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Related Experiment Videos

Imaging activation of two Ras isoforms simultaneously in a single cell.

Anna Peyker1, Oliver Rocks, Philippe I H Bastiaens

  • 1European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Chembiochem : a European Journal of Chemical Biology
|January 8, 2005
PubMed
Summary

This study introduces a new fluorescence imaging method to simultaneously track two protein activations in one cell. This technique reveals distinct Ras protein activation patterns in different cellular compartments.

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Area of Science:

  • Cellular Biology
  • Biophysics
  • Molecular Imaging

Background:

  • Fluorescence resonance energy transfer (FRET) microscopy enables protein interaction studies in living cells.
  • Current FRET methods are limited to measuring single protein interactions due to spectral constraints.
  • Understanding complex cellular signaling networks requires monitoring multiple protein interactions simultaneously.

Purpose of the Study:

  • To develop a novel time-resolved fluorescence imaging method for simultaneous monitoring of two protein activations in a single cell.
  • To adapt a Ras sensor for measuring Ras activation via FRET using distinct fluorescent proteins.
  • To investigate differential Ras isoform and mutant activation profiles in various cellular compartments.

Main Methods:

  • Utilized yellow (YFP) and cyan (CFP) fluorescent proteins as donors and a tandem construct of Heteractis crispa Red (tHcRed) as a common acceptor.

Related Experiment Videos

  • Employed fluorescence-lifetime imaging microscopy (FLIM) to measure YFP and CFP donor lifetimes, enabling discrimination of two independent FRET signals.
  • Developed a Ras sensor based on Ras and its Ras binding domain (RBD) of Raf, where interaction signifies Ras activation detected by FRET.

    • Main Results:

    • Successfully measured two independent FRET signals simultaneously within a single cell.
    • Demonstrated distinct activation profiles for different Ras isoforms and mutants localized to the plasma membrane, Golgi, or both compartments.
    • Observed differential Ras regulation in specific cellular compartments upon growth-factor stimulation.

    Conclusions:

    • The novel time-resolved FRET-FLIM method allows simultaneous monitoring of multiple protein activation states.
    • Ras proteins exhibit compartment-specific activation patterns, indicating differential regulation within the cell.
    • This technique is valuable for studying the spatiotemporal dynamics of protein regulation in complex signaling networks.