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.
Highlights
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase crucial for synaptic plasticity in the central nervous system [1]
This enables activated CaMKII to stay activated, even in the absence of Ca2+, until all subunits are dephosphorylated. This process has been suggested as an underlying mechanism for persistent change in synaptic efficacy, represented by long-term potentiation (LTP)
The elevated CaMKII activity likely remodels the postsynaptic protein complex, which eventually leads to an insertion of new AMPA
Summary
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase crucial for synaptic plasticity in the central nervous system [1]. FLIM imaging detects FRET-induced changes in nanosecond-order fluorescence decay-time of the donor fluorescence after excitation. Quantum yield and extinction coefficient data for GFP, mOrange, mStrawberry, Venus, TagRFP, DsRed monomer from [14]; mRFP from [17]; CFP K26R/N164H by personal communication from Drs Tomoo Ohashi and Harold P.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Biochemical and Biophysical Research Communications
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
