In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors.
Glucose homeostasis is a function of glucose supply, transport across the plasma membrane, and metabolism. To monitor glucose dynamics in individual cells, a glucose nanosensor was developed by flanking the Escherichia coli periplasmic glucose/galactose-binding protein with two different green fluorescent protein variants. Upon binding of substrate the FLIPglu-170n sensor showed a concentration-dependent decrease in fluorescence resonance energy transfer between the attached chromophores with a binding affinity for glucose of 170 nm. Fluorescence resonance energy transfer measurements with different sugars indicated a broad selectivity for monosaccharides. An affinity mutant with a Kd of approximately 600 microM was generated, which showed higher substrate specificity, and thus allowed specific monitoring of reversible glucose dynamics in COS-7 cells in the physiological range. At external glucose concentrations between 0.5 and 10 mM, reflecting typical blood levels, free cytosolic glucose concentrations remained at approximately 50% of external levels. The removal of glucose lead to reduced glucose levels in the cell, demonstrating reversibility and visualizing homeostasis. Glucose levels dropped even in the presence of the transport inhibitor cytochalasin B, indicating rapid metabolism. Consistently, the addition of 2-deoxyglucose, which is not recognized by the sensor, affects glucose uptake and metabolism rates. Within the physiological range, glucose utilization, i.e. hexokinase activity, was not limiting. Furthermore, the results show that in COS-7 cells, cytosolic glucose concentrations can vary over at least two orders of magnitude. The glucose nanosensor provides a novel tool with numerous scientific, medical, and environmental applications.
Fehr
Marcus
M
Zentrum für Molekular biologie der Pflanzen Tübingen, Plant Physiology, Auf der Morgenstelle 1, D-72076 Tübingen, Germany.
Lalonde
Sylvie
S
Lager
Ida
I
Wolff
Michael W
MW
Frommer
Wolf B
WB
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D003847:Deoxyglucose / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D049452:Green Fluorescent Proteins; D006593:Hexokinase / Q000378:metabolism; D006706:Homeostasis; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D036103:Nanotechnology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D001426:Bacterial Proteins; D051272:Glucose Transporter Type 1; D008164:Luminescent Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D003571:Cytochalasin B; D003847:Deoxyglucose; D006593:Hexokinase; D005947:Glucose