Flow Cytometric FRET Analysis of ErbB Receptor Tyrosine Kinase Interaction

Diermeier-Daucher, Simone; Brockhoff, Gero

doi:10.1002/0471142956.cy1214s45

Cited by 4 publications

(7 citation statements)

References 24 publications

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“…There exist several other methods for studying protein interactions in cells by flow cytometry, for instance flow cytometric FRET (FCET) (24, 25), FCCS (7) and BiFC (20). Unfortunately, most of these methods cannot be used on clinical material, since they require expression of modified proteins.…”

Section: Discussionmentioning

confidence: 99%

“…However, FRET has several drawbacks, such as a low signal‐noise ratio for proteins with low expression, the requirement that the labeled antibodies must not only be in close proximity but also correctly oriented for FRET to occur, and the lack of suitability for multiplexed detection. In addition, several correction factors must be calculated, complicating the method (24). For the in situ PLA assay, the fluorophore used for labeling the detection oligonucleotide can be easily replaced, enabling read‐out of different detected protein interactions at separate excitation wavelengths.…”

Section: Discussionmentioning

confidence: 99%

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Flow cytometric in situ proximity ligation analyses of protein interactions and post‐translational modification of the epidermal growth factor receptor family

et al. 2009

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Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo-and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amounts of interactions between EGFR and HER2, but the amount of dimerization was not found to be affected significantly upon stimulation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immunofluorescence staining. In situ PLA was successfully used to study receptor dimerization and activation of the EGF-receptor family with high selectivity and sensitivity. The combination of in situ PLA and flow cytometry provided a statistically powerful way of analyzing protein-protein interactions and post-translational modifications on a singlecell basis. ' 2009 International Society for Advancement of Cytometry

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Flow cytometric in situ proximity ligation analyses of protein interactions and post‐translational modification of the epidermal growth factor receptor family

et al. 2009

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“…1,29 Evaluation of dimerization by FRET methods further confirms homodimer formation in SK-BR-3 cells. 31 In comparison, the A549 cells exhibit 10Â lower levels of HER-2 expression and thus homodimerization is not expected. Indeed, the analysis of the scattering by NPs bound to HER-2 receptors on the A549 cells do not indicate that plasmonic coupling is occurring.…”

Section: Discussionmentioning

confidence: 98%

Monitoring of Receptor Dimerization Using Plasmonic Coupling of Gold Nanoparticles

et al. 2011

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The dimerization of receptors on the cell membrane is an important step in the activation of cell signaling pathways. Several methods exist for observing receptor dimerization, including coimmunoprecipitation, chemical cross-linking, and fluorescence resonance energy transfer (FRET). These techniques are limited in that only FRET is appropriate for live cells, but even that method suffers from photobleaching and bleed-through effects. In this study, we implement an alternative method for the targeting of HER-2 homodimer formation based on the plasmonic coupling of gold nanoparticles functionalized with HER-2 Ab. In the presented studies, SK-BR-3 cells, known to overexpress HER-2, are labeled with these nanoparticles and receptor colocalization is observed using plasmonic coupling. HER-2 targeted nanoparticles bound to these cells exhibit a peak resonance that is significantly red-shifted relative to those bound to similar receptors on A549 cells, which have significantly lower levels of HER-2 expression. This significant red shift indicates plasmonic coupling is occurring and points to a new avenue for assessing dimerization by monitoring their colocalization. To determine that dimerization is occurring, the refractive index of the nanoenvironment of the labels is assessed using a theoretical analysis based on the Mie coated sphere model. The results indicate scattering by single, isolated nanoparticles for the low HER-2 expressing A549 cell line, but the scattering observed for the HER-2 overexpressing SK-BR-3 cell line may only be explained by plasmonic-coupling of proximal nanoparticle pairs. To validate the conformation of nanoparticles bound to HER-2 receptors undergoing dimerization, discrete dipole approximation (DDA) models are used to assess spectra of scattering by coupled nanoparticles. Comparison of the experimental results with theoretical models indicates that NP dimers are formed for the labeling of SK-BR-3 cells, suggesting that receptor dimerization has been observed.

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“…For FRET samples donor and acceptor‐conjugated labeling antibodies were applied in a 1:2 ratio. The single‐labeled samples allowed to determine the spectral overspill/S factors (S 1 = FL3/FL2, S 2 = FL3/FL4, S 3 = FL4/FL2, S 4 = FL2/FL4) and cross excitation caused by the two‐laser flow cytometer FACS Calibur (BD Biosciences, San Jose, CA, USA) we used for Cy3 (488 nm) and Cy5 (635 nm) excitation 15 . Signals derived from 488 nm excitation were detected in fluorescence detection channels FL2 (585/42 band pass filter) and FL3 (670 long pass filter), fluorescence resulting from 635 nm excitation was measured in FL4 (661/16 band pass filter).…”

Section: Methodsmentioning

confidence: 99%

“…Background fluorescence derived from sample (i) was subtracted from measured fluorescence intensities of all other samples of the same treatment modality. Calculation of energy transfer efficiency (E, ) on a cell‐by‐cell basis was done by measuring three fluorescence intensities (I FL2 , I FL3 , and I FL4 ) of each cell and by quantification of donor dye quenching and acceptors sensitized emission of the double‐labeled sample () using the custom‐made ReFlex Software, 15,16 according to the following equations: …”

Section: Methodsmentioning

confidence: 99%

Flow Cytometric FRET Analysis of erbB Receptor Interaction on a Cell‐by‐Cell Basis

Diermeier-Daucher

Hasmann

Brockhoff

2008

Annals of the New York Academy of Sciences

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Lateral interaction of c-erbB family receptors resulting in dimer formation is the key event initiating signal transduction. Consequently cross-activation and intracellular signaling is triggered with immediate impact on cell proliferation, migration, cell survival, and differentiation. In order to elucidate the connection of signal input (receptor activation) and signal output (altered cellular behavior) we dynamically assessed cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. We quantitated c-erbB2 receptor homodimerization upon treatment with the therapeutic monoclonal anti-c-erbB2 antibodies trastuzumab (Herceptin) and pertuzumab by flow cytometric FRET (FCET) measurements on a cell-by-cell basis and calculated the extent of antibody-induced cell cycle exit. The results confirm that trastuzumab does not decrease c-erbB2 homodimers despite its strong potency to drive c-erbB2-overexpressing cells into quiescence. Pertuzumab, however, is able to prevent c-erbB2 homodimerization and thereby enhance the antiproliferative effect of trastuzumab when administered in combination.

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Flow Cytometric FRET Analysis of ErbB Receptor Tyrosine Kinase Interaction

Cited by 4 publications

References 24 publications

Flow cytometric in situ proximity ligation analyses of protein interactions and post‐translational modification of the epidermal growth factor receptor family

Flow cytometric in situ proximity ligation analyses of protein interactions and post‐translational modification of the epidermal growth factor receptor family

Monitoring of Receptor Dimerization Using Plasmonic Coupling of Gold Nanoparticles

Flow Cytometric FRET Analysis of erbB Receptor Interaction on a Cell‐by‐Cell Basis

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