Molecular crowding in synthetic cells

What is it about?

This pioneering research reveals how crowding with a biologically active molecule like ATP enables precise control over reaction environments, opening new possibilities for synthetic biology and beyond. At the heart of their work is an innovative, all-aqueous, template-directed assembly method that allows the team to build "polysaccharidosomes"—membranized microcapsules made from polysaccharides. These tiny structures simulate cellular crowding by harnessing liquid-liquid phase separation (LLPS), a process that cells use to form functional compartments without membranes. This breakthrough in creating LLPS-driven molecular crowding within a scalable and biocompatible system marks a significant advance in developing biomimetic systems.

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Photo by Adrien Converse on Unsplash

Photo by Adrien Converse on Unsplash

Why is it important?

Unlocking the Power of Molecular Crowding Molecular crowding is a defining feature of the densely packed environment inside living cells, where macromolecules like proteins, nucleic acids, and metabolites are jostling for space. This crowding doesn’t just affect the physical arrangement of molecules—it profoundly impacts how biochemical reactions unfold. By altering molecular mobility and reaction kinetics, crowding can boost certain reactions while slowing others. In living cells, this dense packing helps form membraneless organelles like stress granules and nucleoli, which use LLPS to compartmentalize reactions. Mukwaya and his colleagues tapped into this principle by recreating crowded conditions inside their protocells. Using ATP as a molecular crowder, they showed how increasing ATP concentrations leads to different biochemical outcomes. A Revolutionary Assembly Technique Traditional methods of constructing microcapsules often rely on organic solvents, which can compromise the biocompatibility of the encapsulated materials and make scaling up the process difficult. By contrast, this research team developed a streamlined, aqueous-based method that overcomes these issues. Their technique uses CaCO₃ particles as sacrificial templates, which not only simplifies the assembly process but also allows for the precise loading of macromolecules—such as proteins—into well-defined polysaccharide-based microcapsules The team’s approach produces uniform microcapsules (polysaccharidosomes), packed with proteins such as BSA (bovine serum albumin), and capable of controlling molecular crowding. Inside these microcapsules, the introduction of DEAE-Dextran (a diffusive polyelectrolyte) triggers spontaneous LLPS, leading to the formation of liquid-like coacervate droplets. These droplets grow and merge into a stable coacervate pool within the protocells, exhibiting fascinating behaviors like dripping and fluorescence recovery after photobleaching (FRAP) The ATP Effect: Controlling Crowding, Diffusion, and Biochemistry ATP, a key molecule in cellular energy metabolism, plays a critical role in modulating the degree of crowding within these synthetic protocells. The researchers found that at low ATP concentrations (1.25–2.5 mM), the BSA proteins formed liquid-like complexes with high diffusivity. But as the ATP concentration increased beyond 5 mM, local diffusion of fluorescently-labeled BSA slowed down significantly, almost halting at 10 mM ATP. This slowdown mirrored what happens in real cells as crowding increases and molecular movement becomes restricted. Interestingly, in protocells containing BSA/DEAE-Dextran LLPS droplets, molecular diffusion decreased even at low ATP concentrations (0.2–1.0 mM), and further declines at higher ATP levels ultimately stopped diffusion altogether. These observations demonstrate how ATP-induced crowding affects both the structure and function of these synthetic cellular models. The Impact on Enzymatic Activity and DNA Communication The team didn’t stop at crowding and diffusion—they also explored how these changes influence key biological processes, like enzyme activity and DNA-based information exchange. By encapsulating enzymes such as Cy5-labeled horseradish peroxidase (Cy5-HRP) and oligonucleotides, the researchers discovered that moderate levels of ATP-induced crowding actually enhanced enzymatic activity without hindering DNA strand displacement reactions, a key mechanism for genetic information transfer. However, when ATP levels rose even higher, the crowding environment became too dense. Enzymatic activity slowed as substrates struggled to reach the enzyme’s active site, and DNA-based communication was disrupted, with incomplete or blocked formation of DNA duplexes. This finding emphasizes the importance of maintaining the right level of crowding for optimal biochemical performance.

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