Nanoscale Imaging of Protein Molecules at the Postsynaptic Density

Chen, Xiaobing; Winters, Christine A.; Azzam, Rita; Sousa, Alioscka A.; Leapman, Richard D.; Reese, Thomas S.

doi:10.1007/978-1-4614-9179-8_1

Cited by 7 publications

(19 citation statements)

References 82 publications

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“…away from the membrane in a relatively extended configuration, where it is vertically oriented with respect to the membrane (3,4,37). In contrast, neither SAP102 nor SAP97 has palmitoylation sites.…”

Section: Significancementioning

confidence: 99%

“…Here, we have used EM tomography (3,4) to study the structural effects of knocking down the three key MAGUKs at the PSD to develop a molecular model of the organization of the core PSD structure in intact hippocampal spine synapses. PSDs in intact synapses show numerous regularly spaced and membrane-associated vertical filaments containing PSD-95 in extended conformation connecting with NMDAR and AMPAR-type complexes.…”

Section: Significancementioning

confidence: 99%

“…PSDs in intact synapses show numerous regularly spaced and membrane-associated vertical filaments containing PSD-95 in extended conformation connecting with NMDAR and AMPAR-type complexes. These vertical structures in turn contact horizontal elements, resulting in a molecular scaffold supporting a core PSD structure (3,4,37). Thus, vertical filaments appear to be of crucial importance in sustaining the core PSD structure.…”

Section: Significancementioning

confidence: 99%

“…The tomograms were analyzed using established criteria for classifying the main structural elements at the PSD (3,4,37). Measurements on the core classes of structural elements, such as membrane-associated vertical filaments, revealed significant changes in their density and spatial organization following the MAGUK knockdown (Figs.…”

Section: Minimal Structural Changes In Pre-and Postsynaptic Membranesmentioning

confidence: 99%

“…The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2)(3)(4)(5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6)(7)(8)(9)(10) mediate almost all synaptic transmission in the brain (11).…”

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confidence: 99%

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PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Chen

Levy

Hou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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238

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The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-D-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre-or postsynaptic membrane, and depletes the number of membraneassociated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membraneassociated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.T he postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2-5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6-10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14-18). These MAGUK proteins share conserved modular structures consisting of three...

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Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Minimal Structural Changes In Pre-and Postsynaptic Membranesmentioning

confidence: 99%

mentioning

confidence: 99%

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PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Chen

Levy

Hou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

238

201

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Novel Cell Types, Neurosecretory Cells, and Body Plan of the Early-Diverging Metazoan Trichoplax adhaerens

et al. 2014

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Summary Background Trichoplax adhaerens is the best-known member of the Phylum Placozoa, one of the earliest-diverging metazoan phyla. It is a small disk-shaped animal that glides on surfaces in warm oceans to feed on algae. Prior anatomical studies of Trichoplax revealed that it has a simple three-layered organization with four somatic cell types. Results We reinvestigate the cellular organization of Trichoplax using advanced freezing and microscopy techniques to identify localize and count cells. Six somatic cell types are deployed in stereotyped positions. A thick ventral plate, comprising the majority of the cells, includes ciliated epithelial cells, newly identified lipophil cells packed with large lipid granules, and gland cells. Lipophils project deep into the interior where they alternate with regularly spaced fiber cells whose branches contact all other cell types, including cells of the dorsal and ventral epithelium. Crystal cells, each containing a birefringent crystal, are arrayed around the rim. Gland cells express several proteins typical of neurosecretory cells, and a subset of them, around the rim, also expresses an FMRFamide-like neuropeptide. Conclusions Structural analysis of Trichoplax with significantly improved techniques provides an advance in understanding its cell types and their distributions. We find two previously undetected cell types, lipohil and crystal cells, and an organized body plan in which different cell types are arranged in distinct patterns. The composition of gland cells suggests that they are neurosecretory cells and could control locomotor and feeding behavior.

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Transsynaptic Assemblies Link Domains of Presynaptic and Postsynaptic Intracellular Structures across the Synaptic Cleft

Reese

Cole

2023

J. Neurosci.

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The chemical synapse is a complex machine separated into three parts: presynaptic, postsynaptic, and cleft. Super-resolution light microscopy has revealed alignment of presynaptic vesicle release machinery and postsynaptic neurotransmitter-receptors and scaffolding components in synapse spanning nanocolumns. Cryo-electron tomography confirmed that postsynaptic glutamate receptor-like structures align with presynaptic structures in proximity to synaptic vesicles intotranssynaptic assemblies. In our electron tomographic renderings, nearly all transcleft structures visibly connect to intracellular structures through transmembrane structures to form transsynaptic assemblies, potentially providing a structural basis for transsynaptic alignment. Here, we describe the patterns of composition, distribution, and interactions of all assemblies spanning the synapse by producing 3D renderings of all visibly connected structures in excitatory and inhibitory synapses in dissociated rat hippocampal neuronal cultures of both sexes prepared by high-pressure freezing and freeze-substitution. The majority of transcleft structures connect to material in both pre- and postsynaptic compartments. We found several instances of assemblies connecting to both synaptic vesicles and postsynaptic density scaffolding. Each excitatory synaptic vesicle within 30 nm of the active zone contacts one or more assembly. Further, intracellular structures were often shared between assemblies, entangling them to form larger complexes orassociation domains, often in small clusters of vesicles. Our findings suggest that transsynaptic assemblies physically connect the three compartments, allow for coordinated molecular organization, and may combine to form specialized functional association domains, resembling the light-level nanocolumns.SIGNIFICANCEA recent tomographic study uncovered that receptor-like cleft structures align across the synapse. These aligned structures were designated astranssynaptic assembliesand demonstrate the coordinated organization of synaptic transmission molecules between compartments. Our present tomographic study expands upon the definition of transsynaptic assemblies by analyzing the 3D distribution and connectivity of all cleft-spanning structures and their connected intracellular structures. While one-to-one component alignment occurs across the synapse, we find that many assemblies share components, leading to a complex entanglement of assemblies, typically around clusters of synaptic vesicles. Transsynaptic assemblies appear to form domains which may be the structural basis for alignment of molecular nanodomains into synapse spanning nanocolumns described by super-resolution light microscopy.

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Nanoscale Imaging of Protein Molecules at the Postsynaptic Density

Cited by 7 publications

References 82 publications

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Novel Cell Types, Neurosecretory Cells, and Body Plan of the Early-Diverging Metazoan Trichoplax adhaerens

Transsynaptic Assemblies Link Domains of Presynaptic and Postsynaptic Intracellular Structures across the Synaptic Cleft

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