# iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

## TL;DR

The paper introduces iPAR, a new tool to study protein aggregation in yeast cells using fluorescent markers, enabling detailed observation of aggregate behavior in live cells.

## Contribution

The novel contribution is the development of iPAR, an inducible fluorescent reporter system for studying cytoplasmic protein aggregation dynamics in live cells.

## Key findings

- iPAR enables quantitative tracking of cytoplasmic aggregates using fluorescence microscopy.
- Cytoplasmic aggregates are mobile and contain tens to hundreds of iPAR molecules per aggregate.
- Proteotoxic aggregates are not inherited by daughter cells, unlike nuclei and vacuoles.

## Abstract

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 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 ∆ssCPY* aggregation biomarker, with expression controlled under the copper-regulated CUP1 promoter

Monomeric tags overcome challenges associated with non-physiological reporter aggregation, whilst CUP1 provides more precise control of protein production. We show that iPAR and the associated bioimaging methodology enables quantitative study of cytoplasmic aggregate kinetics and inheritance features in 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, we show directly, for the first time, 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.

The online version contains supplementary material available at 10.1186/s44330-025-00023-w.