The genetically encoded biosensor FEOX is a molecular gauge for cellular iron environment dynamics at single cell resolution.

Iron-sensing is evolutionarily conserved among life on Earth, and mammalian cells are known to sense cellular iron through mechanisms similar to bacteria and marine invertebrates. While iron regulatory proteins (IRPs) function as RNA-binding proteins during iron-limiting conditions and reflect cellular iron status, we lack genetically-encodeable tools to quantify bioavailable intracellular iron status over developmental time and at single-cell resolution. In order to better understand the cellular environment that supports or restricts IRP-active conditions, particularly during complex and dynamic changes leading to differentiated states, we present a ratiometric genetically-encoded biosensor called FEOX. FEOX is a ratiometric fluorescent biosensor of the cellular iron environment based on a mammalian hemerythrin-like domain, acting as an iron-dependent ligand-based regulatory switch. Compared to increased IRP activity in response to iron-limitation, FEOX dynamics demonstrate decreased ratiometric fluorescence representing decreased cellular iron during iron-limitation. Using FEOX we are able to quantify the dynamics of the bioavailable cellular iron environment during early stem cell differentiation by ratiometric measurements at single-cell resolution. Results from FEOX and from an IRP activity sensor during stem cell pluripotency transition and early differentiation provide orthogonal support for an increased iron demand state. Using these genetically-encodeable tools will allow greater insight into cellular iron homeostasis within mammalian systems. IRPs function as RNA-binding proteins during iron-limiting conditions, regulating the stability and translation of messenger RNAs that are essential for cellular iron homeostasis. However, to better understand and study the cell and molecular regulators of the iron-limiting conditions required for IRP RNA-binding activity, new tools are needed to monitor iron-responsive alterations in the intracellular environment at single-cell resolution and over developmental time. We devised a genetically-encoded biosensor called FEOX that depends on molecular iron interactions independent of IRP function and directly gauges bioavailable cellular iron.