研究目的
To develop single fluorescent protein-based glucose indicators for monitoring glucose dynamics in living cells and in vivo, and to apply them for dual-color imaging and understanding energy metabolism.
研究成果
The developed Green Glifons are effective tools for visualizing glucose dynamics across a wide range of concentrations and in various species. They enable dual-color imaging and provide insights into glucose metabolism, such as the effects of artificial sweeteners on glucose uptake in pancreatic β cells. These indicators will advance research in energy metabolism by facilitating high spatiotemporal resolution studies.
研究不足
The indicators have pH sensitivity similar to other GFP-based sensors, which may affect measurements under non-physiological conditions. The dynamic range in live cells is lower than in vitro due to basal glucose levels. The specificity includes response to galactose, which could interfere in certain contexts.
1：Experimental Design and Method Selection:
The study involved designing glucose indicators by inserting a bacterial glucose-binding domain into a fluorescent protein (Citrine GFP variant), optimizing linkers and mutations for dynamic range and EC50 values, and applying these indicators in various biological contexts. Theoretical models include fluorescence spectroscopy and imaging techniques.
2：Sample Selection and Data Sources:
Samples included purified recombinant proteins, HeLa cells, MIN6 m9 β cells, C. elegans, and mouse blood plasma. Selection criteria were based on cell types relevant to glucose metabolism.
3：List of Experimental Equipment and Materials:
Equipment included spectro-fluorophotometers, microscopes, cameras, plate readers, and various reagents and plasmids. Materials included culture media, buffers, enzymes, and dyes.
4：Experimental Procedures and Operational Workflow:
Steps included plasmid construction, protein expression and purification, in vitro spectroscopy, cell culture and transfection, live-cell imaging, C. elegans imaging, and data analysis. Procedures involved glucose stimulation, inhibitor treatments, and dual-color imaging.
5：Data Analysis Methods:
Data were analyzed using ImageJ for dose-response curves, MetaMorph for fluorescence intensity measurements, and GraphPad Prism for statistical tests (Welch's t-test, ANOVA).
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spectro-fluorophotometer
F-2500
Hitachi
Measure excitation and emission spectra of purified proteins.
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ultraviolet-visible spectrophotometer
UV-1800
Shimadzu
Measure absorption spectra of purified proteins.
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fluorescence plate reader
Varioskan LUX
Thermo Fisher Scientific
Measure fluorescence intensity in 96-well plates for blood glucose level measurements.
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fluorescence inverted microscope
IX-71
Olympus
Perform live-cell imaging of HeLa cells and C. elegans.
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fluorescence inverted microscope
Axio Observer D1
Carl Zeiss
Perform live-cell imaging of MIN6 m9 cells.
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inverted microscope
IX50
Olympus
Used for microinjection into C. elegans gonads.
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96-well plate
OptiPlate-96 F
PerkinElmer
Hold samples for fluorescence measurements in plate reader.
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glucose oxidase-based sensor
ACCU-CHEK ST Meter
Roche
Measure blood glucose levels for comparison with Green Glifon measurements.
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manipulator
MN-4
Narishige
Assist in microinjection procedures.
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micromanipulator
MMO-203
Narishige
Assist in microinjection procedures.
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microinjector
FemtoJet 4i
Eppendorf
Perform microinjection of plasmids into C. elegans.
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Ni-NTA beads
QIAGEN
Purify recombinant proteins using affinity chromatography.
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PD-10 gel filtration column
GE Healthcare
Remove imidazole from purified proteins.
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Lipofectamine 2000 Transfection Reagent
Life Technologies
Transfect plasmids into mammalian cells.
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Rhod2
Sigma-Aldrich
Dye for calcium imaging in dual-color experiments.
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