Cyclophospholipids Increase Protocellular Stability to Metal Ions.

Model protocells have long been constructed with fatty acids, because these lipids are prebiotically plausible and can, at least theoretically, support a protocell life cycle. However, fatty acid protocells are stable only within a narrow range of pH and metal ion concentration. This instability is particularly problematic as the early Earth would have had a range of conditions, and extant life is completely reliant on metal ions for catalysis and the folding and activity of biological polymers. Here, prebiotically plausible monoacyl cyclophospholipids are shown to form robust vesicles that survive a broad range of pH and high concentrations of Mg2+ , Ca2+ , and Na+ . Importantly, stability to Mg2+ and Ca2+ is improved by the presence of environmental concentrations of Na+ . These results suggest that cyclophospholipids, or lipids with similar characteristics, may have played a central role during the emergence of Darwinian evolution.

Artificial photosynthetic cell producing energy for protein synthesis.

Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.

Thylakoid Containing Artificial Cells for the Inhibition Investigation of Light-Driven Electron Transfer during Photosynthesis.

The fabrication of artificial cells containing nature components is challenging. Herein we construct a thylakoid containing artificial cell (TA-cell) by forming multicompartmental structure inside giant unilamellar vesicles (GUVs) using osmotic stress. The thylakoids are selectively loaded inside each compartment in GUVs to mimic "chloroplast". The TA-cells are able to carry out photosynthesis upon light on. The TA-cells keep their 50% functionality of electron transfer for 12 days, which is twice of those of free thylakoids. Using TA-cells the inhibition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and heavy metal ions (Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+) on the electron transfer process in TA-cells is systematically investigated. Their half maximal inhibitory concentration (IC50) values are 36.23 ± 1.87, 0.02 ± 0.01, 0.42 ± 0.08, 0.82 ± 0.12, 1.97 ± 0.21, and 4.08 ± 0.18 µM, respectively. Hg2+ is the most toxic ion for the photosynthesis process among these five heavy metal ions. This biomimetic system can be expanded to study other processes during the photosynthesis. The TA-cells pave a way to fabricate more complicated nature component containing artificial cells.

Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways.

In the discipline of bottom-up synthetic biology, vesicles define the boundaries of artificial cells and are increasingly being used as biochemical microreactors operating in physiological environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymatic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chemical pathway to be separated in space, this platform bridges the gap between table-top chemistry and chemistry that is performed within vesicles.

Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour.

Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.

Spontaneous structuration in coacervate-based protocells by polyoxometalate-mediated membrane assembly.

Molecularly crowded, polyelectrolyte/ribonucleotide-enriched membrane-free coacervate droplets are transformed into membrane-bounded sub-divided vesicles by using a polyoxometalate-mediated surface-templating procedure. The coacervate to vesicle transition results in reconstruction of the coacervate micro-droplets into novel three-tiered micro-compartments comprising a semi-permeable negatively charged polyoxometalate/polyelectrolyte outer membrane, a sub-membrane coacervate shell, and an internal aqueous lumen. We demonstrate that organic dyes, ssDNA, magnetic nanoparticles and enzymes can be concentrated into the interior of the micro-compartments by sequestration into the coacervate micro-droplets prior to vesicle formation. The vesicle-encapsulated proteins are inaccessible to proteases in the external medium, and can be exploited for the spatial localization and coupling of two-enzyme cascade reactions within single or between multiple populations of hybrid vesicles dispersed in aqueous media.

Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes.

BACKGROUND: Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs) with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. METHODS: To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs) for the direct and rapid expansion of TILs isolated from primary cancer specimens. RESULTS: TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. CONCLUSION: Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.