Background: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. Methods: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and b2-microglobulin with antibodies conjugated with fluorescein-and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. Results: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye-and instrument-
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, b1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of b1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of b1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-bypixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between b1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between b1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of b1-integrinErbB2 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 b1-integrins. '
International Society for Analytical CytologyKey terms receptor tyrosine kinase; ErbB2, b1-integrin; CD44; trastuzumab resistance; laser scanning confocal microscopy; fluorescence resonance energy transfer; tsFRET; dbFRET; abFRET UNDERSTANDING the molecular mechanisms of signal transduction processes requires the elucidation of assembling and/or disassembling of protein complexes including the conformational changes accompanying these processes. One of the best approaches for studying these molecular rearrangements is fluorescence resonance energy transfer (FRET), which is a sensitive method for measuring intra-or intermolecular distances. FRET-based methods are capable of resolving molecular associations and conformational changes in the 1-10 nm range far exceeding the diffraction limit of the conventional microscope. FRET is a nonradiative process in which energy is transferred from the first excited electronic state of a donor molecule (D) to a nearby acceptor molecule (A) in a dipole-dipole interaction under favorable conditions, resulting in the quenching of donor fluorescence accompanied by the increase (also known as sensitization) of acceptor fluorescence (1-3). For mapping the physical associations of various membrane molecules, flow cytometric and microscopic
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