Abstract
Recently Ca2+-Calmodulin-dependent protein kinase II (CaMKII), an abundant synaptic protein, was shown to be important in the organization of actin filaments at synapses. We report the binding properties and mechanistic role of βCaMKII as an actin binding protein in vitro. A particular case is the ability of βCaMKII to bundle actin filaments, which we corroborated by cryo-electron tomography. In addition, a pyrene-actin fluorescent polymerization assay was used to determine that the enzyme reduced the rate of polymerization by ∼ 80%, suggesting that βCaMKII either serves as a capping protein or binds monomeric actin reducing the amount of freely available monomers to nucleate polymer assembly. Finally, by means of fluorescent cross correlation spectroscopy we determined that the β isoform of CaMKII does bind to monomeric actin, reaching saturation at a stoichiometry of 6:1 actin mononmers per βCaMKII holoenzyme with a binding affinity of ∼2 μM. In conclusion, βCaMKII has a dual functional role; it can sequester monomeric actin to reduce actin polymerization and can also bundle actin filaments. Together, these effects would impact both the dynamics of assembly of actin filaments and enhance the structural rigidity of the filaments once formed, significantly impacting the structure of synapses.
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