title: Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity.
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.
authors.affiliation: 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.
authors.forename: Showming
authors.initials: S
authors.lastname: Kwok
authors.forename: Claudia
authors.initials: C
authors.lastname: Lee
authors.forename: Susana A
authors.initials: SA
authors.lastname: Sánchez
authors.forename: Theodore L
authors.initials: TL
authors.lastname: Hazlett
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Hayashi
mesh_terms: 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
chemical_list: D049452:Green Fluorescent Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2