analysis to reveal millimeter wave (MMW)-induced effects on the movement of the vesicles. We found that in the lipid vesicles not containing MNPs, an exposure to MMW induced collective reorientation of vesicle motion occurring at the onset of MMW switch "on." Instead, no marked changes in the movements of lipid vesicles containing MNPs were observed at the onset of first MMW switch on, but, importantly, by examining the course followed; once the vesicles are already irradiated, a directional motion of vesicles was induced. The latter vesicles were characterized by a planar motion, absence of gravitational effects, and having trajectories spanning a range of deflection angles narrower than vesicles not containing MNPs. An explanation for this observed delayed response could be attributed to the possible interaction of MNPs with components of lipid membrane that, influencing, e.g., phospholipids density and membrane stiffening, ultimately leads to change vesicle movement.

On the way towards 'basic autonomous agents': stochastic simulations of minimal lipid-peptide cells.

In this paper, we apply a recently developed stochastic simulation platform to investigate the dynamic behaviour of minimal 'self-(re-)producing' cellular systems. In particular, we study a set of preliminary conditions for appearance of the simplest forms of autonomy in the context of lipid vesicles (more specifically, lipid-peptide vesicles) that enclose an autocatalytic/proto-metabolic reaction network. The problem is approached from a 'bottom-up' perspective, in the sense that we try to show how relatively simple cell components/processes could engage in a far-from-equilibrium dynamics, staying in those conditions thanks to a rudimentary but effective control of the matter-energy flow through it. In this general scenario, basic autonomy and, together with it, minimal agent systems would appear when (hypothetically pre-biological) cellular systems establish molecular trans-membrane mechanisms that allow them to couple internal chemical reactions with transport processes, in a way that they channel/transform external material-energetic resources into their own means and actively regulate boundary conditions (e.g., osmotic gradients, inflow/outflow of different compounds, ...) that are critical for their constitution and persistence as proto-metabolic cells. The results of our simulations indicate that, before that stage is reached, there are a number of relevant issues that have to be carefully analysed and clarified: especially the immediate effects that the insertion of peptide chains (channel precursors) in the lipid bilayer may have in the structural properties of the membrane (elasticity, permeability, ...) and in the overall dynamic behaviour of the cell.

Extrinsic stochastic factors (solute partition) in gene expression inside lipid vesicles and lipid-stabilized water-in-oil droplets: a review.

The encapsulation of transcription-translation (TX-TL) machinery inside lipid vesicles and water-in-oil droplets leads to the construction of cytomimetic systems (often called 'synthetic cells') for synthetic biology and origins-of-life research. A number of recent reports have shown that protein synthesis inside these microcompartments is highly diverse in terms of rate and amount of synthesized protein. Here, we discuss the role of extrinsic stochastic effects (i.e. solute partition phenomena) as relevant factors contributing to this pattern. We evidence and discuss cases where between-compartment diversity seems to exceed the expected theoretical values. The need of accurate determination of solute content inside individual vesicles or droplets is emphasized, aiming at validating or rejecting the predictions calculated from the standard fluctuations theory. At the same time, we promote the integration of experiments and stochastic modeling to reveal the details of solute encapsulation and intra-compartment reactions.

Do protocells preferentially retain macromolecular solutes upon division/fragmentation? A study based on the extrusion of POPC giant vesicles.

A key process of protocell behaviour is their recursive growth and division. In order to be sustainable, the latter must be characterized by an even and homogeneous partition of the solute molecules initially present in the parent protocell among the daughter ones. Here we have investigated, by means of an artificial division model (extrusion of giant lipid vesicles) and confocal microscopy, the fate of solutes when a large vesicle fragments into many smaller vesicles. Solutes of low- and high-molecular weight such as pyranine, calcein, albumin-FITC, dextran-FITC and carbonic anhydrase have been employed. Although the vesicle extrusion brings about a release of their inner content in the environment, the results shown in this initial report indicate that macromolecules can be partially retained when compared with low-molecular weight ones. Results are discussed from the viewpoint of the life cycle of primitive cells. In particular, the findings suggest that a similar mechanism operating during the critical step of vesicle growth-division could have contributed to primitive evolution.