In cell measurement of fluorescence lifetime imaging microscopy revealed C-terminal conformation changes of Ferroportin upon addition of Mn2+

Abstract

Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy (FLIM), which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin (FPN), a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on C-terminal of FPN (FPN-eGFP) and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn2+ treatment, suggesting a preference conformation of FPN in Mn2+ exposure. It is also observed that the lifetime (rather than intensity) measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn2+ treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.