To understand metabolic networks, fluxes and regulation, it is crucial to be able to determine the cellular and subcellular levels of metabolites. Methods such as PET and NMR imaging have provided us with the possibility of studying metabolic processes in living organisms. However, at present these technologies do not permit measuring at the subcellular level. The cameleon, a fluorescence resonance energy transfer (FRET)-based nanosensor uses the ability of the calcium-bound form of calmodulin to interact with calmodulin binding polypeptides to turn the corresponding dramatic conformational change into a change in resonance energy transfer between two fluorescent proteins attached to the fusion protein. The cameleon and its derivatives were successfully used to follow calcium changes in real time not only in isolated cells, but also in living organisms. To provide a set of tools for real-time measurements of metabolite levels with subcellular resolution, protein-based nanosensors for various metabolites were developed. The metabolite nanosensors consist of two variants of the green fluorescent protein fused to bacterial periplasmic binding proteins. Different from the cameleon, a conformational change in the binding protein is directly detected as a change in FRET efficiency. The prototypes are able to detect various carbohydrates such as ribose, glucose and maltose as purified proteins in vitro. The nanosensors can be expressed in yeast and in mammalian cell cultures and were used to determine carbohydrate homeostasis in living cells with subcellular resolution. One future goal is to expand the set of sensors to cover a wider spectrum of metabolites by using the natural spectrum of bacterial periplasmic binding proteins and by computational design of the binding pockets of the prototype sensors.
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February 2005
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Conference Article|
February 01 2005
Development and use of fluorescent nanosensors for metabolite imaging in living cells
M. Fehr;
M. Fehr
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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S. Okumoto;
S. Okumoto
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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K. Deuschle;
K. Deuschle
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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I. Lager;
I. Lager
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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L.L. Looger;
L.L. Looger
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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J. Persson;
J. Persson
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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L. Kozhukh;
L. Kozhukh
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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S. Lalonde;
S. Lalonde
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
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W.B. Frommer
W.B. Frommer
1
1Carnegie Institution, Plant Biology, 260 Panama St., Stanford, CA 94305, U.S.A.
1To whom correspondence should be addressed (email wfrommer@stanford.edu).
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Publisher: Portland Press Ltd
Received:
September 17 2004
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2005 The Biochemical Society
2005
Biochem Soc Trans (2005) 33 (1): 287–290.
Article history
Received:
September 17 2004
Citation
M. Fehr, S. Okumoto, K. Deuschle, I. Lager, L.L. Looger, J. Persson, L. Kozhukh, S. Lalonde, W.B. Frommer; Development and use of fluorescent nanosensors for metabolite imaging in living cells. Biochem Soc Trans 1 February 2005; 33 (1): 287–290. doi: https://doi.org/10.1042/BST0330287
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