Optical Analysis of Hypoxia Inducible Factor (HIF)-1 Complex Assembly: Imaging of Cellular Oxygen Sensing
Hypoxia is a common phenomenon that occurs in a variety of diseases such as cardiovascular ischemia, anemia, and cancer. Cellular oxygen sensors measure changes in tissue oxygenation and induce responses aimed at restoring sufficient supply with oxygen. Genetic adaptation to hypoxia is under control of hypoxia-inducible factors (HIFs), of which two highly homologous subunits HIF-1α and HIF-2α are regulated by oxygen tension. Together with HIF-1β (=ARNT; aryl hydrocarbon receptor nuclear translocator) they form transcriptionally active complexes under hypoxia which drive the expression of hypoxia inducible genes. The meaning of different HIF complexes, i.e., HIF-1α/ARNT versus HIF-2α/ARNT with respect to target gene or tissue specificity has not been fully resolved. We applied modern microscopic methods like fluorescence resonance energy transfer (FRET) to elucidate protein–protein interactions and fluorescence recovery after photo-bleaching (FRAP) to study mobility of HIF proteins inside the nuclei of living cells. We found differences both in nuclear mobility and the assembly of HIF-1 versus HIF-2 which might help to better understand the assembly of HIF complexes.
KeywordsHIF-1 assembly Dimerization Mobility
Parts of this work were supported by grants from the Deutsche Forschungsgemeinschaft GRK1431 and the Fachhochschule Dortmund.
- 6.Carnot P, Deflandre C. Sur l’activité hémopoiétique du sérum au cours de la régénération du sang. C R Acad Sci Paris. 1906;143:384–6.Google Scholar
- 9.Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 2001;107(1):43–54.CrossRefPubMedGoogle Scholar
- 16.Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468–72.CrossRefPubMedGoogle Scholar
- 20.Makino Y, Uenishi R, Okamoto K, Isoe T, Hosono O, Tanaka H, Kanopka A, Poellinger L, Haneda M, Morimoto C. Transcriptional up-regulation of inhibitory PAS domain protein gene expression by hypoxia-inducible factor 1 (HIF-1): a negative feedback regulatory circuit in HIF-1-mediated signaling in hypoxic cells. J Biol Chem. 2007;282(19):14073–82.CrossRefPubMedGoogle Scholar