Nanoscopy of the cellular response to hypoxia by means of fluorescence resonance energy transfer (FRET) and new FRET software

Fluorescence resonance energy transfer (FRET) was established to determine the assembly of the HIF-1 complex and to study the interaction of the α-subunit of HIF-1 with the O2-sensing hydroxylase. New software was developed to improve the quality and reliability of FRET measurements.FRET revealed close proximity between the HIF-1 subunits in multiple cells. Data obtained by sensitized FRET in this study were fully compatible with previous work using acceptor bleaching FRET. Interaction between the O2-sensing hydroxylase PHD1 and HIF-1α was demonstrated and revealed exclusive localization of O2-sensing in the nucleus. The new software FRET significantly improved the quality and speed of FRET measurements.FRET measurements do not only allow following the assembly of the HIF-1 complex under hypoxic conditions but can also provide important information about the process of O2-sensing and its localisation within a cell.MCS codes: 92C30, 92C05, 92C40Oxygen deprivation of the tissues endangers energy supply and thus function and survival of the cells. Hypoxia, defined as a state of reduced oxygen tension (PO2), develops when the demand for oxygen exceeds supply. To restore oxygen and thus energy homeostasis an adequate response requires the coordinated expression of genes that control tissue perfusion and oxygen capacity of the blood, glucose uptake and anaerobic glycolysis [1]. The key transcriptional regulator of the genomic response to hypoxia is the transcription factor complex Hypoxia-Inducible Factor-1 (HIF-1) which supposedly regulates about 5% of the human genome [2]. HIF-1 is formed as a dimer of oxygen-regulated α-subunits (HIF-1α or HIF-2α) and a constitutive nuclear subunit HIF-1β [3]. Under well oxygenated conditions (normoxia) three enzymes containing prolyl hydroxylase domains (PHD1, PHD2 and PHD3) posttranslationally modify HIF-α subunits. The hydroxylated α-subunits are recognized by the von Hippel-Lindau protein (pVHL) which recruits an E3 ubiquitin ligas