Two-sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy

Zsolt Fazekas, Miklós Petrás, Ákos Fábián, Zsuzsanna Pályi-Krekk, Péter Nagy, Sándor Damjanovich, György Vereb, János Szöllosi

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, β1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of β1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of β1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-by-pixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between β1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between β1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of β1-integrin-ErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with β1-integrins.

Original languageEnglish
Pages (from-to)209-219
Number of pages11
JournalCytometry Part A
Volume73
Issue number3
DOIs
Publication statusPublished - Mar 1 2008

Keywords

  • CD44
  • ErbB2
  • Fluorescence resonance energy transfer
  • Laser scanning confocal microscopy
  • Receptor tyrosine kinase
  • Trastuzumab resistance
  • abFRET
  • dbFRET
  • tsFRET
  • β1-integrin

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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