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Abstract 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. Here, a protein‐based platform termed TEV Protease‐mediated Releasable Actin‐binding Protein (TRAP) is designed and constructed for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell‐penetrating peptide membrane translocation. TRAP's efficacy in facilitating light‐activated secretion of both fluorescent and luminescent proteins is demonstrated. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli‐responsive biomaterials, versatile synthetic cell‐based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics. 

Creating artificial cells with a dynamic cytoskeleton, akin to those in living cells, is a major goal in bottom‐up synthetic biology. In this study, we demonstrate the in situ polymerization of microtubules encapsulated in giant polymer‐lipid hybrid vesicles (GHVs) composed of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine and an amphiphilic block copolymer. The block copolymer is comprised of poly(cholesteryl methacrylate‐co‐butyl methacrylate) as the hydrophobic block and either poly(6‐O‐methacryloyl‐D‐galactopyranose) or poly(carboxyethyl acrylate) as the hydrophilic extension. Depending on the concentrations of guanosine triphosphate (GTP) or its slowly hydrolyzable analog, guanosine‐5′‐[(α,β)‐methyleno]triphosphate (GMPCPP), different microtubule morphologies are observed, including encapsulated microtubule networks, spike protrusions, as well as membrane‐associated or aggregated microtubules. Overall, this work represents a step forward in mimicking the cellular cytoskeletons and uncovering the influence of membrane composition on microtubule morphologies.