Tracking Intracellular Labile Iron with a Genetically Encoded Fluorescent Reporter System Based on Protein Stability

Here, we developed IronFist, a genetically encoded fluorescent reporter that enables dynamic tracking of labile ferrous ions (Fe²⁺) in live cells. IronFist is a bicistronic system combining the iron-sensitive hemerythrin-like (Hr) domain from the F-box and leucine-rich repeat protein 5 (FBXL5), fused to the bright fluorescent protein (FP) mNeonGreen, alongside mCherry as a reference FP signal. When labile iron levels are low, Hr-mNeonGreen undergoes ubiquitination and degradation, leading to a low green-to-red fluorescence ratio. Conversely, as cytosolic Fe²⁺ levels rise and Fe²⁺ ions bind to Hr, the green fluorescence is stabilized, increasing the IronFist ratio signal. Using IronFist for end point measurements, we observed that most cells maintain low basal labile iron levels. However, upon treatment with iron(II) sulfate or iron carbohydrate nanoparticles, we detected significant elevations in the cellular labile iron pool (LIP). Cells responded faster and more strongly to iron(II) sulfate, whereas responses to iron carbohydrate nanoparticles were slower and weaker. Time-lapse imaging further revealed substantial cell-to-cell heterogeneity in iron handling. We conclude that IronFist fills a critical methodological gap in assessing cellular iron homeostasis and related pathologies by enabling high-content tracking of iron dynamics at the single-cell level.