High-throughput, fluorescent analysis of reactive oxygen species in C. elegans after knockdown of MRCK-1

Abstract

Reactive oxygen species (ROS) produced in living organisms can generate oxidative stress, which contributes to cell damage associated with neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Studying responses to oxidative stress in model organisms, like the nematode worm Caenorhabditis elegans, may provide insight into basic underlying cellular mechanisms shared with humans. MRCK-1 (myotonic dystrophy kinase-related Cdc42 binding kinase), a highly conserved intracellular kinase, is activated by the G-protein, Cdc42, which contains a redox sensitive motif. Previous studies have shown that MRCK-1 regulates cell polarity and cytoskeleton reorganization, particularly during cell division and in response to cellular damage. To determine if MRCK-1 plays a role in other cellular defense responses, we are testing if reduced mrck-1 levels through RNA interference (RNAi) results in the production of increased ROS under stressed conditions. The fluorescent, cell permeable ROS indicator, carboxymethyl dichlorodihydrofluorescein derivative, CM-H2DCFDA, shows significantly increased fluorescence in hydrogen peroxide-treated worms when quantified over a period of 20 hours using the SpectraMax microplate reader. Measuring the fluorescence of worms exposed to dsRNA from two independent mrck-1 RNAi constructs, mrck-m and mrck-s, enabled the quantification of ROS levels when MRCK-1 function is decreased. Results suggest that there is no significant difference between mrck-1 constructs and the empty vector control under stressed conditions over a 5 to 14 hour read (Kruskal-Wallis rank sum test p-value=0.055). Exploring the levels of ROS in RNAi-treated C. elegans may give further insight into the cellular role of MRCK-1 both in worms and in humans.