Monitoring aggregate clearance and formation in cell-based assays

Abstract

Understanding the fundamental mechanism underlying the accumulation and clearance of misfolded proteins can lead to insights into the synthetic and degradative pathways that maintain the homeostasis of proteins in all cells. Given the interconnection between protein homeostasis and cell health, as well as the complexity of aggregate formation and the degradation pathways with which it is intertwined, the design of the tools that are used to examine protein aggregation and accumulation can have a profound impact on the interpretation of results. We rely on two previously published stable cell lines that use conditional expression and the ligand-receptor tag known as HaloTag, to temporally distinguish distinct pools of aggregates, and use a combination of biochemical- and imaging-based methods to measure aggregation of a canonical aggregation-prone protein. We measure aggregate load biochemically using Filter Trap Analysis, which combines a filter trap retardation assay and immunoblotting to measure detergent soluble and insoluble protein levels, and visually, using confocal microscopy to monitor simultaneously aggregate formation and growth events in the background of aggregate clearance. As a secondary screen to more simplistic screen based approaches, this method permits further insight into how aggregate load is affected.