A New Application of Fluorescence Ratio Imaging Technique to In Situ Demonstration of the Protein Phosphorylation Rate

We introduce the methodology of the ratio imaging technique and its new application to in situ demonstration of protein phosphorylation. Ezrin/radixin/moesin (ERM) proteins are known to be cross−linkers between plasma membrane and actin filaments (Factin). Phosphorylation of their COOH−terminal threonine residue was implicated in the regulation of their cross−linking activity. Using a rat mAb 297S specific for COOH−terminally phosphorylated ERM (CPERM) proteins and a mouse mAb CR22 which recognizes total ERM proteins, we detected subcellular distribution of ERM proteins and CPERM proteins in mouse macrophages by indirect immunofluorescence. Total ERM and CPERM proteins were revealed as Texas red− and Alexa 488 (green)−images, respectively, with dual excitation confocal laser microscopy. Obtained Texas red− and Alexa 488−images were saved as TIFF−format files and processed for digital image analysis using a MetaMorph software. The ratio value in each pixel was automatically calculated by dividing an Alexa 488−image by a corresponding Texas red−image, and built a pseudocolor ratio image. The ratio image showed that the phosphorylation rate of ERM proteins in cytoplasm gradually increased toward the roots of filopodia, and was highest in filopodia. Thus, ratio imaging seems to be advantageous to visualize where ERM proteins are phosphorylated in the cells, not being affected by the total amount of ERM proteins or cytoplasmic volume of the cells.