A synthetic cytoskeleton based on peptide-DNA crosslinkers was developed to regulate the function of synthetic cells. The design was inspired by actin-binding proteins and their ability to reversibly crosslink or bundle filaments. Peptide filaments, connected through DNA hybridization, form tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and allows for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide-DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. The modular design of peptide-DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells. The study demonstrates that the spatial organization of the peptide-DNA cytoskeleton, guided by the associated crosslinkers, fine-tunes the passive diffusion of micrometre-sized payloads within the droplets and enables the targeted recruitment of DNA-modified signals onto complementary DNA anchors. Encapsulating peptide-DNA within lipid-coated droplets generates cell-like deformations and filopodia-like protrusions. Environmental stresses such as heat induce the cytoskeletons to reorganize and reshape the droplets. This modular peptide-DNA platform allows the plug and play of alternative peptide assemblies or more complex DNA designs to yield emerging functional morphologies. The study highlights the potential of peptide-DNA architectures to enable the introduction of emergent properties into synthetic cells and enhance their functionality. The findings suggest that the spatial organization of synthetic cytoskeletons can be tuned to control the mechanical and biochemical properties of synthetic cells, similar to natural cells. The results demonstrate the potential of peptide-DNA nanotechnology for the construction of functional, fully artificial cells.A synthetic cytoskeleton based on peptide-DNA crosslinkers was developed to regulate the function of synthetic cells. The design was inspired by actin-binding proteins and their ability to reversibly crosslink or bundle filaments. Peptide filaments, connected through DNA hybridization, form tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and allows for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide-DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. The modular design of peptide-DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells. The study demonstrates that the spatial organization of the peptide-DNA cytoskeleton, guided by the associated crosslinkers, fine-tunes the passive diffusion of micrometre-sized payloads within the droplets and enables the targeted recruitment of DNA-modified signals onto complementary DNA anchors. Encapsulating peptide-DNA within lipid-coated droplets generates cell-like deformations and filopodia-like protrusions. Environmental stresses such as heat induce the cytoskeletons to reorganize and reshape the droplets. This modular peptide-DNA platform allows the plug and play of alternative peptide assemblies or more complex DNA designs to yield emerging functional morphologies. The study highlights the potential of peptide-DNA architectures to enable the introduction of emergent properties into synthetic cells and enhance their functionality. The findings suggest that the spatial organization of synthetic cytoskeletons can be tuned to control the mechanical and biochemical properties of synthetic cells, similar to natural cells. The results demonstrate the potential of peptide-DNA nanotechnology for the construction of functional, fully artificial cells.