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.
SourceAvailable from: Surendra Agrawal[Show abstract] [Hide abstract]
ABSTRACT: Nanotechnology research is rolling worldwide, having an impact on multiple sectors and with a general belief that medical and biological applications will form the greatest area of expansion over the next decade. This field is mainly driven by an endeavour to bring radical solutions to areas of medical need, not fulfilled presently. This article discusses the basic concepts and developments in the field of nanosensors and their applications in pharmaceutical and medicine fields. Various types of nanosensors including optical nanosensors, electrochemical nanosensors, chemical nanosensors, electrometers, biosensors, and deployable nanosensors are described. It describes the progression of this field of research from its birth up to the present, with emphasis on the techniques of sensor construction and their application to biomedical systems.
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ABSTRACT: The use of fluorescent proteins (FPs) in modern cell biology and microscopy has had an extraordinary impact on our ability to investigate dynamic processes in living cells. FPs are unique in that fluorescence is encoded solely by the primary amino acid sequence of the FP and does not require enzymatic modification or cofactors. This genetically encoded fluorescence enables the expression of FPs in diverse cells and organisms and the detection of that fluorescence in living systems. This chapter focuses on microscopy-based applications of FP detection to monitor protein localization, dynamics, interaction, and the cellular environment.Methods in cell biology 01/2013; 114:99-123. DOI:10.1016/B978-0-12-407761-4.00005-1 · 1.44 Impact Factor
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ABSTRACT: Quorum sensing (QS) is involved in many important biological functions such as luminescence, antibiotic production, and biofilm formation. The autoinducer N-(3-oxo-hexanoyl)-L-homoserine lactone (3OC6HSL) plays a significant role in the QS system of the marine bacterium Vibrio fischeri. Tracing 3OC6HSL would be significant in studies related to QS signal transduction. Traditional detection of QS signaling molecules has relied on bacterial reporter strains and high-performance liquid chromatography, which are time consuming and have low sensitivity. Because 3OC6HSL binding to LuxR from V. fischeri causes a conformational change, we developed a genetically encoded biosensor based on Förster resonance energy transfer (FRET) by inserting LuxR between the FRET pair YFP/CFP. The detection limit of the sensor was 100 μM. We attained an optimized sensor with 70 % Δratio increase by screening different hydrophobic linkers, and demonstrated the feasibility of this sensor for visualizing 3OC6HSL both in vitro and in vivo.Bioprocess and Biosystems Engineering 09/2013; 37(5). DOI:10.1007/s00449-013-1055-7 · 1.82 Impact Factor