The FIRE biosensor illuminates iron regulatory protein activity and cellular iron homeostasis.

On Earth, iron is abundant, bioavailable, and crucial for initiating the first catalytic reactions of life from prokaryotes to plants to mammals. Iron-complexed proteins are critical to biological pathways and essential cellular functions. While it is well known that the regulation of iron is necessary for mammalian development, little is known about the timeline of how specific transcripts network and interact in response to cellular iron regulation to shape cell fate, function, and plasticity in the developing embryo and beyond. Here, we present a ratiometric genetically encoded dual biosensor called FIRE (Fe-IRE [iron-responsive element]) to evaluate iron regulatory protein (IRP)-binding activity and cellular iron status in live cells, allowing for the study and dissection of dynamic changes in cellular iron and IRP activity over developmental time. FIRE reveals a previously unrecognized foundational timeline of IRP activity and cellular iron homeostasis during stem cell pluripotency transition and early differentiation.