title: Visualization of maltose uptake in living yeast cells by fluorescent nanosensors.
abstract: Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing protein-based nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications.
authors.affiliation: Zentrum für Molekularbiologie der Pflanzen, Plant Physiology, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Fehr
authors.forename: Wolf B
authors.initials: WB
authors.lastname: Frommer
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lalonde
mesh_terms: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000378:metabolism; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D015336:Molecular Probe Techniques; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013395:Sucrose / Q000378:metabolism
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D013395:Sucrose; D008320:Maltose