Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity.
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.
Kwok
Showming
S
RIKEN-MIT Neuroscience Research Center, The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue 46-4243A, Cambridge, MA 02139, USA.
Lee
Claudia
C
Sánchez
Susana A
SA
Hazlett
Theodore L
TL
Gratton
Enrico
E
Hayashi
Yasunori
Y
D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015336:Molecular Probe Techniques
D049452:Green Fluorescent Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2