iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

Area of Science:

• Cell Biology
• Biochemistry
• Microscopy

Background:

• Cellular protein homeostasis (proteostasis) is crucial for function, and its dysregulation leads to protein aggregation.
• Protein aggregates are implicated in neurodegenerative diseases and compromise cell viability.
• Understanding intracellular aggregate dynamics in vivo is vital for biomedicine and pharmacology.

Purpose of the Study:

• To develop an improved methodology for producing and studying fluorescent protein aggregates in live budding yeast.
• To enable quantitative analysis of intracellular aggregate formation, distribution, and clearance in vivo.
• To investigate the inheritance patterns of protein aggregates in budding yeast.

Main Methods:

• Developed iPAR (inducible Protein Aggregation Reporter), a technology using monomeric fluorescent protein reporters fused to an aggregation biomarker.
• Utilized the copper-regulated CUP1 promoter for controlled protein expression.
• Employed advanced fluorescence microscopy techniques, including epifluorescence, confocal, and single-molecule precise Slimfield microscopy, for live-cell imaging.

Main Results:

• iPAR allows for the detection, tracking, and quantification of cytoplasmic protein aggregates in live yeast cells.
• Cytoplasmic aggregates are mobile and vary in size, containing tens to hundreds of iPAR molecules, with size increasing under osmotic stress.
• Demonstrated quantitative study of aggregate kinetics and inheritance features in vivo.

Conclusions:

• Protein aggregates associate with nuclear and vacuolar compartments but are not inherited by daughter cells.
• The iPAR technology provides a novel platform for studying proteotoxic accumulations and their dynamics.
• iPAR has potential for adaptation to other model systems to study diseases related to protein oligomerization.