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


CONCLUSIONS
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.
Buntru
Alexander
A
Lehrstuhl für Zellbiologie, Universität Konstanz, Konstanz, Germany. Alex.Buntru@gmx.de
Zimmermann
Timo
T
Hauck
Christof R
CR
D000818:Animals; D002272:Carcinoembryonic Antigen / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D054884:Host-Pathogen Interactions; D007668:Kidney / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology; D009344:Neisseria gonorrhoeae / Q000502:physiology; D054730:Protein Interaction Domains and Motifs; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D018909:src Homology Domains / Q000502:physiology
C075729:CEACAM3 protein, human; D002272:Carcinoembryonic Antigen; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck