Analysing the action of bacterial toxins in living cells with fluorescence resonance energy transfer (FRET).
Bacterial toxins represent small molecules produced by microorganisms. Different toxins act on specific target molecules in mammalian cells. Once discovered, bacterial toxins have been providing tools to study cellular functions and often helped the dissection of complex cellular pathways, e.g. endocytic or secretory trafficking or signal transduction, by virtue of the fact that they either block or activate their specific cellular target molecules. Purified bacterial toxins have also allowed to address many basic biological questions and have provided tools for in vitro and in vivo experimental approaches in many fields of modern biology. The understanding of how bacterial toxins act in living cells often depends on our ability to visualize the trafficking and signaling pathways of these molecules. Fluorescence microscopy and other imaging tools are essential to provide insights into the functional changes induced by these pathogens at the level of individual host cells or single target proteins. Inside a single cell we can measure and quantify the effects of bacterial toxins on specific cellular proteins by microscopic and spectroscopic techniques. Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows to study protein-protein interactions. FRET can provide distance information in the range of 3- 7 nm between fluorescently labeled bacterial proteins in the live cell and cellular target proteins expressed as chimeras with green fluorescent protein (GFP), or spectrally shifted variants thereof. The purpose of this review is to introduce readers to the main experimental setups for analyses of protein-protein interactions using FRET as well as some applications.
Majoul
Irina
I
Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. im266@cam.ac.uk
D002772:Cholera Toxin / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry; D021381:Protein Transport; D018000:Receptors, Peptide / Q000378:metabolism*
D005456:Fluorescent Dyes; C064459:KDEL receptor; D008164:Luminescent Proteins; D018000:Receptors, Peptide; D049452:Green Fluorescent Proteins; D002772:Cholera Toxin