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.

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.

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.

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).

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.

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.

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.

Novel directions in molecular systems design: The case of light-transducing synthetic cells.

Important progresses have been achieved in the past years in the field of bottom-up synthetic biology, especially aiming at constructing cell-like systems based on lipid vesicles (liposomes) entrapping both biomolecules or synthetic compounds. These "synthetic cells" mimic the behaviour of biological cells but are constituted by a minimal number of components. One key aspect related to this research is the energetic needs of synthetic cells. Up to now, high-energy compounds have been given in order to drive biochemical reactions inside the vesicle lumen. In order to be autonomous, synthetic cells must produce their own biochemical energy from available energy sources. At this aim we started a long-term research program focused on the construction of photoautotrophic synthetic cells, starting with the reconstitution, in active and highly oriented form, of the photosynthetic reaction centre in giant lipid vesicles (Altamura et al., PNAS 2017, 114, 3837-3842). Here we comment this first milestone by showing the synthetic biology context wherein it is developed, the future steps, and the experimental approach that might allow such an achievement.

Crude phosphorylation mixtures containing racemic lipid amphiphiles self-assemble to give stable primitive compartments.

It is an open question how the chemical structure of prebiotic vesicle-forming amphiphiles complexified to produce robust primitive compartments that could safely host foreign molecules. Previous work suggests that comparingly labile vesicles composed of plausibly prebiotic fatty acids were eventually chemically transformed with glycerol and a suitable phosphate source into phospholipids that would form robust vesicles. Here we show that phosphatidic acid (PA) and phosphatidylethanolamine (PE) lipids can be obtained from racemic dioleoyl glycerol under plausibly prebiotic phosphorylation conditions. Upon in situ hydration of the crude phosphorylation mixtures only those that contained rac-DOPA (not rac-DOPE) generated stable giant vesicles that were capable of encapsulating water-soluble probes, as evidenced by confocal microscopy and flow cytometry. Chemical reaction side-products (identified by IR and MS and quantified by 1H NMR) acted as co-surfactants and facilitated vesicle formation. To mimic the compositional variation of such primitive lipid mixtures, self-assembly of a combinatorial set of the above amphiphiles was tested, revealing that too high dioleoyl glycerol contents inhibited vesicle formation. We conclude that a decisive driving force for the gradual transition from unstable fatty acid vesicles to robust diacylglyceryl phosphate vesicles was to avoid the accumulation of unphosphorylated diacylglycerols in primitive vesicle membranes.

First moves towards photoautotrophic synthetic cells: In vitro study of photosynthetic reaction centre and cytochrome bc1 complex interactions.

Following a bottom-up synthetic biology approach it is shown that vesicle-based cell-like systems (shortly "synthetic cells") can be designed and assembled to perform specific function (for biotechnological applications) and for studies in the origin-of-life field. We recently focused on the construction of synthetic cells capable to converting light into chemical energy. Here we first present our approach, which has been realized so far by the reconstitution of photosynthetic reaction centre in the membrane of giant lipid vesicles. Next, the details of our ongoing research program are presented. It involves the use of the reaction centre, the coenzyme Q-cytochrome c oxidoreductase, and the ATP synthase for creating an autonomous synthetic cell. We show experimental results on the chemistry of the first two proteins showing that they can efficiently sustain light-driven chemical oscillations. Moreover, the cyclic pattern has been reproduced in silico by a minimal kinetic model.

Permeability-driven selection in a semi-empirical protocell model: the roots of prebiotic systems evolution.

The origin-of-life problem has been traditionally conceived as the chemical challenge to find the type of molecule and free-solution reaction dynamics that could have started Darwinian evolution. Different autocatalytic and 'self-replicative' molecular species have been extensively investigated, together with plausible synthetic pathways that might have led, abiotically, to such a minimalist scenario. However, in addition to molecular kinetics or molecular evolutionary dynamics, other physical and chemical constraints (like compartmentalization, differential diffusion, selective transport, osmotic forces, energetic couplings) could have been crucial for the cohesion, functional integration, and intrinsic stability/robustness of intermediate systems between chemistry and biology. These less acknowledged mechanisms of interaction and molecular control might have made the initial pathways to prebiotic systems evolution more intricate, but were surely essential for sustaining far-from-equilibrium chemical dynamics, given their functional relevance in all modern cells. Here we explore a protocellular scenario in which some of those additional constraints/mechanisms are addressed, demonstrating their 'system-level' implications. In particular, an experimental study on the permeability of prebiotic vesicle membranes composed of binary lipid mixtures allows us to construct a semi-empirical model where protocells are able to reproduce and undergo an evolutionary process based on their coupling with an internal chemistry that supports lipid synthesis.

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.

Enzymatic reactions in confined environments.

Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

Experiments on and Numerical Modeling of the Capture and Concentration of Transcription-Translation Machinery inside Vesicles.

Synthetic or semi-synthetic minimal cells are those cell-like artificial compartments that are based on the encapsulation of molecules inside lipid vesicles (liposomes). Synthetic cells are currently used as primitive cell models and are very promising tools for future biotechnology. Despite the recent experimental advancements and sophistication reached in this field, the complete elucidation of many fundamental physical aspects still poses experimental and theoretical challenges. The interplay between solute capture and vesicle formation is one of the most intriguing ones. In a series of studies, we have reported that when vesicles spontaneously form in a dilute solution of proteins, ribosomes, or ribo-peptidic complexes, then, contrary to statistical predictions, it is possible to find a small fraction of liposomes (<1%) that contain a very large number of solutes, so that their local (intravesicular) concentrations largely exceed the expected value. More recently, we have demonstrated that this effect (spontaneous crowding) operates also on multimolecular mixtures, and can drive the synthesis of proteins inside vesicles, whereas the same reaction does not proceed at a measurable rate in the external bulk phase. Here we firstly introduce and discuss these already published observations. Then, we present a computational investigation of the encapsulation of transcription-translation (TX-TL) machinery inside vesicles, based on a minimal protein synthesis model and on different solute partition functions. Results show that experimental data are compatible with an entrapment model that follows a power law rather than a Gaussian distribution. The results are discussed from the viewpoint of origin of life, highlighting open questions and possible future research directions.

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).

Physical Routes to Primitive Cells: An Experimental Model Based on the Spontaneous Entrapment of Enzymes inside Micrometer-Sized Liposomes.

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.

Modelling lipid competition dynamics in heterogeneous protocell populations.

Recent experimental work in the field of synthetic protocell biology has shown that prebiotic vesicles are able to 'steal' lipids from each other. This phenomenon is driven purely by asymmetries in the physical state or composition of the vesicle membranes, and, when lipid resource is limited, translates directly into competition amongst the vesicles. Such a scenario is interesting from an origins of life perspective because a rudimentary form of cell-level selection emerges. To sharpen intuition about possible mechanisms underlying this behaviour, experimental work must be complemented with theoretical modelling. The aim of this paper is to provide a coarse-grain mathematical model of protocell lipid competition. Our model is capable of reproducing, often quantitatively, results from core experimental papers that reported distinct types vesicle competition. Additionally, we make some predictions untested in the lab, and develop a general numerical method for quickly solving the equilibrium point of a model vesicle population.