Dataset Information

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors.

ABSTRACT: Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing protein-based nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications.

SUBMITTER: Fehr M

PROVIDER: S-EPMC125039 | biostudies-literature | 2002 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors.

Fehr Marcus M Frommer Wolf B WB Lalonde Sylvie S

Proceedings of the National Academy of Sciences of the United States of America 20020703 15

Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing protein-based nanosensors for a wide spectrum of solutes, allow ...[more]

PMID: 12097642

Similar Datasets

Project description:Background: HaloTag is a modified bacterial enzyme that binds rapidly and irreversibly to an array of synthetic ligands, including chemical dyes. When expressed in live cells in conjunction with a protein of interest, HaloTag can be used to study protein trafficking, synthesis, and degradation. For instance, sequential HaloTag labeling with spectrally separable dyes can be used to separate preexisting protein pools from proteins newly synthesized following experimental manipulations or the passage of time. Unfortunately, incomplete labeling by the first dye, or labeling by residual, trapped dye pools can confound interpretation. Methods: Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. To that end, we synthesized a non-fluorescent, cell permeable blocker (1-chloro-6-(2-propoxyethoxy)hexane; CPXH), essentially the HaloTag ligand backbone without the reactive amine used to attach fluorescent groups. Results: High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise2 and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 µM at 30 min. No overt effects on cell viability were observed at any concentration or treatment duration. The ability of CPXH to improve the reliability of newly synthesized protein detection was then demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. Practically no labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in live cells using HaloTag technology.

Dataset Information

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors.

Publications

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets