Fig. 3: Accumulation of protein aggregates of scaffold proteins and enzymes in aged condensates.

a Confocal images showing the onset and progression of protein aggregates in catalytic condensates. A 1:9 (v/v) ratio of Cy5-labeled to unlabeled MenH-RIDD was used to visualize the enzymes. The co-localization of enzyme and scaffold signals suggests co-aggregation of the proteins. Protein aggregates are indicated by red arrowheads. Scale bar, 10 μm. b Normalized fluorescent intensities along the white lines in a showing the co-localization of FIB1-GFP-RIAD and MenH fluorescent puncta. c Schematic of the comparative extraction assay. Proteins within the condensates were solubilized using a sequential treatment with different buffers (i.e., 1 M NaCl and 6 M guanidine hydrochloride), followed by SDS-PAGE analysis of the extracted components. SDS-PAGE analysis of proteins extracted from condensates using sequential treatments with varying buffers. e Quantification of the data in (d), illustrating the fractions of extracted proteins by different buffers over time. f TEM images of catalytic condensates at different stages. Particles with increased electron density are observed over time, indicating the formation of protein aggregates (top). Higher magnification images reveal an increase in crystalized areas exhibiting an approximately 3.5 Å lattice spacing characteristic of β-sheet structures (bottom). Protein aggregates are indicated by magenta arrowheads. The white dashed outline the edge of the condensates. Scale bars, 200 nm (top) and 0.5 nm (bottom). g Normalized Thioflavin T (ThT) fluorescence signal over time, demonstrating the accumulation of β-sheet structure within the condensates. The normalized fluorescence values were fitted to the Sigmoid function, with R² values > 0.99. Data are represented as mean ± SD from three independent experiments (n = 3). Source data are provided as a Source Data file.