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

Christoph Wotzlaw^*, Teresa Otto^*, Utta Berchner-Pfannschmidt^*, Eric Metzen, Helmut Acker^* and Joachim Fandrey^*,1

^* Institut für Physiologie, Universität Duisburg-Essen, Essen, Germany; and

Institut für Physiologie, Universität zu Lübeck, Lübeck, Germany

¹Correspondence: Institut für Physiologie, Universität Duisburg-Essen, Hufelandstrasse 55, D-45122 Essen, Germany. E-mail: joachim.fandrey{at}uni-due.de

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-1 and its constitutive ß subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. 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-1 than of HIF-1ß, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between and ß subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits 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.

Key Words: fluorescence resonance energy transfer • fluorescence recovery after photobleaching • hypoxia-inducible factor-1 • oxygen sensing

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