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

Richard N. Day, Ammasi Periasamy, Fred Schaufele

Research output: Contribution to journalArticle

172 Scopus citations

Abstract

Cells respond to environmental cues by modifying protein complexes in the nucleus to produce a change in the pattern of gene expression. In this article, we review techniques that allow us to visualize these protein interactions as they occur in living cells. The cloning of genes from marine organisms that encode fluorescent proteins provides a way to tag and monitor the intracellular behavior of expressed fusion proteins. The genetic engineering of jellyfish green fluorescent protein (GFP) and the recent cloning of a sea anemone red fluorescent protein (RFP) have provided fluorescent tags that emit light at wavelengths ranging from the blue to the red spectrum. Several of these color variants can be readily distinguished by fluorescence microscopy, allowing them to be used in combination to monitor the behavior of 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 interacting, however, requires spatial resolution that exceeds the optical limit of the light microscope. This degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). The application of FRET microscopy to detect the interactions between proteins labeled with the color variants of GFP and the limitations of the FRET approach are discussed. The use of different-color fluorescent proteins in combination with FRET offers the opportunity to study the complex behavior of key regulatory proteins in their natural environment within the living cell.

Original languageEnglish (US)
Pages (from-to)4-18
Number of pages15
JournalMethods
Volume25
Issue number1
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

Keywords

  • Colocalization
  • Energy transfer
  • Fluorescence microscopy
  • Green fluorescent protein
  • Transcription factors

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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