Fluorescent Reporter-Based Paralysis Assay: A Technique to Assess Age-Associated Progressive Formation of Polyglutamine Fluorescent Reporter and Associated Paralysis in Caenorhabditis elegans
내레이션 대본
Cellular protein homeostasis, or proteostasis, involves the regulation of protein folding and function, along with degradation of misfolded proteins. During aging, proteostasis machinery declines, causing misfolded proteins to accumulate and further aggregate, resulting in cellular dysfunction.
To determine age-associated proteostasis decline, begin with developmentally synchronized, adult transgenic Caenorhabditis elegans.
The muscle cells of these worms express proteins with abnormally-expanded polyglutamine, or polyQ, repeats, fused with a fluorescent tag. PolyQ proteins are prone to misfolding, and hence upon accumulation, can form aggregates.
Transfer the worms onto a microscope slide comprising an agarose pad and immobilize with suitable chemical for live-cell imaging. Using a fluorescence microscope, visualize the worms' body wall muscles over a period of time to determine the bright, fluorescent foci signals, corresponding to the fluorescent polyQ aggregates.
Within days, owing to age-related proteostasis decline, progressive polyQ aggregate accumulation occurs, leading to higher number of fluorescent foci. The increased polyQ aggregates in muscle cells cause toxicity and disrupt muscle cell function, resulting in paralysis.
Under a microscope, record the number of live, paralyzed worms lacking movement upon mechanical stimuli. Increased worm paralysis rate suggests age-associated proteostasis decline in muscle tissue in the worms.
