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. Th… Show more
“…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.…”
Section: Resultsmentioning
confidence: 66%
“…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).…”
Section: Resultsmentioning
confidence: 99%
“…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 …”
Section: Resultsmentioning
confidence: 99%
“…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).…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington’s disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.
“…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.…”
Section: Resultsmentioning
confidence: 66%
“…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).…”
Section: Resultsmentioning
confidence: 99%
“…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 …”
Section: Resultsmentioning
confidence: 99%
“…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).…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington’s disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.
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