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.
Mesh Terms:
Fluorescence Recovery After Photobleaching, Fluorescence Resonance Energy Transfer, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Protein Subunits, Tumor Cells, Cultured
Fluorescence Recovery After Photobleaching, Fluorescence Resonance Energy Transfer, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Protein Subunits, Tumor Cells, Cultured
FASEB J.
Date: Mar. 01, 2007
PubMed ID: 17197389
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