Genetically Encoded Probe for Fluorescence Lifetime Imaging of CaMKII Activity

Biochem Biophys Res Commun. 2008 May 2;369(2):519-25. doi: 10.1016/j.bbrc.2008.02.070. Epub 2008 Feb 25.

Abstract

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism*
  • Cell Line
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins / genetics*
  • Humans
  • Kidney / metabolism*
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Molecular Probe Techniques*

Substances

  • Green Fluorescent Proteins
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2