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 CaMKIIα, mapping its spatial and kinetic heterogeneity at high resolution. We found that CaMKIIα exhibits at least three kinetic subpopulations, even within individual spines. Latrunculin application or coexpression of CaMKIIβ 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.

Keywords: NMDA receptors; kinase; long-term synaptic plasticity; postsynaptic density; super-resolution imaging; synapse.

Figure 1.

Super-resolved CaMKII distribution and dynamic subpopulations in living neurons. A, Cells expressing mEos2-CaMKIIα were imaged by single-molecule tracking PALM at 50 Hz. The intensity of the images acquired was averaged to construct a diffraction-limited image (left), and all localizations were binned to create a density map (right). Scale bar, 2.5 μm. B, Histogram of effective resolution calculated from R_eff = $\sqrt{{\text{r}}_{nn}^2+{\sigma }^2}$, where r_nn is the mean nearest neighbor distance of molecules within the region analyzed and σ the mean SD of the localizations (Gould et al., 2009). C, Cumulative frequency of the width of spine necks and filopodia. D, Example tracks of mEos2-CaMKIIα, with green and red points indicating the first and last localized position in the track. Scale bar, 500 nm. E, Histogram of track length (n = >20,000 tracks per cell for 4 neurons). F, Mean MSD from different groups (red represents live cells; black represents fixed cells) were plotted as a function of elapsed time to construct MSD versus time plot. A linear fit of the first three points of the MSD versus plot was used to calculate D_eff (n = 4 neurons). G, Example MSD versus time plots from molecules tracked for 4 frames. H, Histogram of D_eff derived from tracks with at least 4 frames, using a weighted fit of the MSD versus time, including zero as described in Materials and Methods (red and black) or a fit excluding zero (grays) (n = 4 neurons). I, Median D_eff from F and H. J, Single example of histogram of D_eff fitted with a two-component Gaussian: black represents original data; green represents Gaussian peak fitted; blue represents sum of individual Gaussians. K, Single example of histogram of D_eff fitted with a three-component Gaussian. L, The proportion of different subpopulations was quantified based on the characteristics of the fit. Area₁: 9.3%; Area₂: 35.9%; Area₃: 54.8% (n = 11). M, Mean D_eff of the subpopulations. D_eff1: 0.0025 μm²/s; D_eff2: 0.0173 μm²/s; D_eff3: 0.1903 μm²/s. N, Histogram of D_eff from simulated molecules (black) and experimental results (red). O, Correlation between spine size and mean D_eff (n = 61 spines, 11 neurons).

Figure 2.

Actin cytoskeleton regulates CaMKII mobility. A, Histogram of D_eff of cells transfected with mEos2-CaMKIIα or mEos2-CaMKIIα/Cer3-CaMKIIβ (1:1) (n = 13 neurons for mEos2-CaMKIIα and n = 17 neurons for mEos2-CaMKIIα/Cer3-CaMKIIβ). B, Histograms of D_eff were fit as in Figure 1K, and the proportion of each kinetic subpopulation was quantified. D_eff1: 0.0025 μm²/s (9.3 ± 3.3%); D_eff2: 0.0173 μm²/s (35.9 ± 5.4%); D_eff3: 0.1903 ± 0.045 μm²/s (54.8 ± 4.9%) in the mEos2-CaMKIIα expressing cells (n = 11 neurons). D_eff1: 0.0025 μm²/s (16.3 ± 3.6%); D_eff2: 0.011 μm²/s (46.3 ± 5.7%); D_eff3: 0.161 μm²/s (37.4 ± 3.8%) in the cells transfected with mEos2-CaMKIIα/Cer3-CaMKIIβ (n = 15 neurons). *p < 0.05 (two-way ANOVA repeated measures with one factor repeated). C, Histogram of D_eff of cells transfected with mEos2-CaMKIIα/Cer3-CaMKIIβ before and after Lat A (n = 3 neurons). D, D_eff distributions before or after Lat A treatment (n = 3 neurons). *p < 0.01 before versus after lat A (two-way ANOVA repeated measures with two factors repeated).

Figure 3.

The spatial distribution of CaMKII mobility is heterogeneous within cells. A, D_eff was calculated and color-coded to construct a spatial map of molecular mobility. Scale bar: left top, 1.5 μm. Enlarged view of the spine marked from the left. Scale bar: right top, 1 μm. B, Histogram of D_eff from spines or dendrites (K-S test, p < 0.001, n = 5). C, D_eff in spines or dendrites was fit as in Figure 1K, and the proportion of each kinetic subpopulation was quantified (n = 4).

Figure 4.

Activated CaMKII concentrates in spines both at the PSD and away from the PSD after NMDA receptor stimulation. A, Cells transfected with GFP-CaMKIIα and PSD-95-mCherry were imaged by confocal microscopy before and after NMDAR stimulation. Scale bar, 2 μm. B, Enlarged view from A (left) and other experiments before (right top) and after 0 Mg²⁺/Gly stimulation (right bottom). Scale bar, 1 μm. C, CaMKII localizations were binned (25 nm) to construct a map of CaMKII density under basal conditions and after Glu/Gly stimulation. Scale bar, 1 μm. D, CaMKII enrichment at the PSD was quantified in different cells under basal conditions and after Glu/Gly stimulation. *p < 0.05 (K-S test, n = 52 synapses for basal, n = 22 synapses for Glu/Gly). E, For basal condition and after Glu/Gly stimulation, image (top) shows density map of the spine from C (green) superimposed on deconvolved widefield image of PSD95-Cerulean3 (red). Lines indicate profiles along which intensity was measured, as shown in the graph (bottom). F, Quantification of the intensity of phospho-T286 immunostaining under basal conditions and after Glu/Gly stimulation. *p < 0.001 (K-S test, n = 11 cells). G, Example of phospho-T286 CaMKII (red) with the plasma membrane labeled with DiI (green) resolved by two-color localization microscopy. H, Example of phospho-T286 CaMKII as in G (red) with the PSD resolved by PALM of PSD-95-mEos2 (green). Scale bar, 1 μm.

Figure 5.

CaMKII is immobilized away from the PSD following NMDAR activation. A, D_eff map of mEos2-CaMKIIα in the same neuron before (left) and after (right) Glu/Gly stimulation. Scale bar, 2 μm. B, Histograms of D_eff of mEos2-CaMKIIα (left) or cytosolic mEos2 (right) before and after Glu/Gly stimulation (mEos2-CaMKIIα: K-S test, p < 0.001, n = 5 cells; mEos2: K-S test, p > 0.1, n = 5 cells). C, CaMKII mobility map superimposed on deconvolved widefield image of PSD-95-Cerulean3 (black). Scale bar, 500 nm. D, Mean D_eff either within or outside of the PSD border (n = 54 synapses for basal condition, n = 22 synapses for Glu/Gly, two groups were from different cells). *p < 0.001 (two-way ANOVA repeated measures with one factor repeated). E, Mean D_eff calculated in spatial bins either within or every 100 nm outside the PSD (n = 52 spines for basal condition, n = 22 spines for Glu/Gly). *p < 0.001 (two-way ANOVA).