Crowder Induced Phase Separation Modulates the Huntingtin Protein Aggregation Landscape

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Macromolecular crowding is crucial in cellular environments, influencing protein aggregation dynamics.
• Understanding these effects is vital for comprehending cellular processes and disease mechanisms.

Purpose of the Study:

• To investigate the impact of mixed macromolecular crowders on the aggregation of Huntingtin exon-1 protein (HD39Q).
• To explore how varying crowder combinations and concentrations modulate protein aggregation pathways and localization.

Main Methods:

• Utilized thioflavin-T (ThT) kinetics to monitor aggregation rates.
• Employed confocal imaging with labeled proteins and crowders to visualize phase separation and protein distribution.
• Studied both binary and ternary crowder systems to mimic cellular complexity.

Main Results:

• Binary crowder mixtures (PEG8, Dextran, Ficoll 70) induced biphasic aggregation kinetics, deviating from simple models.
• Crowder mixtures led to phase separation, influencing aggregation pathways distinctly based on composition.
• Ternary crowder systems formed hierarchical droplet architectures, further altering aggregation and protein localization.
• Aggregation and partitioning were dependent on biomolecule properties and the specific phase composition.

Conclusions:

• Mixed macromolecular crowding significantly reshapes protein aggregation landscapes in vitro.
• These complex crowding environments add spatial and temporal complexity, offering more physiologically relevant models.
• Findings provide insights into disease mechanisms involving protein aggregation, such as Huntington's disease.