Molecular Imaging
Fluorescence resonance energy transfer (FRET) combined with confocal laser microscopy is a powerful tool to analyze protein?protein interaction in vivo. We have applied this combination to study the assembly of the hypoxia-inducible factor (HIF) complex in living cells under hypoxic conditions. In hypoxia, the basic helix-loop-helix/Period/ARNT/Single-minded (PAS) proteins HIF-1? and HIF-2? accumulate and are translocated into the nucleus. Here, HIF-1? and HIF-2? dimerize with HIF-1?, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), to form HIF-1/HIF-2 complexes, which control the expression of specific target genes. Therefore, a new Java-based analyzing program was developed at our institute to calculate the nanometer distance between ? and ? subunits of the transcriptionally active HIF-1/-2 complex bound to DNA. Fusion proteins of HIF subunits and variants of green fluorescent proteins (cyan and yellow fluorescent proteins) were expressed in living cells and protein?protein interactions were imaged in vivo by means of FRET.
fluorescence resonance energy transfer (fret) hypoxia-inducible factor (hif) fusion proteins green fluorescence protein in vivo imaging gateway cloning u2os cells