Multiple spatial and kinetic subpopulations of CaMKII in spines and dendrites as resolved by single-molecule tracking PALM

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII) is essential for synaptic plasticity underlying memory formation. The existing hypothesis is that with stimuli that induce LTP, activated CaMKII translocates to the PSD and phosphorylates synaptic proteins and thus increases synaptic strength. However, many known CaMKII substrates that regulate synaptic transmission lie away from the PSD, and it is unlikely that PSD-bound CaMKII can phosphorylate such targets. Therefore, I propose an alternative hypothesis that CaMKII interacts with binding partners both at and away from the PSD upon NMDAR stimulation. It has been difficult to study protein-protein interaction in living cells. I considered one way to do this would be to measure CaMKII mobility as an indication of its interaction with the substrate. Confocal imaging does not achieve the resolution necessary to distinguish subpopulations within the small volumes of a spine. Here, I used photo-activated localization microscopy (PALM) to track single molecules of CaMKIIα in live neurons, mapping their spatial distribution and kinetic heterogeneity at high resolution. I found that CaMKIIα exhibits at least three kinetic subpopulations, even within individual spines. Latrunculin application, which depolymerizes actin filaments, or co-expression of CaMKIIβ containing its actin-binding domain, strongly modulated CaMKII diffusion, indicating that a major subpopulation is regulated by the actin cytoskeleton. CaMKII in spines was typically more slowly mobile than in dendrites, consistent with the presence of a higher density of binding partners or obstacles. Importantly, NMDA receptor stimulation that triggered CaMKII activation prompted the immobilization and presumed binding of CaMKII in spines not only at PSDs but also at other points up to several hundred nanometers away, suggesting that activated kinase does not target only the PSD. Consistent with this, single endogenous activated CaMKII molecules detected via STORM immunocytochemistry were concentrated in spines both at the PSD and at points distant from the synapse. Together, these results indicate that CaMKII mobility within spines is determined by association with multiple interacting proteins that could potentially activate different downstream signals, even outside the PSD, The results lend greater support to the alternative hypothesis proposed above, and suggest diverse mechanisms by which CaMKII may regulate synaptic transmission.