Hwang, S.-W. | Search bioRxiv

bioRxiv2024

Light-based juxtacrine signaling between synthetic cells

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.

synthetic biology↗

bioRxiv2022

Engineering functional membrane-membrane interface by InterSpy

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 SpyTag/SpyCatcher interaction. Inclusion of split fluorescent protein fragments in the designed InterSpy toolkit allows tracking the formation of membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. We first demonstrate InterSpy by testing split protein designs using a mammalian cell-free expression system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with a desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, since InterSpy is fully genetically encoded, the engineered system is adapted to cells and showcased. InterSpy demonstrates the power of cell-free expression systems in functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful for synthetic biology where cell-cell contacts and communication are recreated in a controlled manner using minimal components.

synthetic biology↗