Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling

Buntru, Alexander; Zimmermann, Timo; Hauck, Christof R.

Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling

Dateien

Buntru_opus-111550.pdfGröße: 674.55 KBDownloads: 291

Datum

2009

Autor:innen

Buntru, Alexander

Zimmermann, Timo

Hauck, Christof R.

Open Access-Veröffentlichung

Open Access Gold

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

BMC Biology. 2009, 7, 81. ISSN 1741-7007. eISSN 1741-7007. Available under: doi: 10.1186/1741-7007-7-81

Zusammenfassung

BACKGROUND: Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET) to visualize pathogen-initiated signaling events in human cells. RESULTS: Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3). In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2) domain of Hck (Hck-SH2) was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet)-Hck-SH2) and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet) and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. CONCLUSION: These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Zitieren

ISO 690

BUNTRU, Alexander, Timo ZIMMERMANN, Christof R. HAUCK, 2009. Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling. In: BMC Biology. 2009, 7, 81. ISSN 1741-7007. eISSN 1741-7007. Available under: doi: 10.1186/1741-7007-7-81

BibTex

@article{Buntru2009Fluor-7650,
  year={2009},
  doi={10.1186/1741-7007-7-81},
  title={Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling},
  volume={7},
  issn={1741-7007},
  journal={BMC Biology},
  author={Buntru, Alexander and Zimmermann, Timo and Hauck, Christof R.},
  note={Article Number: 81}
}

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