Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells

  title={Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells},
  author={Fred Silvester Wouters and Philippe I. H. Bastiaens},
  journal={Current Biology},

Imaging Phosphorylation Dynamics of the Epidermal Growth Factor Receptor*

Data show that EGFR is under tonic phosphatase suppression maintaining the receptor in an unphosphorylated (silent) state and is deph phosphorylated at endomembranes after ligand-mediated endocytosis.

Fluorescence Lifetime Imaging Microscopy of Signal Transduction Protein Reactions in Cells

The encoding of a priori knowledge about the states of proteins in a global analysis of the FLIM data is essential for successfully recovering the population images.

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.

A FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another, implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled camera.

Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

This protocol describes both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis and shows how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei.

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

This review describes the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals and uses the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells.

Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques.

The implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope demonstrates that FLIM allows sensitive measurements of protein-protein interactions on a spatial scale less than 10 nm using commercially available components.



Imaging protein kinase Calpha activation in cells.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently

Imaging Protein Kinase Cα Activation in Cells

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently

Endocytosis of Functional Epidermal Growth Factor Receptor-Green Fluorescent Protein Chimera*

This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible.

Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor.

The combined PFA/methanol protocol is universally applicable for the fixation of transmembrane and soluble cytoplasmic proteins and preserves the fluorescence of GFP.

Lifetime‐selective fluorescence imaging using an rf phase‐sensitive camera

We report the creation of two‐dimensional fluorescence lifetime images, based on a sinusoidally modulated image intensifier that is operated as a radio‐frequency phase‐sensitive camera, synchronized

Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin.

The results demonstrate vesicular transport of the holotoxin from the plasma membrane to the Golgi compartment with subsequent separation of the CTA and CTB subunits.

Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase.

The hypothesis that full-length receptor aggregation itself, induced by ligand binding to the extracellular domain, results in intracellular domain interactions and the activation of kinase activity is supported.

Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation.

It is proposed that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylated, probably facilitated by receptor oligomerization.