Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin

@article{Miyawaki1997FluorescentIF,
  title={Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin},
  author={A. Miyawaki and J. Llopis and R. Heim and J. McCaffery and Joseph A. Adams and M. Ikura and R. Tsien},
  journal={Nature},
  year={1997},
  volume={388},
  pages={882-887}
}
Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators ‘cameleons’. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP),, calmodulin, the calmodulin-binding peptide M13… Expand

Paper Mentions

Imaging intracellular free Ca2+ concentration using yellow cameleons.
TLDR
The use of this cameleon to image rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)]i) within HeLa cells is described and is amenable to emission ratioing, which is more quantitative than single-wavelength monitoring. Expand
Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum
TLDR
Red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have been developed and ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+, in a reliable manner. Expand
FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule
TLDR
A new cameleon is rationally designed that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 μM. Expand
GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles
TLDR
GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg2+, tunable Ca2+ affinity, uncomplicated calibration, and targetability to five distinct organelles. Expand
Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum
TLDR
GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging engineered to have Kd values of 24 μM and 12 μM are reported, which can be used to image mitochondrial and ER Ca 2+ dynamics in several cell types. Expand
Calcium measurements in organelles with Ca2+-sensitive fluorescent proteins.
TLDR
This chapter will review the progress made in the design and use of genetic Ca2+ indicators, and discuss how these new tools provide an opportunity to challenge several unsolved questions in Ca2- signaling. Expand
Ca2+ as a Second Messenger: New Reporters for Calcium (Cameleons and Camgaroos)
TLDR
Ca 2+ transients have traditionally been measured using synthetic fluorescent chelators or recombinant aequorin, but GFP family proteins have been observed to form obligate dimers and may thereby generate false-positive FRET signals. Expand
Calcium indicators based on calmodulin-fluorescent protein fusions.
TLDR
This chapter describes the methods involved in cloning chameleons, characterizing their biochemical and biophysical properties, and imaging them in single cells using a digital fluorescence microscope. Expand
Targeting of calcium-sensing protein cameleon from cytoplasm to nucleus
  • A. Grover
  • Biology
  • Journal of Plant Biochemistry and Biotechnology
  • 2014
TLDR
To combine the brightness of fluorescent indicators with the targetability of a biosynthetic indicator, new fluorescent indicators for Ca2+ that are genetically encoded without cofactors have been constructed and are called cameleons. Expand
Measuring calcium signaling using genetically targetable fluorescent indicators
TLDR
This protocol details how to set up and conduct a Ca2+-imaging experiment, accomplish offline data processing and convert the observed FRET ratio changes to Ca2+, and highlights some of the challenges in observing organellar Ca2+. Expand
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