Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus

Cells respond to environmental cues by modifying protein complexes In the n ucleus to produce a change in the pattern of gene expression. In this artic le, we review techniques that allow us to visualize these protein Interacti ons as they occur in living cells. The cloning of genes from marine organis ms that encode fluorescent proteins provides a way to tag and monitor the I ntracellular behavior of expressed fusion proteins. The genetic engineering of jellyfish green fluorescent protein (GFP) and the recent cloning of a s ea anemone red fluorescent protein (RFP) have provided fluorescent tags tha t emit light at wavelengths ranging from the blue to the red spectrum. Seve ral of these color variants can be readily distinguished by fluorescence mi croscopy, allowing them to be used In combination to monitor the behavior o f two or more independent proteins in the same living cell. We describe the use of this approach to examine where transcription factors are assembled in the nucleus. To demonstrate that these labeled nuclear proteins are Inte racting, however, requires spatial resolution that exceeds the optical limi t of the light microscope. This degree of spatial resolution can be achieve d with the conventional light microscope using the technique of fluorescenc e resonance energy transfer (FRET). The application of FRET microscopy to d etect the interactions between proteins labeled with the color variants of GFP and the limitations of the FRET approach are discussed. The use of diff erent-color fluorescent proteins in combination with FRET offers the opport unity to study the complex behavior of key regulatory proteins in their nat ural environment within the living cell. (C) 2001 Academic Press.