Development of a Genetically Encoded Live-Cell Iron Mapping System

Any disturbances in cellular iron regulation can lead to dysfunctions and various diseases. However, our current understanding of iron dynamics in living cells is hampered by the lack of high-resolution iron reporter systems capable of monitoring the iron status of individual cells dynamically. To address this challenge, we have developed the Live-Cell Iron Mapping System (LIMS), which consists of two distinct genetically encoded fluorescent iron reporter systems: the rFeRepS and IronFist. The rFeRepS (ratiometric Iron Reporter Systems) code for two genetically encoded constructs that translate the iron status of individual cells into a ratiometric fluorescent signal. The ratio signal reflects the cellular iron status, based on endogenous iron-responsive proteins. A high rFeRepS ratio indicates ferritin-based cellular iron storage, while a low ratio implies transferrin receptor (TfR)-based active cellular iron uptake. The IronFist (Iron Fluorescent Indicators based on Stability and Translation) is derived from the hemerythrin-like domain (Hr) of the F-box and leucine-rich protein 5 (FXBL5), which undergoes a structural rearrangement upon binding to ferrous iron, preventing its ubiquitination and subsequent degradation. In the IronFist design, we fused either the blue or green fluorescent protein (FP) variant, mTagBFP2 or mNeonGreen, to the C-terminus of Hr and co-expressed mCherry via a ribosomal skipping sequence as a reference protein for normalization. Thus, IronFist dynamically translates fluctuations of the labile iron pool into a ratiometric fluorescent signal. We demonstrated the functionality of both iron reporter systems by treating cells with different iron formulations including iron carbohydrate nanoparticles. Our results show clear cell-to-cell variations in the response to high iron treatments.