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1. Light-based juxtacrine signaling between synthetic cells

   https://doi.org/10.1101/2024.01.05.574425  

   Moghimianavval, Hossein; Loi, Kyle J; Hwang, Sung-Won; Bashirzadeh, Yashar; Liu, Allen P (January 2024, bioRxiv)

   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. 

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2. Engineering Functional Membrane–Membrane Interfaces by InterSpy

   https://doi.org/10.1002/smll.202202104  

   Moghimianavval, Hossein; Patel, Chintan; Mohapatra, Sonisilpa; Hwang, Sung‐Won; Kayikcioglu, Tunc; Bashirzadeh, Yashar; Liu, Allen P.; Ha, Taekjip (May 2022, Small)

   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. 

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3. Strategies and applications of synthetic cell communication

   https://doi.org/10.1038/s41589-025-02002-2  

   Moghimianavval, Hossein; Newell, Charlie; Parvizian, Parsa; Booth, Michael J; Liu, Allen P (August 2025, Nature Chemical Biology)

   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. 

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4. Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling

   https://doi.org/10.1021/acsbiomaterials.4c00885  

   Brien, Hannah J; Lee, Joanne C; Sharma, Jhanvi; Hamann, Catherine A; Spetz, Madeline R; Lippmann, Ethan S; Brunger, Jonathan M (October 2024, ACS Biomaterials Science & Engineering)

   The emerging field of synthetic morphogenesis implements synthetic biology tools to investigate the minimal cellular processes sufficient for orchestrating key developmental events. As the field continues to grow, there is a need for new tools that enable scientists to uncover nuances in the molecular mechanisms driving cell fate patterning that emerge during morphogenesis. Here, we present a platform that combines cell engineering with biomaterial design to potentiate artificial signaling in pluripotent stem cells (PSCs). This platform, referred to as PSC-MATRIX, extends the use of programmable biomaterials to PSCs competent to activate morphogen production through orthogonal signaling, giving rise to the opportunity to probe developmental events by initiating morphogenetic programs in a spatially constrained manner through non-native signaling channels. We show that the PSC-MATRIX platform enables temporal and spatial control of transgene expression in response to bulk, soluble inputs in synthetic Notch (synNotch)-engineered human PSCs for an extended culture of up to 11 days. Furthermore, we used PSC-MATRIX to regulate multiple differentiation events via material-mediated artificial signaling in engineered PSCs using the orthogonal ligand green fluorescent protein, highlighting the potential of this platform for probing and guiding fate acquisition. Overall, this platform offers a synthetic approach to interrogate the molecular mechanisms driving PSC differentiation that could be applied to a variety of differentiation protocols. 

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5. Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles

   https://doi.org/10.1038/s41467-022-29338-w  

   Jones, Ross D.; Qian, Yili; Ilia, Katherine; Wang, Benjamin; Laub, Michael T.; Del Vecchio, Domitilla; Weiss, Ron (December 2022, Nature Communications)

   Abstract Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks. 

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