FRET microscopy to visualize transcription factor dimerization in the nucleus of the living cell

Richard N. Day, Ammasi Periasamy

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Cells respond to environmental cues or developmental programs by modifying protein complexes in the nucleus to alter patterns of gene transcription. Recent advances in digital imaging coupled with the development of new fluorescent probes provide the tools to begin to study where and when changes in protein interactions take place in the nucleus of the living cell. Here, we describe the application of fluorescence resonance energy transfer (FRET) using both steady-state and two-photon excitation (TPE) microscopy to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPα) in living pituitary cells. The efficiency of FRET will be improved if the overlap of the donor emission spectra with the absorption spectra for the acceptor is increased. The trade off for this improved efficiency, however, is that there will be an increase in the background signal from which the weak sensitized acceptor emission must be extracted. Here, we compare and contrast the FRET signals obtained from dimerized C/EBPα proteins fused to several different color variants of the jellyfish green fluorescent protein (GFP). We use both wide-field and 2P FRET microscopy to characterize the spectral cross-talk and FRET signals for each of the donor and acceptor pairs.

Original languageEnglish (US)
Pages (from-to)163-170
Number of pages8
JournalProceedings of SPIE - The International Society for Optical Engineering
Volume4262
DOIs
StatePublished - Jan 1 2001

    Fingerprint

Keywords

  • Colocalization
  • Fluorescence resonance energy transfer (FRET) microscopy
  • Green fluorescent protein
  • Transcription factors
  • Two-photon excitation

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics
  • Computer Science Applications
  • Applied Mathematics
  • Electrical and Electronic Engineering

Cite this