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

BACKGROUND Cellular oxygen sensing is fundamental to all mammalian cells to adequately respond to a shortage of oxygen by increasing the expression of genes that will ensure energy homeostasis. The transcription factor Hypoxia-Inducible-Factor-1 (HIF-1) is the key regulator of the response because it coordinates the expression of hypoxia inducible genes. The abundance and activity of HIF-1 are controlled through posttranslational modification by hydroxylases, the cellular oxygen sensors, of which the activity is oxygen dependent. METHODS Fluorescence resonance energy transfer (FRET) was established to determine the assembly of the HIF-1 complex and to study the interaction of the alpha-subunit of HIF-1 with the O2-sensing hydroxylase. New software was developed to improve the quality and reliability of FRET measurements. RESULTS 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-1alpha 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. CONCLUSION 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, 92C40.