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Background <p>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&#xa0;<i>in vivo</i>&#xa0;is therefore of strong interest for biomedicine and pharmacology. However, factors involved in formation, distribution and clearance of intracellular aggregates are not fully understood</p> Methods <p>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&#xa0;∆ssCPY*&#xa0;aggregation biomarker, with expression controlled under the copper-regulated&#xa0;<i>CUP1</i>&#xa0;promoter</p> Results <p>Monomeric tags overcome challenges associated with non-physiological reporter aggregation, whilst&#xa0;<i>CUP1</i>&#xa0;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&#xa0;<i>in vivo</i>. We demonstrate that iPAR can be used with traditional epifluorescence and confocal microscopy as well as single-molecule precise Slimfield millisecond microscopy.&#xa0;Our results indicate that cytoplasmic aggregates are mobile and contain a broad&#xa0;range of number of&#xa0;iPAR molecules, from tens to several hundred per aggregate, whose mean value increases with extracellular hyperosmotic stress</p> Discussion <p>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.</p>

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iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月1日

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  • Sarah Lecinski,
  • Jamieson A. L. Howard,
  • Chris MacDonald,
  • Mark C. Leake

摘要

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, 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.