iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

Abstract

1.AbstractCells 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 dynamicsin vivois therefore of strong interest for biomedicine and pharmacology. However, factors involved in formation, distribution and clearance of intracellular aggregates are not fully 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-regulatedCUP1promoter. Monomeric tags overcome challenges associated with non-physiological reporter aggregation, whilstCUP1provides more precise control of protein production. We show that iPAR and the associated bioimaging methodology enables quantitative study of cytoplasmic aggregate kinetics and inheritance featuresin vivo. We demonstrate that iPAR can be used with traditional epifluorescence and confocal microscopy as well as single-molecule precise Slimfield millisecond microscopy. Our results indicate that cytoplasmic aggregates are mobile and contain a broad range of number of iPAR molecules, from tens to several hundred per aggregate, whose mean value increases with extracellular hyperosmotic stress. Time lapse imaging shows that although larger iPAR aggregates associate with nuclear and vacuolar compartments, and for the first time we show directly that these proteotoxic accumulations are not inherited by daughter cells, unlike nuclei and vacuoles. If suitably adapted, iPAR offers new potential for studying diseases relating to protein oligomerization processes in other model cellular systems.