Genetically encoded sensors for metabolites

Deuschle, Karen; Fehr, Marcus; Hilpert, Melanie; Lager, Ida; Lalonde, Sylvie; Looger, Loren L.; Okumoto, Sakiko; Persson, Jörgen; Schmidt, Anja; Frommer, Wolf B.

doi:10.1002/cyto.a.20119

Cited by 54 publications

(36 citation statements)

References 57 publications

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“…Besides genetic differences, several other sources for population heterogeneity exist, of which several are also known to cause metabolic differences. Today, methods capable of resolving differences in metabolite levels on the single cell level are provided, within limits, by molecular sensors such as FRET sensors [6,7] or aptamer-based technology [8 ,9]. Both types of molecular sensors, however, are difficult to develop, are limited to specific analytes, and quantitative analyses (e.g.…”

Section: Introductionmentioning

confidence: 99%

Single cell metabolomics

Heinemann

Zenobi

2011

Current Opinion in Biotechnology

116

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Recent discoveries suggest that cells of a clonal population often display multiple metabolic phenotypes at the same time. Motivated by the success of mass spectrometry (MS) in the investigation of population-level metabolomics, the analytical community has initiated efforts towards MS-based single cell metabolomics to investigate metabolic phenomena that are buried under the population average. Here, we review the current approaches and illustrate their advantages and disadvantages. Because of significant advances in the field, different technologies are now at the verge of generating data that are useful for exploring and investigating metabolic heterogeneity. IntroductionQuantitative metabolomics, the technology for large-scale quantification of intracellular metabolite concentrations, is a powerful tool in systems biology research that has recently led to a series of interesting findings (e.g. [1][2][3][4]). Because of the metabolome's chemical diversity, mass spectrometry (MS) is the analytical method of choice [5]. In addition to analytical challenges, quantitative metabolomics as required for addressing (systems) biology questions poses significant challenges in sample processing. One important challenge is the need to preserve the original metabolome during sample processing, which is often difficult because of the presence of enzymes in the sample and the fast metabolic turnover rates.For sensitivity reasons, current metabolomics methods require samples that contain a large number of cells. However, cell populations are not necessarily homogeneous. Besides genetic differences, several other sources for population heterogeneity exist, of which several are also known to cause metabolic differences. Today, methods capable of resolving differences in metabolite levels on the single cell level are provided, within limits, by molecular sensors such as FRET sensors [6,7] or aptamer-based technology [8 ,9]. Both types of molecular sensors, however, are difficult to develop, are limited to specific analytes, and quantitative analyses (e.g. in terms of mol/L) are hardly possible with them. Laserinduced fluorescence, as introduced by Dovichi for single cell proteomics, is limited to fluorescent compounds or labelled species [10,11]. In addition, all these existing methods share the limitation that they can never be extended to the ''-omics'' level, that is to measuring a large number of metabolites at the same time. They will thus not be applicable to discovery type research and research that requires a large number of metabolites to be measured in the same cell.Because of the success of mass spectrometry in population-level metabolome analyses, the analytical community has recently made great strides towards single cell level metabolite analyses (for a review, see [12]). So far, however, hardly any new biological insight has been generated from these endeavours. In this Current Opinion paper, we will thus not only review the current status of MS-based single cell metabolomics but also discuss which of the differ...

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Section: Introductionmentioning

confidence: 99%

Single cell metabolomics

Heinemann

Zenobi

2011

Current Opinion in Biotechnology

116

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“…The role for high glucose in signaling is an extraordinarily complicated problem to solve, because it must take into consideration that d-glucose is rapidly taken up, a high concentration of sucrose occurs in apoplastic transport and must encounter localized invertases within their cell walls, cells may have desensitization mechanisms operating as a function of concentration and exposure time, and there are no physiological assays for d-glucose responses with response times in minutes to assess this (current sugar sensitivity assays are in days using organ growth acclimated in applied glucose concentrations). In vivo extracellular sensors for d-glucose (Deuschle et al, 2005) are required to answer unequivocally this question, but these are not yet available. However, indirect measurements have been applied to address this problem, and estimates of apoplastic levels of d-glucose at 150 mM (3%) or higher have been suggested (McLaughlin and Boyer, 2004;Makela et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

A Golgi-localized Hexose Transporter Is Involved in Heterotrimeric G Protein-mediated Early Development inArabidopsis

X.¹,

Weerasinghe²,

Perdue³

et al. 2006

MBoC

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Signal transduction involving heterotrimeric G proteins is universal among fungi, animals, and plants. In plants and fungi, the best understood function for the G protein complex is its modulation of cell proliferation and one of several important signals that are known to modulate the rate at which these cells proliferate is D-glucose. Arabidopsis thaliana seedlings lacking the ␤ subunit (AGB1) of the G protein complex have altered cell division in the hypocotyl and are D-glucose hypersensitive. With the aim to discover new elements in G protein signaling, we screened for gain-of-function suppressors of altered cell proliferation during early development in the agb1-2 mutant background. One agb1-2-dependent suppressor, designated sgb1-1 D for suppressor of G protein beta1 (agb1-2), restored to wild type the altered cell division in the hypocotyl and sugar hypersensitivity of the agb1-2 mutant. Consistent with AGB1 localization, SGB1 is found at the highest steady-state level in tissues with active cell division, and this level increases in hypocotyls when grown on D-glucose and sucrose. SGB1 is shown here to be a Golgi-localized hexose transporter and acts genetically with AGB1 in early seedling development. INTRODUCTIONAn evolutionarily ancient mechanism for sensing extracellular signals involves the heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits. Heterotrimeric G protein complexes link ligand perception via seven-transmembrane (7TM), G protein-coupled receptors (GPCRs) to downstream effectors. Genes that encode G protein signaling elements have been identified in amoebae, fungi, plants, and animals, but among all multicellular eukaryotes, plants have the simplest repertoire of G protein elements to date. Specifically, the Arabidopsis genome encodes a single canonical G␣ and G␤ (AGB1) subunit and two G␥ subunits and a single regulator of G signaling (RGS1) protein (Jones and Assmann, 2004). There are as yet no plant GPCRs having confirmed ligands, although plants do have a limited set of predicted 7TM proteins (Moriyama and Jones, unpublished data). Similarly, there are few known downstream effectors that physically interact with either the plant G␣ subunit or the G␤␥ dimer. One example is a pirin protein (Lapik and Kaufman, 2003), known to serve as a transcriptional cofactor in humans, but with unknown function in Arabidopsis. Based on either genetic or biochemical tests, G␣ effectors in plants also include phospholipase D (Mishra et al., 2006) and ion channels (Aharon et al., 1998;Wang et al., 2001). Recently, we reported that a plant interactor and putative effector to G␣ is an outer membrane plastid protein designated THF1, and this protein together with G␣ comprises part of a dglucose signaling network (Huang et al., 2006).In animals and yeast, heterotrimeric G proteins couple a diverse set of signals such as photons, ions, small molecules, sugars, peptides, and protein ligands (Jones and Assmann, 2004) to control a broad range of physiology (Csaszar and Abel, 2001;Rosenkilde et al., 2001;Roc...

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“…GFP mutants with shifted wavelengths of excitation or emission have been used as FRET donors and acceptors (Miyawaki, 2003;Miyawaki et al, 1997). GFP-based sensors can be expressed in situ by gene transfer techniques, and have therefore been applied, in particular, for cellular imaging of physiological activity in complex systems, a field extensively reviewed (Ehrhardt, 2003;Deuschle et al, 2005;Miyawaki, 2003;Fehr et al, 2005;Chudakov et al, 2005). As an example, calmodulin which undergoes a conformational change upon binding to calcium, was used to induce FRET between a donor and acceptor variant of attached GFP.…”

Section: Methods For Labeling Bindersmentioning

confidence: 99%

Fluorescence sensing of intermolecular interactions and development of direct molecular biosensors

2006

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Molecular biosensors are devices of molecular size that are designed for sensing different analytes on the basis of biospecific recognition. They should provide two coupled functions - the recognition (specific binding) of the target and the transduction of information about the recognition event into a measurable signal. The present review highlights the achievements and prospects in design and operation of molecular biosensors for which the transduction mechanism is based on fluorescence. We focus on the general strategy of fluorescent molecular sensing, construction of sensor elements, based on natural and designed biopolymers (proteins and nucleic acids). Particular attention is given to the coupling of sensing elements with fluorescent reporter dyes and to the methods for producing efficient fluorescence responses.

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Genetically encoded sensors for metabolites

Cited by 54 publications

References 57 publications

Single cell metabolomics

Single cell metabolomics

A Golgi-localized Hexose Transporter Is Involved in Heterotrimeric G Protein-mediated Early Development inArabidopsis

Fluorescence sensing of intermolecular interactions and development of direct molecular biosensors

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