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-1alpha and its constitutive beta 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-1alpha than of HIF-1beta, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between alpha and beta 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
Christoph
C
Institut für Physiologie, Universität Duisburg-Essen, Hufelandstrasse 55, D-45122 Essen, Germany.
Otto
Teresa
T
Berchner-Pfannschmidt
Utta
U
Metzen
Eric
E
Acker
Helmut
H
Fandrey
Joachim
J
D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis* / Q000737:chemistry; D021122:Protein Subunits / Q000032:analysis; D014407:Tumor Cells, Cultured
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D021122:Protein Subunits