Assessment of the flagellar redox potential in Chlamydomonas reinhardtii using a redox-sensitive fluorescent protein, Oba-Qc.

ABSTRACT

Cellular reducing-oxidizing (redox) potential is mainly determined by the concentration ratio between reduced and oxidized glutathiones. It is normally kept at a moderately reduced state but affected to some extent by metabolic activities such as respiration and/or photosynthesis. Changes in redox potential induce many cellular activities collectively called redox responses. For an understanding of the dynamics of the cellular redox responses, redox potential must be accurately assessed in vivo. In this study, we developed a method to measure the in vivo redox potential in the green alga Chlamydomonas reinhardtii, using Oba-Qc, a recently developed redox-monitoring protein. Taking advantage of the periodic flagellar assembly, we introduced Oba-Qc molecules into the flagella at a constant density. Fluorescence signals from flagella in live cells, calibrated against the fluorescence from the samples in buffers of known redox potentials, determined the redox potential to be ∼-250 mV in the light and ∼-280 mV in the dark. Introduction of a sensor protein fused with a structural protein that assembles at a constant density will be also applicable for measurements of various kinds cellular signals in flagella.