Intercellular Signaling and Intracellular Signal Processing: Looking Through the Lens of Synthetic Biology

Abstract

Communication enables high-level behaviors such as cooperation or social relationships across all scales of life. The apparent emergent advantages of intercellular signaling and its crucial role in evolution of multicellular life have made development of signaling and communication for synthetic cells---cell-sized compartments often enclosed by a phospholipid bilayer with programmable biological functions---a clear goal. Over the past two decades, cumulative efforts have transformed synthetic cells into powerful, smart bioreactors. However, many potential applications of synthetic cells in both basic and translational science have remained unrealized due to the challenges in developing intercellular signaling for synthetic cells. My thesis work consists of two lines of work in an effort to develop a platform for synthetic cell signaling as well as a general framework for signal processing within synthetic cells.

In Chapter 2, I design a protein-based platform, named InterSpy, for activation of a split-protein exclusively at the contact interface of two membranes. I utilize a peptide-protein pair called SpyTag and SpyCatcher as dimerizing molecules to facilitate split protein reconstitution and activation at membrane-membrane interfaces. I present experimental data that supports InterSpy's application in functionalizing both synthetic and cellular membrane-membrane interfaces with a split fluorescent protein. These results demonstrate InterSpy as a versatile, modular, and general platform for exclusive protein activation at both synthetic and natural cell contact interfaces.

Reconstitution of protein function at the interface of two membranes enables development of intercellular signaling between synthetic cells that are physically in contact, effectively mimicking natural juxtacrine or contact-dependent cell-cell communication. Thus, in Chapter 3, I modify InterSpy as a tool to generate light signals at the membrane-membrane interface of synthetic cells. Instead of a split fluorescent protein, here, InterSpy facilitates reconstitution of a split luciferase, NanoBiT, at the membrane-membrane interface of sender and receiver synthetic cells. To complete the signaling design, I designed receiver cells to encapsulate an optogenetics protein called iLID which dimerizes with its binding partner SspB when it is photoactivated. Experimental data presented in Chapter 3 demonstrates a light-based juxtacrine signaling pathway between synthetic cells utilizing NanoBiT for signal generation and iLID-SspB dimerization for signal detection. Collectively, these results show the potential of InterSpy as a general platform for implementation of various forms of juxtacrine communication between synthetic cells holding potential for biomedical and environmental applications.

Chapter 4 introduces a simple protein-based network capable of demonstrating linear and non-linear input processing. The network is constructed from sigma factors, bacterial transcription factors, and anti-sigma proteins. Dimerization of sigma factors and anti-sigma factors, known as sigma factor sequestration, renders the sigma factor inactive and incapable of driving gene expression. Computational analysis of sigma-based sequestration networks in Chapter 4 demonstrates their ability in creating linear and non-linear input classifier units. In addition, I investigate and determine the effects of physiological resource constraints on the function of sigma-based sequestration networks. Given their simplicity and compatibility with both cellular and cell-free synthetic biology systems, sigma-based sequestration networks hold potential as advanced signal-processing units in synthetic cells.

Overall, the results in my dissertation present a modular synthetic biology platform for reconstitution of protein activity at membrane-membrane interfaces with applications in engineering intercellular signaling between synthetic cells as well as a general design of a protein-based network for intracellular signal processing.