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Published on: May 19, 2017
Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin
1Department of Pharmacology, University of California, San Diego, La Jolla 92093-0647, USA.
Nature
|August 28, 1997
Summary
Researchers developed genetically encoded fluorescent calcium (Ca2+) indicators called cameleons. These cameleons allow visualization of localized Ca2+ signals within organelles and cytosol in live cells.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Localized calcium (Ca2+) signals are crucial for cellular processes but difficult to measure.
- Existing methods struggle to capture the spatial and temporal dynamics of intracellular Ca2+.
Purpose of the Study:
- To develop novel, genetically encoded fluorescent indicators for measuring localized Ca2+ signals.
- To visualize Ca2+ dynamics in specific subcellular compartments like the cytosol, nucleus, and endoplasmic reticulum.
Main Methods:
- Construction of 'cameleons', which are tandem fusions of fluorescent proteins (GFP variants), calmodulin, and M13 peptide.
- Utilizing fluorescence resonance energy transfer (FRET) between GFP variants to detect Ca2+ binding to calmodulin.
- Genetically encoding cameleons for targeted expression and visualization in live HeLa cells.
Main Results:
- Cameleons successfully visualized localized Ca2+ dynamics in the cytosol, nucleus, and endoplasmic reticulum.
- Resting Ca2+ concentrations in the endoplasmic reticulum were measured between 60-400 microM.
- Mobilized Ca2+ concentrations in the endoplasmic reticulum ranged from 1-50 microM.
- FRET also demonstrated the ability to monitor protein heterodimerization.
Conclusions:
- Cameleons are effective tools for measuring localized Ca2+ signals and protein interactions in live cells.
- The developed indicators enable detailed studies of Ca2+ signaling in various subcellular compartments.
- This technology advances the understanding of cellular Ca2+ dynamics and function.
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