Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors.

Area of Science:

• Cellular Biology
• Biochemistry
• Biomedical Engineering

Background:

• Cellular iron is crucial for numerous biochemical processes.
• Accurate measurement of bioavailable iron is challenging due to technical limitations and lack of functional readouts.
• Existing methods struggle with precise localization and assessing iron's availability for metalloproteins.

Purpose of the Study:

• To introduce and validate geNOps, a genetically encoded fluorescent biosensor, for measuring bioavailable iron in cellular locales.
• To demonstrate geNOps's utility in assessing cellular iron absorption, distribution, and availability.
• To compare the efficacy of different iron formulations in activating the biosensor.

Main Methods:

• Utilized geNOps, a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor.
• Exploited nitrosylation-dependent fluorescence quenching of geNOps as a readout for cellular iron dynamics.
• Applied geNOps to intact cells treated with various iron compounds (ferrous iron salts, iron(III)-HBED, iron sucrose, ferric carboxymaltose).

Main Results:

• geNOps successfully measured available ferrous iron (II) in cultured cells at the subcellular level.
• The iron(III)-HBED complex activated the iron (II)-dependent NO probe in intact cells.
• Iron sucrose rapidly activated the biosensor in cytosol and mitochondria within 20 minutes.
• Ferric carboxymaltose did not functionalize the probe even after 2 hours of treatment.

Conclusions:

• The geNOps biosensor provides a direct readout for cellular iron absorption, distribution, and availability.
• geNOps enables functional assessment of bioavailable iron (II) at the subcellular level.
• The findings highlight differential cellular uptake and functionalization of various iron formulations, with implications for iron therapy.