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Cell signalling and communication are fundamental to living cellular communities. For the past two decades, there has been continuous development of bottom-up engineered synthetic cells, which have become more and more similar to their natural counterparts. However, we are only scratching the surface with the development of synthetic cellular communities and their integration into natural tissues. Here, we review different intercellular communication mechanisms engineered for synthetic cells and classify them based on their resemblance to natural cell signalling mechanisms: autocrine, paracrine, and juxtacrine. In particular, we highlight recent advances in molecular tools for intercellular communication designs and discuss potential applications of engineering synthetic cellular communities and synthetic cell-natural cell communication. With further advances in this area, synthetic cellular communities will be powerful tools for understanding and manipulating cellular functions, thus unlocking potential applications in biosensing, cellular reprogramming, and sustainability. 

Abstract Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli‐responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non‐specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, a protein‐based platform termed TEV Protease‐mediated Releasable Actin‐binding Protein (TRAP) is designed and constructed for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell‐penetrating peptide membrane translocation. TRAP's efficacy in facilitating light‐activated secretion of both fluorescent and luminescent proteins is demonstrated. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli‐responsive biomaterials, versatile synthetic cell‐based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics. 

Cell signaling through direct physical cell–cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact‐dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with spatial responses. Herein, a light‐activated contact‐dependent communication scheme for synthetic cells is designed and demonstrated. A split luminescent protein is utilized to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. The modular design not only demonstrates contact‐dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact‐dependent signaling mechanisms. 

Constructing molecular classifiers that enable cells to recognize linear and nonlinear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effects of biological constraints such as resource limitation and competitive binding on the function of those classifiers have been left unexplored. Here, we first demonstrate the design of a sigma factor-based perceptron as a molecular classifier working based on the principles of molecular sequestration between the sigma factor and its antisigma molecule. We then investigate how the output of the biomolecular perceptron, i.e., its response pattern or decision boundary, is affected by the competitive binding of sigma factors to a pool of shared and limited resources of core RNA polymerase. Finally, we reveal the influence of sharing limited resources on multilayer perceptron neural networks and outline design principles that enable the construction of nonlinear classifiers using sigma-based biomolecular neural networks in the presence of competitive resource-sharing effects. 

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While expression of soluble proteins is usually successful in common CFE systems, reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful co-translational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells. 

Constructing molecular classifiers that enable cells to recognize linear and non-linear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effect of biological constraints such as resource limitation and competitive binding on the function of those classifiers has been left unexplored. Here, we first demonstrate the design of a sigma factor-based perceptron as a molecular classifier working on the principles of molecular sequestration between the sigma factor and its anti-sigma molecule. We then investigate how the output of the biomolecular perceptron,i.e., its response pattern or decision boundary, is affected by the competitive binding of sigma factors to a pool of shared and limited resources of core RNA polymerase. Finally, we reveal the influence of sharing limited resources on multi-layer perceptron neural networks and outline design principles that enable the construction of non-linear classifiers using sigma-based biomolecular neural networks in the presence of competitive resource-sharing effects. 

Cell signaling through direct physical cell-cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with different types of responses. Here, we design and demonstrate a light-activated contact-dependent communication tool for synthetic cells. We utilize a split bioluminescent protein to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. Our modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms. 

In the pursuit of understanding life, model membranes made of phospholipids were envisaged decades ago as a platform for the bottom-up study of biological processes. Micron-sized lipid vesicles have gained great acceptance as their bilayer membrane resembles the natural cell membrane. Important biological events involving membranes, such as membrane protein insertion, membrane fusion, and intercellular communication, will be highlighted in this review with recent research updates. We will first review different lipid bilayer platforms used for incorporation of integral membrane proteins and challenges associated with their functional reconstitution. We next discuss different methods for reconstitution of membrane fusion and compare their fusion efficiency. Lastly, we will highlight the importance and challenges of intercellular communication between synthetic cells and synthetic cells-to-natural cells. We will summarize the review by highlighting the challenges and opportunities associated with studying membrane–membrane interactions and possible future research directions. 

Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane–membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane–membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell-free expression (CFE) system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane–membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful where cell-cell contacts and communication are recreated in a controlled manner using minimal components.