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

Abstract

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 α-subunit of HIF-1 with the O 2-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 O 2-sensing hydroxylase PHD1 and HIF-1α was demonstrated and revealed exclusive localization of O 2-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 O 2-sensing and its localisation within a cell.MCS codes: 92C30, 92C05, 92C40. © 2010 Wotzlaw et al.