HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 21, 19127-19133, May 23, 2003

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 278/21/19127    most recent M301333200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fehr, M. Articles by Frommer, W. B. Search for Related Content PubMed PubMed Citation Articles by Fehr, M. Articles by Frommer, W. B. Social Bookmarking                      What's this?

In Vivo Imaging of the Dynamics of Glucose Uptake in the Cytosol of COS-7 Cells by Fluorescent Nanosensors^*,

Marcus Fehr , Sylvie Lalonde, Ida Lager, Michael W. Wolff and Wolf B. Frommer  

From the Zentrum für Molekular biologie der Pflanzen Tübingen, Plant Physiology, Auf der Morgenstelle 1, D-72076 Tübingen, Germany

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 K_d of 600 µM 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 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.

Received for publication, February 6, 2003 , and in revised form, March 12, 2003.

To whom correspondence should be addressed. Tel.: 650-325-1521; Fax: 650-325-6857; E-mail: frommer{at}andrew2.stanford.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J.-S. Ha, J. J. Song, Y.-M. Lee, S.-J. Kim, J.-H. Sohn, C.-S. Shin, and S.-G. Lee Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in Living Saccharomyces cerevisiae Cells Appl. Envir. Microbiol., November 15, 2007; 73(22): 7408 - 7414. [Abstract] [Full Text] [PDF]

				L. E. Meyer, L. B. Machado, A. P. S. A. Santiago, W. S. da-Silva, F. G. De Felice, O. Holub, M. F. Oliveira, and A. Galina Mitochondrial Creatine Kinase Activity Prevents Reactive Oxygen Species Generation: ANTIOXIDANT ROLE OF MITOCHONDRIAL KINASE-DEPENDENT ADP RE-CYCLING ACTIVITY J. Biol. Chem., December 8, 2006; 281(49): 37361 - 37371. [Abstract] [Full Text] [PDF]

				I. Lager, L. L. Looger, M. Hilpert, S. Lalonde, and W. B. Frommer Conversion of a Putative Agrobacterium Sugar-binding Protein into a FRET Sensor with High Selectivity for Sucrose J. Biol. Chem., October 13, 2006; 281(41): 30875 - 30883. [Abstract] [Full Text] [PDF]

				K. Deuschle, B. Chaudhuri, S. Okumoto, I. Lager, S. Lalonde, and W. B. Frommer Rapid Metabolism of Glucose Detected with FRET Glucose Nanosensors in Epidermal Cells and Intact Roots of Arabidopsis RNA-Silencing Mutants PLANT CELL, September 1, 2006; 18(9): 2314 - 2325. [Abstract] [Full Text] [PDF]

				M. Fehr, H. Takanaga, D. W. Ehrhardt, and W. B. Frommer Evidence for High-Capacity Bidirectional Glucose Transport across the Endoplasmic Reticulum Membrane by Genetically Encoded Fluorescence Resonance Energy Transfer Nanosensors Mol. Cell. Biol., December 15, 2005; 25(24): 11102 - 11112. [Abstract] [Full Text] [PDF]

				S. Okumoto, L. L. Looger, K. D. Micheva, R. J. Reimer, S. J Smith, and W. B. Frommer Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors PNAS, June 14, 2005; 102(24): 8740 - 8745. [Abstract] [Full Text] [PDF]

				L. L. Looger, S. Lalonde, and W. B. Frommer Genetically Encoded FRET Sensors for Visualizing Metabolites with Subcellular Resolution in Living Cells Plant Physiology, June 1, 2005; 138(2): 555 - 557. [Full Text] [PDF]

				M. Zaccolo Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses Circ. Res., April 16, 2004; 94(7): 866 - 873. [Abstract] [Full Text] [PDF]