Identifying C. elegans Lifespan Mutants by Screening for Early-Onset Protein Aggregation

Abstract

Genetic screens have been widely used to identify genetic pathways that control specific biological functions. In C. elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Since aged individuals are unable to generate clonal populations, screens that target post-reproductive phenotypes, such as longevity, are challenging. In this work, we developed an approach that combines microfluidic technologies and image processing to perform a high-throughput, automated screen for mutants with shortened lifespan using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively-active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently labeled PAB-1 as a readout. We identified five mutants with increased aggregation levels, of which two exhibited a reduced lifespan. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently-labeled aggregates in populations that do not require conditional sterilization or manual separation of parental and progeny populations. We further analyzed the morphology of protein aggregates and reveal that patterns of aggregation in naturally-aging animals differ from mutants with increased aggregation, suggesting aggregate growth is time-dependent. This screening approach can be customized to other non-developmental phenotypes that appear during adulthood, as well as to other aging markers to identify additional longevity-regulating genetic pathways.