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1. Engineering transmembrane signal transduction in synthetic membranes using two-component systems

   https://doi.org/10.1073/pnas.2218610120  

   Peruzzi, Justin A.; Galvez, Nina R.; Kamat, Neha P. (May 2023, Proceedings of the National Academy of Sciences)

   Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step toward this goal, we investigated how bacterial two-component systems (TCSs) can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the TCS can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing. 

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2. Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

   https://doi.org/10.3791/66595  

   Loi, Kyle J; Moghimianavval, Hossein; Liu, Allen P (March 2024, Journal of Visualized Experiments)

   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. 

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3. Light-guided actin polymerization drives directed motility in protocells

   https://doi.org/10.1101/2024.10.14.617543  

   Matsubayashi, Hideaki T; Razavi, Shiva; Rock, T Willow; Nakajima, Daichi; Nakamura, Hideki; Kramer, Daniel A; Matsuura, Tomoaki; Chen, Baoyu; Murata, Satoshi; Nomura, Shinichiro M; et al (October 2024, bioRxiv)

   Motility is a hallmark of life’s dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, recapitulating this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 µm/min, comparable to typical adherent mammalian cells. Notably, our findings reveal a synergistic interplay between branched and linear actin forms in promoting membrane protrusions, highlighting the cooperative nature of these cytoskeletal elements. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials. 

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4. Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications

   https://doi.org/10.1021/acsabm.3c01267  

   Smith, Amanda J.; Larsen, Theodore R.; Zimmerman, Harmony K.; Virolainen, Samuel J.; Meyer, Joshua J.; Keranen Burden, Lisa M.; Burden, Daniel L. (March 2024, ACS Applied Bio Materials)

   Artificial lipid bilayers have revolutionized biochemical and biophysical research by providing a versatile interface to study aspects of cell membranes and membrane-bound processes in a controlled environment. Artificial bilayers also play a central role in numerous biosensing applications, form the foundational interface for liposomal drug delivery, and provide a vital structure for the development of synthetic cells. But unlike the envelope in many living cells, artificial bilayers can be mechanically fragile. Here, we develop prototype scaffolds for artificial bilayers made from multiple chemically linked tiers of actin filaments that can be bonded to lipid headgroups. We call the interlinked and layered assembly a multiple minimal actin cortex (multi-MAC). Construction of multi-MACs has the potential to significantly increase the bilayer’s resistance to applied stress while retaining many desirable physical and chemical properties that are characteristic of lipid bilayers. Furthermore, the linking chemistry of multi-MACs is generalizable and can be applied almost anywhere lipid bilayers are important. This work describes a filament-by-filament approach to multi-MAC assembly that produces distinct 2D and 3D architectures. The nature of the structure depends on a combination of the underlying chemical conditions. Using fluorescence imaging techniques in model planar bilayers, we explore how multi-MACs vary with electrostatic charge, assembly time, ionic strength, and type of chemical linker. We also assess how the presence of a multi-MAC alters the underlying lateral diffusion of lipids and investigate the ability of multi-MACs to withstand exposure to shear stress. 

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5. Turn-on protein switches for controlling actin binding in cells

   https://doi.org/10.1038/s41467-024-49934-2  

   Effiong, Unyime M; Khairandish, Hannah; Ramirez-Velez, Isabela; Wang, Yanran; Belardi, Brian (December 2024, Nature Communications)

   Abstract Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP’s influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality and multiplexing. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into structures to control cell and tissue shape and behavior. 

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