Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells.

The reconstitution of basic cellular functions in micrometer-sized liposomes has led to a surge of interest in the construction of synthetic cells. Microscopy and flow cytometry are powerful tools for characterizing biological processes in liposomes with fluorescence readouts. However, applying each method separately leads to a compromise between information-rich imaging by microscopy and statistical population analysis by flow cytometry. To address this shortcoming, we here introduce imaging flow cytometry (IFC) for high-throughput, microscopy-based screening of gene-expressing liposomes in laminar flow. We developed a comprehensive pipeline and analysis toolset based on a commercial IFC instrument and software. About 60 thousands of liposome events were collected per run starting from one microliter of the stock liposome solution. Robust population statistics from individual liposome images was performed based on fluorescence and morphological parameters. This allowed us to quantify complex phenotypes covering a wide range of liposomal states that are relevant for building a synthetic cell. The general applicability, current workflow limitations, and future prospects of IFC in synthetic cell research are finally discussed.

Gene-Directed FtsZ Ring Assembly Generates Constricted Liposomes with Stable Membrane Necks.

Mimicking bacterial cell division in well-defined cell-free systems has the potential to elucidate the minimal set of proteins required for cytoskeletal formation, membrane constriction, and final abscission. Membrane-anchored FtsZ polymers are often regarded as a sufficient system to realize this chain of events. By using purified FtsZ and its membrane-binding protein FtsA or the gain-of-function mutant FtsA* expressed in PURE (Protein synthesis Using Reconstituted Elements) from a DNA template, it is shown in this study that cytoskeletal structures are formed, and yield constricted liposomes exhibiting various morphologies. However, the resulting buds remain attached to the parental liposome by a narrow membrane neck. No division events can be monitored even after long-time tracking by fluorescence microscopy, nor when the osmolarity of the external solution is increased. The results provide evidence that reconstituted FtsA-FtsZ proto-rings coating the membrane necks are too stable to enable abscission. The prospect of combining a DNA-encoded FtsZ system with assisting mechanisms to achieve synthetic cell division is discussed.

Min waves without MinC can pattern FtsA-anchored FtsZ filaments on model membranes.

Although the essential proteins that drive bacterial cytokinesis have been identified, the precise mechanisms by which they dynamically interact to enable symmetrical division are largely unknown. In Escherichia coli, cell division begins with the formation of a proto-ring composed of FtsZ and its membrane-tethering proteins FtsA and ZipA. In the broadly proposed molecular scenario for ring positioning, Min waves composed of MinD and MinE distribute the FtsZ-polymerization inhibitor MinC away from mid-cell, where the Z-ring can form. Therefore, MinC is believed to be an essential element connecting the Min and FtsZ subsystems. Here, by combining cell-free protein synthesis with planar lipid membranes and microdroplets, we demonstrate that MinDE drive the formation of dynamic, antiphase patterns of FtsA-anchored FtsZ filaments even in the absence of MinC. These results suggest that Z-ring positioning may be achieved with a more minimal set of proteins than previously envisaged, providing a fresh perspective about synthetic cell division.

Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles.

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes.

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

De novo synthesized Min proteins drive oscillatory liposome deformation and regulate FtsA-FtsZ cytoskeletal patterns.

The Min biochemical network regulates bacterial cell division and is a prototypical example of self-organizing molecular systems. Cell-free assays relying on purified proteins have shown that MinE and MinD self-organize into surface waves and oscillatory patterns. In the context of developing a synthetic cell from elementary biological modules, harnessing Min oscillations might allow us to implement higher-order cellular functions. To convey hereditary information, the Min system must be encoded in a DNA molecule that can be copied, transcribed, and translated. Here, the MinD and MinE proteins are synthesized de novo from their genes inside liposomes. Dynamic protein patterns and accompanying liposome shape deformation are observed. When integrated with the cytoskeletal proteins FtsA and FtsZ, the synthetic Min system is able to dynamically regulate FtsZ patterns. By enabling genetic control over Min protein self-organization and membrane remodeling, our methodology offers unique opportunities towards directed evolution of bacterial division processes in vitro.