Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection

Abstract

This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for the analytical ultracentrifuge. Further, new sedimentation velocity methods, such as the multi-speed method for data capture and wide distribution analysis for data analysis, are applied to improve the resolution of the measures of heterogeneity over a wide range of sizes. The focus here is to test the ability to measure sedimentation of polyglutamine aggregates in complex mixtures as a prelude to future studies that will explore the effects of genetic manipulation and environment on aggregation and toxicity. Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging from robust analysis of the monomer species through an intermediate and quite heterogeneous population of oligomeric species, and all the way up to detecting species that likely represent intact inclusion bodies based on comparison to an analysis of fluorescent puncta in living worms by confocal microscopy. Our results support the hypothesis that misfolding of expanded polyglutamine tracts into insoluble aggregates involves transitions through a number of stable intermediate structures, a model that accounts for how an aggregation pathway can lead to intermediates that can have varying toxic or protective attributes. An understanding of the details of intermediate and large-scale aggregation for polyglutamine sequences, as found in neurodegenerative diseases such as Huntington's Dis-

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans

“…This presumed limited aggregation may be due to aggregation of the GFP itself within the fusion construct, as has been noted in the literature, 13,20,23,53–55 or alternatively, sequestration of other factors (see Discussion). Our previous work, studying polyglutamine aggregation in a worm model, showed evidence of significant aggregation of GFP by itself, in both SDD-AGE (~400 kDa) and SV experiments (running at ~80 S), with the sedimentation boundary being present even in the polyglutamine fusion constructs.…”

“…23 This validation approach was also performed here. To detect a putative monomer population, data were collected at 50000 rpm and analyzed using a continuous distribution function, c ( s ) (Figure 2).…”

“…An average of the weighted s values for D. melanogaster HttQ97 is 439120 S, while in C. elegans HttQ74, it is 403900 S. Simple size calculations based on the range of s values reported suggest that they are similar in size to that of the puncta in the corresponding microscopy images (Figures 4 and 5), assuming a spheroidal shape. 23 …”

“…23 It has been shown by several different groups that the N-terminal flanking sequence of Htt induces limited aggregation (4–12 subunits) through helical pairing interactions in in vitro experiments 5 and may be the explanation for this feature in the MSM–WDA experiments. The distinction between the upper end of the 5–30 S pool and the 40–500 S pool is less well-defined, and as suggested in the Results, some of this aggregation may be due to intrinsic aggregation of the fluorescent proteins themselves, as described in our earlier work and as suggested by the low-molecular weight species in the SDD-AGE experiment (Figure 1).…”

“…Technical hurdles for such studies, using fluorescence detection in the analytical ultra-centrifuge, have been discussed in earlier work from our laboratory 23 and another. 22 The research described here advances the use of this method, in complementation with other biochemical and microscopy methods, to offer a comparative study of polyglutamine aggregation using two different model systems.…”