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. Some functions of CaMKII are mediated by interactions with synaptic proteins, and activity-triggered translocation of CaMKII to synapses has been heavily studied. However, CaMKII actions away from the postsynaptic density (PSD) remain poorly understood, in part because of the difficulty in discerning where CaMKII binds in live cells. We used photoactivated localization microscopy (PALM) in rat hippocampal neurons to track single molecules of CaMKII␣, mapping its spatial and kinetic heterogeneity at high resolution. We found that CaMKII␣ exhibits at least three kinetic subpopulations, even within individual spines. Latrunculin application or coexpression of CaMKII␤ carrying 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 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 quite distant from the synapse. Together, these results indicate that CaMKII mobility within spines is determined by association with multiple interacting proteins, even outside the PSD, suggesting diverse mechanisms by which CaMKII may regulate synaptic transmission.

A Temporary Gating of Actin Remodeling during Synaptic Plasticity Consists of the Interplay between the Kinase and Structural Functions of CaMKII

“…To test whether the phosphorylation influences CaMKIIβ mobility, we analyzed the motion of individual CaMKIIβ molecules using single-particle tracking photoactivated localization microscopy (sptPALM) (Manley et al, 2008; Frost et al, 2010; Lu et al, 2014). The distribution of localized molecules of mEos3-CaMKIIβ WT and All A again revealed a greater enrichment within spines compared to that of the All D mutant (Fig.…”

Psychiatric Risk Factor ANK3/Ankyrin-G Nanodomains Regulate the Structure and Function of Glutamatergic Synapses

“…Ankyrin-G nanodomains were not located directly within the PSD, but surrounding the PSD, or within the spine neck away from the synapse. PALM imaging has recently identified a similar localization for CamKII, a key protein involved in synaptic plasticity (Lu et al, 2014). Our SIM localization data suggest a novel function for ankyrin-G that differs from a ‘classical’ PSD scaffolding protein.…”

“…The advent of super-resolution microscopy has facilitated imaging beyond the diffraction limit of light, providing unprecedented insight into subcellular architecture (Tonnesen and Nagerl, 2013). Several recent studies have used these techniques to reveal that AMPARs, PSD95, actin and CaMKII are localized to subsynaptic domains (Frost et al, 2010; Lu et al, 2014; MacGillavry et al, 2013; Nair et al, 2013). Super-resolution approaches have not yet been used to investigate synaptic function of newly identified psychiatric risk genes.…”

Subsynaptic spatial organization as a regulator of synaptic strength and plasticity

“…2f), for instance CaMKII on specific NMDARs [26]. Alternatively, regulation of receptor-anchoring PSD proteins may be required to drive receptor exchange in local regions of the synapse.…”