Ion A. Ioannou

Synthetic cells are moving beyond compartmentalised microreactors to become integrated components of biological networks that physically and chemically merge with their hosts. Existing reviews classify this field by mode of emission, by chemistry, or by architecture, and so describe the construct rather than its effect. This review takes the receiver's side, treating the functional adaptation of the living host as the readout of bio-hybrid function: how living cells probe their synthetic partners mechanically and adapt to them metabolically. We consider the bio-hybrid interface by how far the feedback loop (sensing-actuation) is closed and set out the evidence that would place a study in each of four tiers: open-loop, half-loop, partial feedback, and closed-loop. Read against these criteria, sixteen of the eighteen studies surveyed here are half-loop, two reach partial feedback, and none is closed-loop. Both partial-feedback systems work the same way, by metabolic interception, which the existing taxonomies have no category for. The one construct that comes closest to closed-loop operation contains no living cell at all. We then trace how single-cell coupling is scaling into hybrid prototissues that act as discovery platforms for pathological microenvironments, and we outline the translational barriers these constructs must survive to reach the clinic.

Synthetic cells are moving beyond compartmentalised microreactors to become integrated components of biological networks that physically and chemically merge with their hosts. Existing reviews classify this field by mode of emission, by chemistry, or by architecture, and so describe the construct rather than its effect. This review takes the receiver's side, treating the functional adaptation of the living host as the readout of bio-hybrid function: how living cells probe their synthetic partners mechanically and adapt to them metabolically. We consider the bio-hybrid interface by how far the feedback loop (sensing-actuation) is closed and set out the evidence that would place a study in each of four tiers: open-loop, half-loop, partial feedback, and closed-loop. Read against these criteria, fifteen of the eighteen studies surveyed here are half-loop, three reach partial feedback, and none is closed-loop. All three partial-feedback systems work the same way, by metabolic interception, which the existing taxonomies have no category for. The one construct that comes closest to closed-loop operation contains no living cell at all. We then trace how single-cell coupling is scaling into hybrid prototissues that act as discovery platforms for pathological microenvironments, and we outline the translational barriers these constructs must survive to reach the clinic.

Eukaryotic cells are characterized by multiple chemically distinct compartments, one of the most notable being the nucleus. Within these compartments, there is a continuous exchange of information, chemicals, and signaling molecules, essential for coordinating and regulating cellular activities. One of the main goals of bottom–up synthetic biology is to enhance the complexity of synthetic cells by establishing functional compartmentalization. There is a need to mimic autonomous signaling between compartments, which in living cells, is often regulated at the genetic level within the nucleus. This advancement is key to unlocking the potential of synthetic cells as cell models and as microdevices in biotechnology. However, a technological bottleneck exists preventing the creation of synthetic cells with a defined nucleus-like compartment capable of genetically programmed intercompartment signaling events. Here, we present an approach for creating synthetic cells with distinct nucleus-like compartments that can encapsulate different biochemical mixtures in discrete compartments. Our system enables in situ protein expression of membrane proteins, enabling autonomous chemical communication between nuclear and cytoplasmic compartments, leading to downstream activation of enzymatic pathways within the cell.

The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of actin networks and changes in their dynamics are related to various diseases, from actin myopathies to uncontrolled cell growth and tumorigenesis. Importantly, the biophysical and mechanical properties of actin and its assemblies are deeply intertwined with the biological functions of the cytoskeleton. Novel tools to study actin and its associated biophysical features are, therefore, of prime importance. Here we develop a new approach which exploits fluorescence lifetime imaging microscopy (FLIM) and environmentally sensitive fluorophores termed molecular rotors, acting as quantitative microviscosity sensors, to monitor dynamic viscoelastic properties of both actin structures and lipid membranes. In order to reproduce a minimal actin cortex in synthetic cell models, we encapsulated and attached actin networks to the lipid bilayer of giant unilamellar vesicles (GUVs). Using a cyanine-based molecular rotor, DiSC₂(3), we show that different types of actin bundles are characterized by distinct packing, which can be spatially resolved using FLIM. Similarly, we show that a lipid bilayer-localized molecular rotor can monitor the effects of attaching cross-linked actin networks to the lipid membrane, revealing an increase in membrane viscosity upon actin attachment. Our approach bypasses constraints associated with existing methods for actin imaging, many of which lack the capability for direct visualization of biophysical properties. It can therefore contribute to a deeper understanding of the role that actin plays in fundamental biological processes and help elucidate the underlying biophysics of actin-related diseases.

The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.