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Related Experiment Videos

Dynamic and quantitative Ca2+ measurements using improved cameleons.

A Miyawaki1, O Griesbeck, R Heim

  • 1Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA.

Proceedings of the National Academy of Sciences of the United States of America
|March 3, 1999
PubMed
Summary

New cameleon biosensors offer improved calcium (Ca2+) measurements by reducing pH interference. These genetically encoded indicators are valuable tools for cellular research despite potential calcium buffering effects.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Imaging

Background:

  • Cameleons are genetically encoded fluorescent indicators for Ca2+ using green fluorescent protein variants and calmodulin (CaM).
  • Original cameleons exhibited significant pH interference and uncharacterized Ca2+-buffering and cross-reactivity.
  • Previous limitations hindered Ca2+ monitoring in various biological contexts.

Purpose of the Study:

  • To reduce the pH sensitivity of cameleon biosensors.
  • To improve the accuracy of Ca2+ measurements in biological systems.
  • To characterize the Ca2+-buffering and signaling interactions of cameleons.

Main Methods:

  • Introduced V68L and Q69K mutations into the acceptor yellow green fluorescent protein of cameleons.
  • Developed genetically encoded fluorescent indicators for Ca2+.

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  • Utilized one- and two-photon excitation microscopy for imaging.

    • Main Results:

    • Significantly reduced pH sensitivity in the modified cameleons.
    • Enabled accurate Ca2+ measurements even during cytosolic acidification.
    • Observed that elevated Ca2+ promotes intramolecular interaction within cameleons, reducing interaction with endogenous CaM.

    Conclusions:

    • Modified cameleons provide more reliable Ca2+ measurements by minimizing pH interference.
    • Overexpression of cameleons primarily increases Ca2+ buffering rather than perturbing CaM-dependent signaling pathways.
    • Cameleons are promising tools for monitoring Ca2+ in diverse biological environments.