Lecinski, Sarah, Howard, Jamieson Anthony Leyland orcid.org/0000-0002-4694-5427, MacDonald, Chris orcid.org/0000-0002-7450-600X et al. (1 more author) (2025) iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation. BMC Methods. 5. ISSN 3004-8729
Abstract
Background: 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. Methods: 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. Results: 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. Discussion: 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.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © The Author(s) 2025 |
Keywords: | Saccharomyces cerevisiae,Protein aggregation,inheritance,cell ageing,Confocal microscopy,Single-molecule |
Dates: |
|
Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Physics (York) The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Pure (York) |
Date Deposited: | 21 Feb 2025 12:30 |
Last Modified: | 03 Apr 2025 15:50 |
Published Version: | https://doi.org/10.1186/s44330-025-00023-w |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1186/s44330-025-00023-w |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:223653 |
Download
Filename: s44330-025-00023-w.pdf
Description: iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation
Licence: CC-BY 2.5