Image-based quantification of mitochondrial iron uptake via Mitoferrin-2

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Biology

Background:

• Iron is essential for cellular functions, including mitochondrial processes like heme and iron-sulfur cluster synthesis.
• Mitochondrial iron homeostasis is regulated by transporters such as Mitoferrin-1 (SLC25A37) and Mitoferrin-2 (SLC25A28).
• Mitoferrin-2, ubiquitously expressed, is linked to diseases like cancer and cardiovascular conditions, making it a potential therapeutic target.

Purpose of the Study:

• To develop a robust method for quantifying mitochondrial iron uptake mediated by Mitoferrin-2 in living cells.
• To establish a medium-throughput screening assay for identifying Mitoferrin-2 modulators.
• To facilitate drug discovery efforts targeting Mitoferrin-2.

Main Methods:

• Utilized HEK293 cells with inducible Mitoferrin-2 expression.
• Developed an assay measuring iron-induced quenching of rhodamine B[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA) fluorescence.
• Validated the assay for medium-throughput screening.

Main Results:

• Successfully quantified mitochondrial iron uptake mediated by Mitoferrin-2.
• Established a validated assay suitable for medium-throughput screening.
• Demonstrated the potential of the assay for identifying Mitoferrin-2 modulators.

Conclusions:

• A novel assay for measuring Mitoferrin-2-mediated mitochondrial iron uptake has been developed.
• This assay is validated for medium-throughput screening, enabling the identification of Mitoferrin-2 modulators.
• The assay holds promise for advancing drug discovery for diseases associated with Mitoferrin-2 dysfunction.