Light-triggered protease-mediated release of actin-bound cargo from synthetic cells.

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.

Engineering Sequestration-Based Biomolecular Classifiers with Shared Resources.

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.

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

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 the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging.

A Mammalian-Based Synthetic Biology Toolbox to Engineer Membrane-Membrane Interfaces.

Intercellular membrane-membrane interfaces are compartments with specialized functions and unique biophysical properties that are essential in numerous cellular processes including cell signaling, development, and immunity. Using synthetic biology to engineer or to create novel cellular functions in the intercellular regions has led to an increasing need for a platform that allows generation of functionalized intercellular membrane-membrane interfaces. Here, we present a synthetic biology platform to engineer functional membrane-membrane interfaces using a pair of dimerizing proteins in both cell-free and cellular environments.

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.

Hybrid Vesicles Enable Mechano-Responsive Hydrogel Degradation.

Stimuli-responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano-responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano-responsive behavior on hydrogels.

Calcium-triggered DNA-mediated membrane fusion in synthetic cells.

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.

Calcium-triggered DNA-mediated membrane fusion in synthetic cells.

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.

Synthesis and Reconstitution Using Mammalian Cell-Free Lysates Enables the Systematic Study of the Regulation of LINC Complex Assembly.

Understanding the structure and structure-function relationships of membrane proteins is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins , we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional inner nuclear membrane Sad1/UNC-84 (SUN) proteins (i.e.

Engineering Functional Membrane-Membrane Interfaces 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 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.

Encapsulated actomyosin patterns drive cell-like membrane shape changes.

Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as α-actinin and fascin as well as actin nucleator Arp2/3 complex largely determine actin network architecture and, consequently, membrane shape changes. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that depending on vesicle size and concentrations of α-actinin and fascin actomyosin networks assemble into ring and aster-like patterns.

In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins.

Membrane proteins are essential components in cell membranes and enable cells to communicate with their outside environment and to carry out intracellular signaling. Functional reconstitution of complex membrane proteins using cell-free expression (CFE) systems has been proved to be challenging mainly due to the lack of necessary machinery for proper folding and translocation of nascent membrane proteins and their delivery to the supplied synthetic bilayers. Here, we provide protocols for detergent-free, cell-free reconstitution of functional membrane proteins using HeLa-based CFE system and outline assays for studying their membrane insertion, topology, and their orientation upon incorporation into the supported lipid bilayers or bilayers of giant unilamellar vesicles as well as methods to isolate functional translocated cell-free produced membrane proteins.

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions.

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.

Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture.

The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size.

Engineering spatiotemporal organization and dynamics in synthetic cells.

Constructing synthetic cells has recently become an appealing area of research. Decades of research in biochemistry and cell biology have amassed detailed part lists of components involved in various cellular processes. Nevertheless, recreating any cellular process in vitro in cell-sized compartments remains ambitious and challenging.