ArticleNature protocols2011
Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast.
Article in Nature protocols, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 40 papers.
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Who cites it
40 citing papers in PubMed, 80 citations in OpenAlex.
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- Plant SWEET Family of Sugar Transporters: Structure, Evolution and Biological Functions.Biomolecules · 2022Review
- Kinetic Modeling ofMetabolites · 2022Review
- Sensors for the quantification, localization and analysis of the dynamics of plant hormones.The Plant journal : for cell and molecular biology · 2021Review
- Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow.International journal of molecular sciences · 2020Review
- Affinity Purification of GO-Matryoshka Biosensors fromBio-protocol · 2020Article
- One-pot HTST synthesis of responsive fluorescent ZnO@apo-enzyme composite microgels for intracellular glucometry.RSC advances · 2020Article
- Resolving subcellular plant metabolism.The Plant journal : for cell and molecular biology · 2019Article
- FRET Microscopy in Yeast.Biosensors · 2019Review
Corrections and comments
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Authors and funding
5 authors at 1 institution in 2 countries.
Funding
Abstract
Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded Förster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (K(d)) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.