Optical analysis of the HIF‐1 complex in living cells by FRET and FRAP

Hypoxia‐inducible factor‐1 (HIF‐1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia‐inducible genes that ensure an adequate energy supply. Assembly of the HIF‐1 complex by its oxygen‐regulated subunit HIF‐1a and its constitutive β subunit also known as ARNT is the key event of the cellular genetic response to hyp‐oxia. By two‐photon microscopy, we studied HIF‐1 assembly in living cells and the mobility of fluorophore‐labeled HIF‐1 subunits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF‐1α than of HIF‐1β, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between α and β subunits of the transcriptionally active HIF‐1 complex bound to DNA. Both N termini of the fluorophore‐labeled HIF‐1 sub‐units were localized as close as 6.2 nm, but even the N and C terminus of the HIF‐1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3‐dimensional organization of the HIF complex than described so far by 2‐dimensional models.—Wotzlaw, C., Otto, T., Berchner‐Pfannschmidt, U., Metzen, E., Acker, H., Fandrey, J. Optical analysis of the HIF‐1 complex in living cells by FRET and FRAP. FASEB J. 21, 700–707 (2007)

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