Research Article

High‐Throughput, Fluorescent Analysis of Reactive Oxygen Species in C. elegans after Knockdown of mrck‐1

Published: 2017-4

Journal: The FASEB Journal

DOI: 10.1096/fasebj.31.1_supplement.770.10

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 wormCaenorhabditis 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 RNA interference (RNAi) ofmrck‐1results in the production of more ROS under stressed conditions. The cell permeable fluorescent ROS indicator, carboxymethyl dichlorodihydrofluorescein derivative, CM‐H2DCFDA, shows significantly increased fluorescence in hydrogen peroxide‐treated worms when quantified using the SpectraMax microplate reader. Oxidative stress conditions were induced with hydrogen peroxide (H2O2) in the presence of CM‐H2DCFDA, and fluorescence was measured over a period of 20 hours. Measuring the fluorescence of worms exposed to dsRNA from two independentmrck‐1RNAi constructs,mrck‐mandmrck‐s, enables the quantification of ROS levels when MRCK‐1 function is decreased. Exploring the levels of ROS in RNAi‐treatedC. elegansmay give further insight into the cellular role of MRCK‐1 both in worms and in humans. Support or Funding Information Special acknowledgments to the Salisbury University Department of Biological Sciences, the Henson Undergraduate Research Award, and the National Science Foundation MRI Acquisition Award DBI‐1337534 for support.

Faculty Members

  • Patti Erickson - Salisbury University Salisbury MD
  • Matthew Russo - Salisbury University Salisbury MD
  • Stephen Rigoulot - Virginia Tech Blacksburg VA
  • Callista Brown - Salisbury University Salisbury MD
  • Ruth Heying - Salisbury University Salisbury MD

Themes

  • Neurodegenerative diseases
  • Model organisms in biomedical research
  • Reactive oxygen species (ROS)
  • Oxidative stress
  • Cellular defense mechanisms
  • Role of MRCK-1 in cellular processes

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