Abstract
BackgroundBacteria-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.ResultsLive-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.ConclusionThese 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.
Highlights
Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens
In cells expressing carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) ΔCT, a mutant form of the receptor that lacks the complete cytoplasmic domain and that is not phosphorylated upon bacterial infection, Hck-Src homology 2 (SH2) is distributed evenly in the cytoplasm, even though the bacteria bind to the extracellular domain (Figure 1b and Additional file 2)
It is interesting to note that cells expressing CEACAM3 WT seem to polarize with regard to bacterial uptake: Hck-SH2 is recruited to one side of the cell, where efficient receptor clustering and internalization takes place
Summary
Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. BMC Biology 2009, 7:81 http://www.biomedcentral.com/1741-7007/7/81 tion, signaling processes in the host cell are usually studied in molecular detail Both biochemical and genetic approaches have shed light on protein-protein interactions and signaling connections that occur in infected eukaryotic cells. Widely used biochemical approaches to investigate protein-protein interactions such as glutathione S-transferase (GST)-pull-down assays or coimmunoprecipitation from cell lysates have two major drawbacks: firstly, it is always possible that the two associated proteins are not directly interacting, but rather are linked by a third protein; secondly, biochemical approaches disrupt the cellular context and lack spatial resolution Genetic methods such as yeast two-hybrid screens, applicable even in a high-throughput format, do not provide any information on where these processes occur under physiological conditions at the subcellular level. There is a need for methods that combine the power of biochemical studies to pinpoint molecular interactions with the ability to study the subcellular context as provided by fluorescence microscopy
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