Stimulation-induced changes in diffusion and structure of calmodulin and calmodulin-dependent protein kinase II proteins in neurons

•CaM and CaMKII diffusion was measured with FCS in HEK293 cells and cultured neurons.•A simultaneous multiple-point FCS recording system was developed.•CaM showed activity-dependent change in diffusion properties in neurons.•FCS detected holoenzymatic forms of CaMKIIα and β and degradation by stimulation. Calcium/calmodulin-dependent protein kinase II (CaMKII) and calmodulin (CaM) play essential roles in synaptic plasticity, which is an elementary process of learning and memory. In this study, fluorescence correlation spectroscopy (FCS) revealed diffusion properties of CaM, CaMKIIα and CaMKIIβ proteins in human embryonic kidney 293 (HEK293) cells and hippocampal neurons. A simultaneous multiple-point FCS recording system was developed on a random-access two-photon microscope, which facilitated efficient analysis of molecular dynamics in neuronal compartments. The diffusion of CaM in neurons was slower than that in HEK293 cells at rest, while the diffusion in stimulated neurons was accelerated and indistinguishable from that in HEK293 cells. This implied that activity-dependent binding partners of CaM exist in neurons, which slow down the diffusion at rest. Diffusion properties of CaMKIIα and β proteins implied that major populations of these proteins exist as holoenzymatic forms. Upon stimulation of neurons, the diffusion of CaMKIIα and β proteins became faster with reduced particle brightness, indicating drastic structural changes of the proteins such as dismissal from holoenzyme structure and further fragmentation.