Studying Selective Autophagy of Protein Aggregates Using Particles Induced by Multimerization (PIMs)

Selective autophagy of protein aggregates, called aggrephagy, is vital for maintaining cellular homeostasis. Classically, studying aggrephagy has been challenging due to the infrequent occurrence of autophagic events and the lack of control over the specificity and timing of protein aggregation. We previously reported two variants of a PIM (particles induced by multimerization) assay that enable the formation of chemically induced, fluorescently labeled protein aggregates in cells. PIMs are recognized by the selective autophagy machinery and are subsequently degraded in the lysosome. By making use of pH-sensitive fluorescent proteins, such as GFP or mKeima, the PIM assay allows for direct visualization of aggregate clearance in cells. Here, we describe a protocol for the use of the PIM assay to study aggrephagy in live and fixed cells.