A recent study published in Nature Chemistry introduces an artificial cytoskeleton designed to provide mechanical support and regulate membrane dynamics in synthetic cell platforms. This system consists of amylose-based coacervates, stabilized by a terpolymer membrane, and a cytoskeleton made from polydiacetylene (PDA) fibrils bundled through electrostatic interactions with a positively charged amylose derivative.
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Background
The cytoskeleton in mammalian cells provides structural support by regulating mechanical properties and stabilizing the cell membrane. It also acts as a scaffold, enabling processes such as cell division and motility. These functions have made it a key focus in artificial cell research.
Artificial cell research uses bottom-up self-assembly to create compartments with cell-like features. This approach offers insights into the organization and behavior of biological systems. Consequently, significant progress has been made in developing artificial or reconstituted cytoskeletons.
Researchers in this study developed an artificial cytoskeleton to examine its role in regulating the mechanical properties of synthetic cells.
Methods
An amylose-based coacervate platform was used to create an artificial cell system with cytoskeletal functionality. Coacervates were formed by combining quaternized amylose (Q-Am), a positively charged derivative, with negatively charged carboxymethylated amylose (Cm-Am). A terpolymer was added to create a semi-permeable layer that stabilizes the structure.
A polydiacetylene (PDA) fibrous network was incorporated as the cytoskeleton. Self-assembly positioned the diacetylenes (DAs) in PDA for topochemical polymerization, resulting in covalent stabilization of the fibrous structures. Carboxylate end groups enabled PDA integration into the positively charged coacervates, forming filamentous structures.
These nanometer-sized fibrils aggregated into micrometer-scale bundles via interactions between the negatively charged DA and Q-Am. Confocal laser scanning microscopy (CLSM) confirmed these entanglements.
Fluorometric analysis quantified encapsulation efficiencies across coacervates with different PDA concentrations. Bioluminescence measurements were performed using a monochromator. Fluorescence recovery after photobleaching (FRAP) assessed cytoskeletal recovery in artificial cells (20–30 µm), monitored in three-second intervals. Real-time deformability cytometry (RT-DC) was used to evaluate the impact of the cytoplasmic network on mechanical properties.
Results and Discussion
The self-assembled polymer-based fibrous network mimicked the structure and function of a natural cytoskeleton. The coacervate platform was compatible with various artificial cell architectures and supported functions such as membrane regulation, biomolecule scaffolding, and mechanical properties similar to those of living cells.
The cytoskeleton showed a strong affinity for biomolecules through reversible and irreversible interactions, enabling precise control over biomolecule concentration and organization. Protein scaffolding within 10 nm proximity was achieved by reconstituting luciferase from its domains, and signal transduction was demonstrated using bioluminescence resonance energy transfer between luciferase and mNeonGreen.
The fluidity and immobile domains in cell membranes could be adjusted by varying the extent of PDA coverage in the cytoskeleton. This control over membrane properties supported biological processes such as cell shape regulation, tissue integrity, and signal transduction.
Unlike traditional platforms like giant unilamellar vesicles, which have liquid-like interiors, the terpolymer membrane-stabilized coacervates formed soft droplets with an elastic shell. Incorporating the biomimetic cytoskeleton created artificial cells with more life-like characteristics, capable of tolerating higher mechanical stresses through even force distribution.
Conclusion
The researchers successfully incorporated a cytoskeleton into artificial cells, achieving precise control over its intracellular positioning. The cytoskeleton provided scaffolding, enabling regulation of mechanical properties and enhancing the functionality of the artificial cells.
The proposed cytoskeleton was unique in its ability to mimic various cell membrane features. However, it lacked dynamic behavior compared to other artificial cytoskeleton systems. Future efforts will focus on developing a more dynamic version, allowing reversible assembly of the fibril network in response to environmental cues.
The current bundling process relies on electrostatic interactions, making disassembly challenging without conditions such as high salt or acidity, which also disrupt the coacervates. To address this, the bundling mechanism needs to be redesigned for reversibility under physiological conditions.
Journal Reference
Novosedlik, S., et al. (2025). Cytoskeleton-functionalized synthetic cells with life-like mechanical features and regulated membrane dynamicity. Nature Chemistry. DOI: 10.1038/s41557-024-01697-5, https://www.nature.com/articles/s41557-024-01697-5
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