Crowder Induced Phase Separation Modulates the Huntingtin Protein Aggregation Landscape

Macromolecular crowding is a defining feature of cellular environments and plays an important role in modulating protein aggregation. Here, we examine how mixed macromolecular crowders influence the aggregation of Huntingtin exon-1 protein with a pathogenic polyglutamine length (HD39Q). Using thioflavin-T (ThT) kinetics, we show that the binary mixtures of polyethylene glycol (PEG8), Dextran (40 and 70), and Ficoll 70 produce biphasic aggregation kinetics that deviate from classical single-step behavior. Confocal imaging using labeled proteins and crowders reveals that not only do the binary combinations of crowders give rise to phase separated conditions, but also these drive the aggregation in subtle but distinct ways based on the respective combinations. Moving beyond binary mixtures for a better in-cell mimic, ternary crowder systems were also studied. These generated hierarchically organized droplet architectures thereby further modulating aggregation pathways and associated protein localization. Comparative imaging of the aggregation of a few model proteins (bovine serum albumin, β-lactoglobulin, and lysozyme) demonstrates that aggregation and partitioning behavior depend strongly on both the properties of the biomolecule and phase composition. These findings highlight how mixed macromolecular crowding can reshape the aggregation landscape by adding complexity at both the spatial and temporal levels, thus making these more relevant for physiologically relevant processes.