Surface-catalyzed liquid-liquid phase separation and amyloid-like assembly in microscale compartments

Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid-liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process.