Development and use of fluorescent nanosensors for metabolite imaging in living cells

Abstract

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.

Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors

“…The FPs attached to the different lobes are expected to undergo a significant relative rearrangement in concert with the conformational movement. The sign of the change in FRET efficiency for each existing sensor was consistent with the change in interfluorophore distance predicted from the crystal structure (9)(10)(11)(12)35), demonstrating the importance of donor-acceptor separation in transfer efficiency. Interestingly, however, the magnitude of the FRET change of the various FLIP sensors is constant despite different absolute distance changes of the termini, suggesting that rotational averaging serves to dampen the distance effect and to change the value of the orientation factor 2 (36).…”

“…Fluorescent indicator protein (FLIP) nanosensors have thus far been developed by using type I periplasmic binding proteins (9)(10)(11)(12)19), with fluorophores attached to the N-and C-termini of the recognition element present on the two different lobes. The FPs attached to the different lobes are expected to undergo a significant relative rearrangement in concert with the conformational movement.…”

“…Recently, a number of bacterial periplasmic binding proteins, which upon ligand binding undergo a venus flytrap-like closure of their two lobes, have successfully been used as scaffolds to develop FRET nanosensors for maltose, ribose, and glucose (9-11). Because they are encoded genetically, the sensors can be used in vivo and targeted to subcellular compartments to specifically analyze concentration changes within a specific compartment of an intact live cell (12).…”

Biosensor Recognition Elements

“…Nanosensors will enable compartmental analyses of metabolite levels and metabolic activity which will drive "diagnostic methodologies." Nanosensor prototypes have been expressed in Yeast and in mammalian cell cultures for determination of carbohydrate homeostasis in living cells with subcellular resolution (Fehr et al, 2005). Nanosensors can be selectively expressed under the control of tissue specific promoters.…”

Engineered Derivatives of Maltose-Binding Protein

“…Frommer and coworkers constructed enhanced cyan fluorescent protein-MBP-enhanced yellow fluorescent protein fusion (CFP-MBP-YFP) biosensors that they characterized in vitro and in vivo in yeast (Fehr et al, 2002, Fehr et al, 2005. The change in fluorescence ratio they observed for these sensors was around 0.1.…”