iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

1.

Cells employ myriad regulatory mechanisms to maintain protein homeostasis, termed proteostasis, to ensure correct cellular function. Dysregulation of proteostasis, which is often induced by physiological stress and ageing, often results in Protein Aggregation in cells. These aggregated structures can perturb normal physiological function, compromising cell integrity and viability, a prime example being early onset of several neurodegenerative diseases. Understanding aggregate dynamics in vivo is therefore of strong interest for biomedicine and pharmacology. However, factors involved in formation, distribution and clearance of intracellular aggregates are poorly understood. Here, we report an improved methodology for production of fluorescent aggregates in model budding yeast which can be detected, tracked and quantified using fluorescence microscopy in live cells. This new openly-available technology, iPAR (inducible Protein Aggregation Reporter), involves monomeric fluorescent protein reporters fused to a ΔssCPY* aggregation biomarker, with expression controlled under the copper-regulated CUP1 promoter. Monomeric tags overcome challenges associated with non-physiological aggregation, whilst CUP1 provides more precise control of protein production. We show that iPAR enables quantitative study of cytoplasmic aggregate kinetics and inheritance features in vivo . If suitably adapted, iPAR offers new potential for studying diseases in other model cellular systems.