Direct visualization of CaMKII at postsynaptic densities by electron microscopy tomography

Fera, Andrea; Döṡemeci, Ayṡe; Sousa, Alioscka A.; Yang, Charlotte; Leapman, Richard D.; Reese, Thomas S.

doi:10.1002/cne.23151

Cited by 13 publications

(16 citation statements)

References 38 publications

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“…Recent data from electron tomography reveals a dense matrix in a ∼25‐nm zone directly apposed to the plasma membrane, coated by a progressively attenuated fringe of electron‐dense material that extends filaments into the cytoplasm of the spine (Burette et al, ); accordingly, we consider this zone to be distinct from the PSD core, and here refer to it as the cytoplasmic fringe of the PSD. Results from a previous immunogold study on biochemically isolated PSDs (Petersen et al, ) are generally consistent with ours, as is a recent high‐resolution electron tomographic study (Fera et al, ) if one bears in mind that the disruption inherent to the PSD isolation in those studies likely caused the loss preferentially of CaMKII that lay farthest from the postsynaptic membrane. CaMKII lying this far from the membrane is poorly positioned to interact with glutamate receptors inserted into the membrane, but could (for example) interact with F‐actin to help mediate activity‐dependent cytoskeletal reorganization.…”

Section: Discussionsupporting

confidence: 90%

“…Although our data from quick-fix material should be taken as an upper bound for the concentration of CaM-KII in the PSD in vivo, the rather modest shifts we found in CaMKII organization after control experiments that included 5 minutes of delay before fixation suggest that the quick-fix estimates of antigen location are not severely impacted by the delay arising from chemical fixation, and may be close to the organization in living brain. However, because our data rely on averaged results, they likely fail to detect the extent of synapseto-synapse variability (which may be considerable; see Fera et al, 2012).…”

Section: Technical Issuesmentioning

confidence: 99%

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Subcellular organization of camkii in rat hippocampal pyramidal neurons

Ding

Kennedy

Weinberg

2013

J of Comparative Neurology

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Calcium/calmodulin-dependent protein kinase II (CaM-KII) plays a key role in N-methyl-D-aspartate (NMDA) receptor-dependent long-term synaptic plasticity; its location is critical for signal transduction, and may provide clues that further elucidate its function. We therefore examined the subcellular localization of CaMKII in CA1 stratum radiatum of adult rat hippocampus, by using immuno-electron microscopy after chemical fixation. When tissue was fixed quickly, the concentration of CaMKIIα (assessed by pre-embedding immunogold) was significantly higher in dendritic shafts than in spine heads. However, when tissue was fixed 5 minutes after perfusion with normal saline, the density of labeling decreased in dendritic shaft while increasing in spine heads, implying rapid translocation into the spine during brief perimortem stress. Likewise, in quickly fixed tissue, CaMKII within spine heads was found at comparable concentrations in the “proximal” half (adjacent to the spine neck) and the “distal” half (containing the postsynaptic density [PSD]), whereas after delayed fixation, label density increased in the distal side of the spine head, suggesting that CaMKII within the spine head moves toward the PSD during this interval. To estimate its distribution at the synapse in vivo, we performed postembedding immunogold staining for CaMKII in quick-fixed tissue, and found that the enzyme did not concentrate primarily within the central matrix of the PSD. Instead, labeling density peaked ∼40 nm inside the postsynaptic membrane, at the cytoplasmic fringe of the PSD. Labeling within 25 nm of the postsynaptic membrane concentrated at the lateral edge of the synapse. This lateral “PSD core” pool of CaMKII may play a special role in synaptic plasticity.

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Section: Discussionsupporting

confidence: 90%

Section: Technical Issuesmentioning

confidence: 99%

Subcellular organization of camkii in rat hippocampal pyramidal neurons

Ding

Kennedy

Weinberg

2013

J of Comparative Neurology

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“…CaMKII is localized in both the core and cytoplasmic surface of the PSD by negative staining EM tomography (Fera et al . ). Nano‐Depth‐Tagging method also supports localization of the enzyme inside the PSD (Liu et al .…”

Section: Discussionmentioning

confidence: 97%

Protein components of post‐synaptic density lattice, a backbone structure for type I excitatory synapses

Suzuki

Kametani

Weiheng

et al. 2017

Journal of Neurochemistry

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It is essential to study the molecular architecture of post-synaptic density (PSD) to understand the molecular mechanism underlying the dynamic nature of PSD, one of the bases of synaptic plasticity. A well-known model for the architecture of PSD of type I excitatory synapses basically comprises of several scaffolding proteins (scaffold protein model). On the contrary, 'PSD lattice' observed through electron microscopy has been considered a basic backbone of type I PSDs. However, major constituents of the PSD lattice and the relationship between the PSD lattice and the scaffold protein model, remain unknown. We purified a PSD lattice fraction from the synaptic plasma membrane of rat forebrain. Protein components of the PSD lattice were examined through immuno-gold negative staining electron microscopy. The results indicated that tubulin, actin, α-internexin, and Ca /calmodulin-dependent kinase II are major constituents of the PSD lattice, whereas scaffold proteins such as PSD-95, SAP102, GKAP, Shank1, and Homer, were rather minor components. A similar structure was also purified from the synaptic plasma membrane of forebrains from 7-day-old rats. On the basis of this study, we propose a 'PSD lattice-based dynamic nanocolumn' model for PSD molecular architecture, in which the scaffold protein model and the PSD lattice model are combined and an idea of dynamic nanocolumn PSD subdomain is also included. In the model, cytoskeletal proteins, in particular, tubulin, actin, and α-internexin, may play major roles in the construction of the PSD backbone and provide linker sites for various PSD scaffold protein complexes/subdomains.

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“…Electron tomography and immunogold labeling were then employed to assess how the structure, protein composition and protein spatial organization differ in individual PSDs from these unique brain regions. We chose to employ electron tomography because of its unique capability to produce 3D structural information of the PSD at the molecular level and because it has been productively employed to visualize PSD structure (Chen et al, 2008, Swulius et al, 2010, Fera et al, 2012, Swulius et al, 2012). 3D structures were produced of cryo-preserved PSD specimens, that avoid artifacts of fixation and staining, providing novel views of the isolated PSD as it exists in a “frozen-hydrated” state.…”

Section: Introductionmentioning

confidence: 99%

Electron tomographic structure and protein composition of isolated rat cerebellar, hippocampal and cortical postsynaptic densities

2015

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Electron tomography and immunogold labeling were used to analyze similarities and differences in the morphology and protein composition of postsynaptic densities (PSDs) isolated from adult rat cerebella, hippocampi, and cortices. There were similarities in physical dimensions and gross morphology between cortical, hippocampal and most cerebellar PSDs, although the morphology among cerebellar PSDs could be categorized into three distinct groups. The majority of cerebellar PSDs were composed of dense regions of protein, similar to cortical and hippocampal PSDs, while others were either composed of granular or latticelike protein regions. Significant differences were found in protein composition and organization across PSDs from the different brain regions. The signaling protein, βCaMKII, was found to be a major component of each PSD type and was more abundant than αCaMKII in both hippocampal and cerebellar PSDs. The scaffold molecule PSD-95, a major component of cortical PSDs, was found absent in a fraction of cerebellar PSDs and when present was clustered in its distribution. In contrast, immunogold labeling for the proteasome was significantly more abundant in cerebellar and hippocampal PSDs than cortical PSDs. Together, these results indicate that PSDs exhibit remarkable diversity in their composition and morphology, presumably as a reflection of the unique functional demands placed on different synapses.

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Direct visualization of CaMKII at postsynaptic densities by electron microscopy tomography

Cited by 13 publications

References 38 publications

Subcellular organization of camkii in rat hippocampal pyramidal neurons

Subcellular organization of camkii in rat hippocampal pyramidal neurons

Protein components of post‐synaptic density lattice, a backbone structure for type I excitatory synapses

Electron tomographic structure and protein composition of isolated rat cerebellar, hippocampal and cortical postsynaptic densities

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