Built using a hemerythrin-like domain from the mammalian FBXL5 protein, FEOX responds to changes in cellular iron by altering its fluorescence signal. This design overcomes the limits of traditional iron sensing tools, which often rely on indirect or delayed markers such as iron regulatory protein (IRP) activity.
Iron's Importance in the Body
Iron plays a central role in cell survival, metabolism, and growth, but its availability must be tightly controlled. IRPs help regulate genes related to iron homeostasis, but their slow, indirect response means they cannot detect rapid or transient changes.
Previous efforts to measure cellular iron with fluorescent tools have faced challenges in specificity, invasiveness, or compatibility with live-cell imaging.
FEOX addresses these challenges with a genetically encoded, ratiometric design that operates inside living cells. It builds on a biological mechanism native to FBXL5, which normally senses iron to regulate IRP2 degradation.
Using this system, the researchers created a fluorescent biosensor that responds dynamically to real-time fluctuations in bioavailable iron.
FEOX: How it Works
The FEOX sensor consists of a synthetic gene cassette encoding a fusion of the FBXL5-derived hemerythrin-like domain and a blue fluorescent protein, mTagBFP2, under a constitutive promoter. A separate cassette expresses mCherry, a red fluorescent protein used as an internal control. Together, the two fluorescent signals allow for ratiometric measurements that correct for variability in expression levels and cell size.
To establish stable expression, the researchers flanked the constructs with piggyBac transposon sequences and integrated them into the genome of mouse embryonic stem cells using lipid-based transfection. Fluorescence-activated cell sorting (FACS) was then used to isolate clones with optimized expression.
Within the sensor design, iron binding stabilizes the FEOX protein, allowing it to accumulate in the cell and emit a stronger fluorescent signal. At the same time, iron depletion triggers proteasomal degradation of the sensor, leading to reduced fluorescence. This direct link between iron availability and protein stability enables a live-cell readout of intracellular iron levels.
Experiments evaluating the sensor's performance confirmed that the FEOX ratio, measured by flow cytometry, reliably tracked changes in bioavailable iron.
Live Tracking Iron
To test FEOX responsiveness, the researchers manipulated intracellular iron levels by treating stem cells with either ferric ammonium citrate (FAC) to increase iron or deferoxamine (DFO) to induce iron depletion.
As expected, FEOX ratios decreased under iron-deficient conditions and rose in response to supplementation. Quantitatively, the increase was modest, up to approximately 11 %, indicating a limited dynamic range for detecting iron overload, though the sensor remains highly sensitive to iron deficiency. This pattern is consistent with the behavior of the FBXL5 hemerythrin-like domain and reflects the biological focus on iron scarcity rather than excess.
Monitoring Iron During Differentiation
FEOX proved especially informative in tracking iron availability during embryonic stem cell differentiation. In both two-dimensional cultures and three-dimensional embryoid bodies, the researchers observed a consistent decline in FEOX ratio over 48 to 72 hours as stem cells transitioned from a naïve pluripotent state to early somatic lineages.
Differentiation was initiated by removing LIF/2i, a cocktail of self-renewal factors, which drives the shift from the naïve to epiblast-like stage. The observed drop in FEOX signal reflects a reduced bioavailable iron during early differentiation, a known phenomenon likely related to increased iron demand and metabolic remodeling.
To compare this signal with conventional iron-sensing pathways, the team used FIRE, an IRP-based biosensor. Unlike FIRE, which reports on IRP activity and responds to iron indirectly, FEOX operates independently of IRPs.
This IRP-independent behavior makes FEOX a complementary tool that provides insights into intracellular iron dynamics, especially useful when used alongside IRP-sensitive systems.
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Strengths and Limitations
FEOX has several advantages. It is compatible with live-cell imaging, genetically encoded, and sensitive enough to detect single-cell differences in iron availability. Its ratiometric format ensures reliable comparisons across experimental conditions and time points, making it well-suited for longitudinal and high-throughput studies.
Despite its strengths, the sensor has some limitations.
It currently lacks subcellular targeting and cannot distinguish between different oxidation states of iron, such as ferric versus ferrous. Its performance depends on the cell’s proteasome activity, and it shows a relatively narrow dynamic range under iron-replete conditions.
Nonetheless, FEOX excels within physiological ranges and during iron-limited states, which are particularly relevant in early development.
A Versatile Tool for Iron Biology
FEOX represents a significant advance in cellular iron sensing.
By providing a direct, real-time readout of bioavailable iron at the single-cell level, it offers a new way to study how cells regulate this essential nutrient during critical processes like stem cell differentiation. Its performance in both 2D and 3D culture systems and its compatibility with other tools, such as FIRE, position it as a versatile system for future studies of iron homeostasis, metabolism, and disease.
Journal Reference
Sangokoya C. (2025). The genetically encoded biosensor FEOX is a molecular gauge for cellular iron environment dynamics at single cell resolution. Scientific Reports 15, 36596. DOI: 10.1038/s41598-025-20428-5