Sedimentation Velocity Analysis of Polyglutamine Assembly in C. elegans using a Fluorescence Detection System

Abstract

The presence of different size protein aggregates is a common feature of neurodegenerative diseases, and which types of aggregates (whether oligomeric or fibrillar) are toxic are still not well defined. using expression of various polyQ expression constructs in C. elegans, we apply analytical ultracentrifugation methods with fluorescence detection capability to determine size distribution of protein aggregation. We find that an increase in intermediate size range pools (80 S-120 S) is a direct result of an increase in polyQ length. Strains of worms expressing polyQ0-YFP, polyQ24-YFP, polyQ35-YFP, polyQ40-YFP, and polyQ40 (no YFP) in muscle wall were synchronized and grown at 20° C and harvested as one-day old adults. PolyQ40 without the fluorescent YFP fusion was labeled using an extrinsic dye, DCVJ, that emits fluorescence when bound to punctates. Quantitative analysis of samples prepared with minimal fractionation for polyQ40-YFP shows evidence of polydisperse aggregates with size range from 30-150 S similar in size to the oligomeric states observed in situ in a mouse neuroblastoma cell line. These intermediates were not observed in polyQ0-YFP control constructs. Our data show that the presence of a polydisperse oligomeric pool, and we suggest that this heterogeneity is associated with the cellular context since such polydispersity is not observed in in vitro model systems.