Distribution of Postsynaptic Density (PSD)-95 and Ca²⁺/Calmodulin-Dependent Protein Kinase II at the PSD

Rapidly processed PSDs contain basal levels of CaMKII

PSDs were isolated by conventional protocols (Carlin et al., 1980) involving Triton X-100 extraction (Fig. 1A). Particular emphasis was placed on rapid homogenization of the brain tissue after decapitation because previous work has shown that a time-dependent increase in the proportion of CaMKII in the PSD fraction develops in the minutes after decapitation (Suzuki et al., 1994). By extrapolation of this time dependence to zero time, it can be estimated that the basal CaMKII content of the PSD fraction is 2.1%. In this study, protein analysis of PSD fraction prepared from brain tissue homogenized within 2 min of decapitation showed that it contained 2% CaMKII (data not shown). Therefore, it can be concluded that the PSDs we analyzed are close to their average basal CaMKII levels.

Cleft and cytoplasmic surfaces of PSDs differ in structure

Viewed in the electron microscope, replicas of PSDs appeared as distinctive, approximately circular disks ranging in diameter from 180 to 750 nm (mean diameter, 313 ± 4 nm; n = 618). These dimensions are consistent with those obtained by serial reconstructions of PSDs at intact synapses (Cohen and Siekevitz, 1978; Harris et al., 1992). Because PSDs are disk-shaped, we expected that they would adhere to the glass substrate in one of two orientations: cleft surface on the glass or cytoplasmic surface on the glass. Only the exposed surface that faced away from the glass would be replicated by rotary platinum shadow, leaving the rest of the PSD invisible, except for any attached immunogold.

PSDs presented two distinct surfaces at approximately equal frequency. One surface was a raised, smooth plateau at a fixed separation from the glass (Fig. 2, left), whereas the other surface had a convoluted contour highly varied in its separation from the glass (Fig. 2, right). These differences in contour were confirmed by examining PSDs in stereo views. Because thin-section views of synapses with prominent PSDs show that the surface of the PSD next to the postsynaptic membrane is planar, whereas the cytoplasmic surface of the PSD extends irregularly into the cytoplasm, it seemed likely that the planar face is the cleft surface, whereas the convoluted face of the PSD is its cytoplasmic surface. Confirmation that the smooth surface of the PSD corresponds to the cleft surface and the convoluted surface to its cytoplasmic aspect is presented below.

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Figure 2.

Cleft and cytoplasmic surfaces of PSDs are different. A, Cleft surface of PSD immunogold-labeled for PSD-95. Immunogold particles appear as white dots (small arrow). Counts of gold label on PSD are shown at the top right. The opening in the central mesh is indicated by the large arrow. B, Cytoplasmic surface of PSD immunogold-labeled for PSD-95. The opening in the central mesh is indicated by an arrow. C, Enlarged area from A. Granular particles are indicated by arrowheads. Underlying filaments of the central mesh are indicated by an arrow. D, Enlarged area of B. Underlying filaments of the central mesh are indicated by an arrow. E, Cleft surface of PSD immunogold-labeled for Shank. Any label present is typically at edge of PSD (arrow). PSD contains a patch of membrane (enlarged in G). F, Cytoplasmic surface of PSD immunogold-labeled for Shank. Material protruding above the central mesh is indicated by an arrow. G, Membrane patch (arrowhead) has typical rim of particles. H, Enlarged area of F. Underlying filaments of central mesh are indicated by an arrow. Scale bars, 100 nm.

The cleft and cytoplasmic surfaces of the PSD were further distinguished by their surface texture. A dense layer of particles, 5-15 nm in diameter, imparting a granular texture, characterized the cleft surface. (Fig. 2A,E, small arrowheads in C) This granular layer covered an underlying lamina composed of 2 nm diameter filaments that formed a central mesh (Fig. 2C, small arrow). Openings 40-80 nm in diameter penetrated the central mesh (Fig. 1A, large arrow). Patches of membrane 50-100 nm in diameter were frequently observed on the planar surfaces of PSDs, appearing integral to and coplanar with their smooth surfaces. These membranes were typically circular and surrounded by a rim of particles (Fig. 2E, arrowhead in G, see Fig. 4C,D). To persist in the isolated PSDs, these membranes must be resistant to Triton X-100 extraction, a characteristic property of the specialized plasma membrane domains known as lipid rafts that are present in synaptosomal preparations (Suzuki, 2002).

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Figure 4.

Variable amounts of CaMKII immunogold label are concentrated on cytoplasmic surfaces of PSDs. A, Cleft surface of PSD immunogold-labeled for CaMKII. The immunogold label appears as white dots. The label (arrows) is present only at the edge of PSD. The count of gold particles is shown at the top right. B, Cytoplasmic surface of PSD immunogold-labeled for CaMKII. The arrow indicates material protruding above the central mesh. C, Cleft surface of PSD immunogold-labeled for CaMKII. The arrowhead indicates the membrane patch. D, Cytoplasmic surface of PSD with moderate amount of CaMKII immunogold label. A membrane patch lying near the glass substrate is visible from the cytoplasmic surface (arrowhead). E, Cleft surface of the PSD showing sparse labeling for CaMKII. F, Cytoplasmic surface of a PSD heavily labeled for CaMKII. Arrows indicate concentrated domains of label on protrusions. Scale bar, 100 nm.

In contrast to the planar and granular characteristics of the cleft surface of PSDs, the cytoplasmic surface had a convoluted appearance caused by irregular protrusions from a layer of material covering the central mesh (Fig. 2B,F). Fine filaments of the central mesh were often visible underlying the protruding material (Fig. 2D,H, arrows).

Openings in the central mesh were seen more frequently from the cytoplasmic surface than the cleft surface (Fig. 2B, arrow), suggesting that the granular layer of the cleft surface covers some openings. Indeed, granular material was visible at the bottom of some openings, but other openings appeared to constitute holes through the PSD, in which case the glass surface was visible at the bottom of an opening. Because platinum was applied at a 20° angle, the protruding material surrounding the openings often blocked platinum from reaching the bottoms of the openings, so it was not always possible to determine whether an opening was a continuous hole through the PSD.

Cleft and cytoplasmic surfaces of PSDs confirmed by protein composition

PSDs attached to glass were labeled with immunogold, freeze-dried, and rotary-shadowed. The distribution of label for various components of the PSD differed on the two surfaces of PSDs. For instance, Shank (Naisbitt et al., 1999), a protein known to lie primarily on the cytoplasmic surface of the PSD by thin-section immunogold-EM (Valtschanoff and Weinberg, 2001) was distributed predominantly on the convoluted surface rather than on the smooth surface (Fig. 2E,F, Table 1). The small amount of labeling present on cleft surfaces oriented away from the glass tended to be at the edges (Fig. 2E, arrow), and stereo views revealed that this label was actually associated with the convoluted surface of the PSD. Thus, the distribution of label for Shank confirmed that the convoluted surface constitutes the cytoplasmic surface of PSDs and faces the cytoplasm at intact synapses.

PSD-95 label distributed evenly across the central mesh of PSDs

In contrast to Shank, the pattern of immunogold label for PSD-95 was similar on cytoplasmic and cleft surfaces (Fig. 2A,B, Table 1). The PSD-95 label followed the contours of the mesh and was present continuously along the mesh. Variability from PSD to PSD was assessed by measuring the density of label per 0.1 μm², the area of a typical PSD (Fig. 3). The average density of label was 30 gold particles per 0.1 μm² (coefficient of variation = 0.45) for both the cleft and cytoplasmic surfaces of the PSD, and every PSD showed some label. These results indicate that PSD-95 is a component of the central mesh, is accessible to immunogold label from both surfaces of the PSD, and is present in all PSDs.

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Figure 3.

CaMKII immunogold label is concentrated on the cytoplasmic surface of PSDs in variable abundance. Frequency histogram comparing the density of immunogold label for CaMKII and PSD-95 on the cleft and cytoplasmic surfaces of unselected PSDs. Immunogold for PSD-95 is normally distributed on both cleft and cytoplasmic surfaces, whereas essentially all label for CaMKII resides on the cytoplasmic surface, where it varies widely between PSDs. Label for CaMKII on cleft surface is negligible (data not shown)

CaMKII label present exclusively at the cytoplasmic surface

Immunogold labeling for CaMKII showed a pattern of labeling markedly different from that of PSD-95 (Fig. 4). CaMKII immunogold label was seven times heavier on the cytoplasmic surface of the PSD than on the cleft surface (Table 1). The small amount of CaMKII label present on the cleft surface was almost exclusively at the edge of the PSD (Fig. 4A, arrows), similar to the edge labeling for Shank. Suspecting that edge labeling is caused by the ability of the antibody to gain access to antigen localized at the edges of the cytoplasmic surface (attached to the glass), the density of label for CaMKII was recomputed after eliminating from consideration a 30 nm wide annular zone at the outer edge of the PSD. After eliminating this potentially distorted edge labeling, the disparity between cleft and cytoplasmic surfaces increased to 40-fold (Table 2). Indeed, when edge label is excluded, the average number of gold particles per unit area on the cleft surface could not be distinguished from the negligible background label on the glass substrate. These results suggest that CaMKII is located exclusively at the cytoplasmic surface of the PSD.

Density of CaMKII label varies more widely than density of PSD-95 label

The density of CaMKII label on cytoplasmic surfaces appeared to vary dramatically from PSD to PSD (Figs. 3, 4B,D,F). Approximately 5% of the PSDs had no gold label and another 20% had only six particles per 0.1 μm², whereas all PSDs showed some label for PSD-95. However, 12% of PSDs had >50 particles per 0.1 μm² (Fig. 3). The coefficient of variation calculated from the randomly acquired data set (Table 1) for the densities of CaMKII was 0.76, substantially larger than that for the densities of PSD-95 labeling (0.45) (Reed et al., 2002). Based on a labeling efficiency of 0.75 (see below), the CV attributable to probabilistic variation in labeling is small (0.1). An F test for comparison of the sizes of the SDs showed that the SD for CaMKII densities was larger than that for PSD-95 densities (p < 0.005; both distributions were normalized to their means to allow comparision). We conclude that there is substantial variability in the density of CaMKII between PSDs and that this is greater than the variability of PSD-95.

CaMKII label concentrates in subregions of PSDs with a regularity of spacing insensitive to average density

In addition to inter-PSD variability in the amount of CaMKII label present on cytoplasmic surfaces, there also existed an intra-PSD variation in density of label for CaMKII. CaMKII was often concentrated in subregions of a PSD, whereas it was entirely lacking in other regions of the same PSD. Regions that did not label for CaMKII typically lacked material protruding from the central mesh (Fig. 5C, asterisks). Hence, the densities of label across the entire cytoplasmic surface of the PSD supplied no information about local densities within individual PSDs. To study local spacing, nearest-neighbor analyses of the distances between immunogold particles within individual PSDs was performed. Histograms of mean nearest-neighbor distances between individual gold particles on a PSD (Fig. 5A) showed the peak nearest-neighbor distances. This peak value was then plotted as a function of the average density of label for that PSD (Fig. 5B). The peak nearest-neighbor distance (30 ± 5.0 nm) was nearly independent of the average density (Fig. 5B). These results suggest that when present, CaMKII packs at a characteristic local density regardless of overall labeling density.

Thickness of central mesh measured by EM tomography

The thickness of the central mesh in four PSDs that were attached to the glass substrate on their cleft side was measured by EM tomography. Virtual sections cut through the z-axis of four PSDs determined that the average thickness of the central mesh was 14 nm, ranging from 11 to 18 nm. Measurement of the thickness of the granular layer was attempted, but it was not possible to differentiate the granular layer from the central mesh confidently, suggesting that the granular layer is exceedingly thin and/or embedded somewhat in the central mesh. Thus, the 14 nm average thickness of the central mesh encompasses the slight contribution to height made by the granular layer.

Distinct layering of CaMKII and PSD-95 label detected by EM tomography

Distances from cleft surfaces of individual gold particles labeling for PSD-95 and CaMKII were compared using EM tomography. Nine complete PSDs labeled for CaMKII (Fig. 6) and three complete PSDs labeled for PSD-95, all adhered to the glass substrate by their cleft surfaces, were reconstructed and compared. Immunogold label (excluding edge label, Table 2) for CaMKII ranged from 14 to 43 nm from the surface of the glass, peaking between 20 and 30 nm (Fig. 7). Thus, essentially all of the CaMKII lies beyond the cytoplasmic surface of the central mesh, which is only 14 nm thick. A small amount of CaMKII label was detected as far as 73 nm from the cleft surface of the PSD. In contrast, immunogold label for PSD-95 peaked at 12 nm from the cleft surface (Fig. 7) and all PSD-95 label lay within 21 nm from the cleft. Thus, PSD-95 should be considered part of the central mesh, whereas CaMKII lies peripheral to it.

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Figure 6.

EM tomography shows distribution of CaMKII label in relation to the cleft surface of the PSD. Surface rendering of tomographic reconstruction of a PSD labeled for CaMKII showing an oblique view from the cytoplasmic surface (above) and edge view (below, from aspect indicated by arrow). Centrally located gold particles are labeled in red and shown in the side view. These particles were selected for measurements. Particles in blue, potentially subjected to distortions at edges, were not included in measurements. Scale bar, 100 nm.

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Figure 7.

Distinct layering of CaMKII and PSD-95 label detected by EM tomography. Distances of individual gold particles from cleft surfaces of PSDs labeled for PSD-95 or for CaMKII were compared using EM tomography. The distance of individual gold particles from the cleft surface of the PSD, where it adhered to the glass substrate, was measured in nine CaMKII-labeled PSDs and in three PSD-95-labeled PSDs. The density of CaMKII label peaked ∼25 nm from the cleft surface of the PSD, whereas the density of PSD-95 peaked at ∼12 nm.

CaMKII label associated with tower-like protrusions visualized by EM tomography

EM tomography allowed the visualization of well defined towers with vertical sides among the protrusions of material from the cytoplasmic side of the central mesh (Fig. 8). Towers were typically labeled by a single CaMKII immunogold. Examination of multiple virtual sections (1 nm thick) through reconstructions from four PSDs (including one in Fig. 6) showed that 82% of 152 towers labeled for CaMKII, and label not associated with towers was <10%. In contrast, no towers labeled for PSD-95 (Fig. 8).

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Figure 8.

CaMKII immunogold label, but not PSD-95 label, is associated with towers on the cytoplasmic surfaces of PSDs. Cross sections of reconstructions of platinum-shadowed PSDs immunogold-labeled for PSD-95 (top two panels) or CaMKII (bottom four panels). Towers ∼20 nm wide (indicated by bars, 20 nm) extend upward from cytoplasmic surfaces of PSDs. After labeling for CaMKII, most towers were labeled (indicated by dark bars), but a few were unlabeled (indicated by white bars). After immunolabeling for PSD-95, towers were unlabeled (white bars), and the label (oblique arrows) was nearer the cleft surface of the PSD. The large black arrows below indicate the plane of the cleft surface on the glass substrate.

Towers varied in height. In three PSDs examined in virtual serial sections (1 nm thick) through the tomographic reconstructions, the mean height of towers was 34 nm measured from the glass, ranging from 20 to 60 nm. Towers appeared to be ∼20 nm in diameter in views generated normal to the plane of the PSD (Fig. 8), consistent with the notion that they represent CaMKII holoenzymes.

PSD-95 is a core structural component of central mesh in all PSDs

The cleft surface of the PSD is covered by a thin, densely packed layer of particles, the molecular identity of which remains unclear but of great interest. Immediately underlying the granular cleft layer is the central mesh that in all PSDs labels abundantly for PSD-95. The positioning of PSD-95 close to the cleft surface of the PSD and postsynaptic membrane is consistent with previous work based on thin-section immunogold studies (Valtschanoff and Weinberg, 2001), as well as with the finding that PSD-95 is membrane associated via palmitoylation of its N terminus (Topinka and Bredt, 1998). Furthermore, finding PSD-95 distributed within the central mesh of all isolated PSDs supports the notion that PSD-95 is a fundamental structural component of most, if not all, excitatory PSDs.

Single layer of CaMKII holoenzymes on cytoplasmic side of central mesh

A CaMKII holoenzyme is a 12 subunit, 20-nm-tall barrel-shaped multimer. Catalytic domains of each subunit extend from the top and bottom of the barrel, resulting in a bipolar distribution of catalytic domains (Kolodziej et al., 2000) to which the antibody used in this study is directed. Because the immunogold complexes can define the position of an epitope within ∼10 nm, a single layer of CaMKII was expected to give rise to, at most, a 40 nm wide distribution of label. Measured by EM tomography, the average distance of CaMKII label from the cleft surface is ∼25 nm. The distance of label from the cleft surface varied from 14 to 43 nm, a range of 29 nm, compatible with the conclusion that most CaMKII holoenzymes reside in a single layer on the cytoplasmic side of the ∼14 nm central mesh. However, a small amount of CaMKII label was detected as far as 73 nm from the cleft surface, indicating that some CaMKII may lie in a second, or even third, layer more distal to the postsynaptic membrane (Fig. 9 and below). Such stacking (see below) is consistent with the known ability of CaMKII holoenzymes to self-associate (Hudmon et al., 2001).

Individual CaMKII holoenzymes on cytoplasmic side of the PSD

Virtual sections generated by EM tomography normal to the plane of the PSD made it possible to visualize towers with vertical walls within the convoluted material protruding from the mesh on the cytoplasmic surface of the PSD. The diameter of the towers is close to that of purified CaMKII (Kolodziej et al., 2000) and almost all towers labeled with antibody against CaMKII. The average height of the towers, 34 nm, minus the 14 nm thickness of the central mesh, matches the 20 nm size of CaMKII holoenzymes. However, a few towers were considerably higher, as if two or three barrel-shaped holoenzymes are stacked in register on top of each other. If towers represent single CaMKII holoenzymes, 30-40 holoenzymes could be attached to an average-size PSD. The consistent images of the towers with vertical sides suggests that the cylindrical axis of most molecules is perpendicular to the membrane, as depicted in Figure 8, but we cannot rule out that some holoenzymes lie on their sides on the central mesh.

CaMKII located in variable amounts on cytoplasmic side of PSD

Comparison of the CaMKII labeling among many PSDs indicates that the density of CaMKII gold label can vary from 0 to >75/0.1 μm². By comparison, the labeling of PSD-95 is more uniform, as reflected by a lower coefficient of variation and a significantly lower SD. As mentioned above, a typical PSD (0.1 μm²) contains ∼30 CaMKII-labeled molecules on its cytoplasmic surface, but this number varies widely. In fact, 6% of PSDs have a density three times the average and would thus have ∼100 holoenzymes attached. Assuming an evenly spaced single layer of kinase, nearest neighbors would be ∼30 nm apart. Thus, holoenzymes would be nearly closely packed, leaving only ∼10 nm between them. These results imply a large variability in the CaMKII content of PSDs from synapse to synapse. One caveat is that although CaMKII is difficult to extract from the PSD (Kennedy, 1997), the possibility of differential loss of CaMKII from individual PSDs during subcellular fractionation cannot be completely excluded.

CaMKII evenly spaced on cytoplasmic side of PSD

The average nearest-neighbor distance (30 nm) of CaMKII label is nearly independent of the overall density of label. The close, regular spacing of label is apparent over regions of the PSD encompassing many holoenzymes and so cannot be attributed to multiple labeling of single holoenzymes. The factors that enforce this close spacing are unclear. The two known binding proteins for CaMKII are densin (Walikonis et al., 2001), a protein of unknown function, and the NMDA channel. Importantly, the NMDA channel has a very large cytoplasmic tail that could well be long enough to extend through the central mesh and bind CaMKII (Kennedy and Manzerra, 2001). Recent work shows that under resting conditions, there is a pool of CaMKII complexed with NMDA channels, as determined by coimmunoprecipitation (Gardoni et al., 2001). Thus, the spacing of CaMKII could be dependent on the process that organizes the spacing of NMDA channels. Although many areas of the cytoplasmic surface of the PSD have evenly spaced label for CaMKII, other areas lack label, and in these areas the central mesh is exposed. This higher-order organization of CaMKII within the PSD (some areas evenly labeled, others not labeled) could also reflect the delivery to the PSD of stable packets that contain multiple CaMKII holoenzymes.

Functional consequences of the molecular structure of PSDs

The localization of PSD-95 close to the membrane places it in a position where it can bind to some of its known integral membrane-protein-binding partners. The more distal localization of CaMKII may be related to binding of soluble CaMKII to the PSD induced by neuronal activity. Studies with green fluorescent protein-CaMKII indicate that a pool of the kinase is initially bound to actin at locations distant from the synapse (Shen et al., 1998). Neural activity that elevates Ca²⁺ causes the binding of Ca²⁺/calmodulin to the kinase, the dissociation of the kinase from actin, the translocation of the kinase to the synaptic region (Shen and Meyer, 1999), specifically to the PSD (Dosemeci et al., 2001), and the binding of the kinase to NMDA channels. Importantly, these processes require enzyme activation but do not require autophosphorylation of the kinase. Once CaMKII is bound to the NMDA channel, it is exposed to the high levels of Ca²⁺ near the NMDA channel that appear to be the trigger for LTP induction. The resulting exposure to sustained high levels of Ca²⁺ near the NMDA channel would favor autophosphorylation of the kinase. Once phosphorylated, CaMKII binds more tightly to the NMDA channel and is persistently active even after Ca²⁺ levels fall. This persistent activity then promotes enzymatic and structural processes that increase the conductance and number of AMPA channels (Lisman et al., 2002). It is these changes that contribute to the enhanced transmission that underlies LTP induction.

The fact that CaMKII binds at high local density is relevant to the mechanism of persistent CaMKII autophosphorylation. The close spacing produces a locally high concentration of phosphorylated sites on the kinase that exceeds the K_m of the PSD phosphatase (PP1) that dephosphorylates CaMKII (Lisman and Zhabotinsky, 2001). The resulting phosphatase saturation should enable CaMKII autophosphorylation activity to keep the molecule phosphorylated and active.

AMPA receptors, like CaMKII molecules, vary greatly in number and density from synapse to synapse. Some synapses, termed silent synapses, lack AMPA receptors, whereas others have high AMPA receptor densities (Nusser et al., 1998, Petralia et al., 1999, Takumi et al., 1999). NMDA channels, in contrast, appear to vary much less (Racca et al., 2000). The variability in AMPA channels is thought to be a factor underlying differences in synaptic strength that reflect the history of synaptic modifications during development and experience (LTP). It has been proposed that persistently phosphorylated CaMKII bound to NMDA channels serves to organize additional anchoring sites for AMPA channels and thereby to strengthen transmission (Lisman and Zhabotinsky, 2001). The variability we have observed in CaMKII density would be consistent with such a role. Therefore, it will be important to determine how the variability in density of CaMKII reported here correlates with the variability in the AMPA receptor content of synapses.