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Am J Physiol Cell Physiol (July 28, 2004). doi:10.1152/ajpcell.00260.2004
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Submitted on May 27, 2004
Accepted on July 16, 2004

Detection of Intracellular Iron by its Regulatory Effect

Jau-Yi Li1, Gita Ram1, Katherine Gast1, Xia Chen1, Kimberly Barasch1, Kiyoshi Mori1, Kai Schmidt-Ott1, Jianjun Wang2, Hung-Chieh Kuo3, Cathy Savage-Dunn2, Michael D Garrick3, and Jonathan Barasch1*

1 Medicine, College of Physicians and Surgeons of Columbia University, New York, NY, USA
2 Queens College and the Graduate Schoold and University Center of CUNY, Flushing, NY, USA
3 Biochemistry, SUNY, Buffalo, NY, USA

* To whom correspondence should be addressed. E-mail: jmb4{at}columbia.edu.

Intracellular iron regulates gene expression by inhibiting the interaction of Iron Regulatory Proteins (IRP) with RNA motifs called Iron Responsive Elements (IRE). To assay this interaction in living cells we have developed two fluorescent IRE based reporters that rapidly, reversibly, and specifically respond to changes in cellular iron status as well as signaling that modifies IRP activity. The reporters were also sufficiently sensitive to distinguish apo- from holo- transferrin in the media, to detect the effect of modifiers of the transferrin pathway, such as HFE, and to detect the donation or chelation of iron by siderophores bound to the lipocalin Ngal. In addition, alternative configurations of the IRE motif either enhanced or repressed fluorescence, permitting a ratio analysis of the iron dependent response. These characteristics make it possible to visualize iron-IRP-IRE interactions in vivo.




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