Super-Resolution Imaging Reveals That AMPA Receptors Inside Synapses Are Dynamically Organized in Nanodomains Regulated by PSD95

Nair, Deepak; Hosy, Eric; Petersen, Jennifer D.; Constals, Audrey; Giannone, Grégory; Choquet, Daniel; Sibarita, Jean‐Baptiste

doi:10.1523/jneurosci.2381-12.2013

Cited by 512 publications

(775 citation statements)

References 70 publications

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“…Combining the measured local diffusion maps with a mathematical model, the authors found that AMPA receptor accumulation is generated by interactions of the receptor with the membrane rather than molecular aggregation. Interestingly, in a third AMPA receptor superresolution study undertaken in part by the same authors, both uPAINT and sptPALM data of AMPA receptors were recorded and compared (Figure 3) [27 ]. Although in this study sptPALM visualized overexpressed AMPA subunits and uPAINT monitored endogenous AMPA receptors tagged with an antibody that did not label all surface receptors, both techniques yielded a comparable distribution of receptors.…”

Section: Imaging Postsynaptic Proteinsmentioning

confidence: 90%

“…This close agreement was further confirmed with STED and electron microscopy, showing that AMPA receptors are not uniformly organized in the synapse. AMPA receptor nanodomains were also shown to change in shape in a highly dynamic fashion, often colocalizing with the scaffold protein PSD95 [27 ]. Another recent work applied similar experimental approaches to the adenosine triphosphate (ATP)-gated P2X7 receptors, members of the purinergic receptor family, labeled with Dendra2 and studied with sptPALM [28].…”

Section: Imaging Postsynaptic Proteinsmentioning

confidence: 99%

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Recent applications of superresolution microscopy in neurobiology

Willig

Barrantes

2014

Current Opinion in Chemical Biology

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Chemical synapses in brain are structural differentiations where excitatory or inhibitory signals are vectorially transmitted between two neurons. Excitatory synapses occur mostly on dendritic spines, submicron sized protrusions of the neuronal dendritic arborizations. Axons establish contacts with these tiny specializations purported to be the smallest functional processing units in the central nervous system. The minute size of synapses and their macromolecular constituents creates an inherent difficulty for imaging but makes them an ideal object for superresolution microscopy. Here we discuss some representative examples of nanoscopy studies, ranging from quantification of receptors and scaffolding proteins in postsynaptic densities and their dynamic behavior, to imaging of synaptic vesicle proteins and dendritic spines in living neurons or even live animals.

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Section: Imaging Postsynaptic Proteinsmentioning

confidence: 90%

Section: Imaging Postsynaptic Proteinsmentioning

confidence: 99%

Recent applications of superresolution microscopy in neurobiology

Willig

Barrantes

2014

Current Opinion in Chemical Biology

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“…PSD-95 controls the number of AMPARs at the PSD through interactions with auxiliary proteins, such as Stargazin/TARPs in complex with AMPARs (23)(24)(25). Single-particle tracking of AMPARs provides evidence that AMPAR/ Stargazin complexes are stabilized by PSD-95 at the membrane (26), where PSD-95 is thought to provide hot spots for accumulating AMPARs at synapses (27,28). Germ-line knockout of PSD-95 reduces AMPAR transmission with little effects on NMDARs (29), whereas acute loss of single members of the MAGUK family decreases primarily AMPAR-mediated synaptic transmission (30)(31)(32), and removal of multiple MAGUKs results in greater losses of transmission mediated by both AMPARs and NMDARs (30).…”

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confidence: 99%

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.

225

201

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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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“…New studies using superresolution light microscopy observed that AMPARs (21,22) and palmitoylated PSD95 (22,23) within PSDs were clustered in nanodomains. Using EM tomography, Chen et al (1,2) found NMDAR clusters were separate from AMPARs.…”

mentioning

confidence: 99%

Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation

Jeyifous

Lin

Chen

et al. 2016

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

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Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via transmembrane AMPAR regulatory protein subunits) or NMDARs [via glutamate ionotropic receptor NMDA-type subunit 2B (GluN2B) subunits] only in its palmitoylated and extended conformation. In contrast, in its extended conformation, SAP97 associates with NMDARs, but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation, and its interactions are dynamic when associated with AMPARs and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains.P ostsynaptic densities (PSDs) at glutamatergic synapses organize and hold NMDA receptors (NMDARs), AMPA receptors (AMPARs), and other signaling molecules in place, apposed to sites of neurotransmitter release. Just below the PSD plasma membrane lies a latticework of vertical and parallel filaments that provides a structural scaffold to stabilize synaptic signaling molecules within PSDs (1, 2). Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are members of a family of membrane-associated guanylate kinases (MAGUKs) (3). PSD95 is the most abundant scaffold protein in adult synapses, with ∼300 PSD95 molecules (2.3% of the mass of the PSD) in the average PSD, and is part of the lattice forming the core of the PSD (4). SAP97 is also a component of the PSD lattice. Estimates of its PSD copy numbers range from 90 SAP97 molecules per average PSD [0.9% of the mass of the PSD (4)] to lower values (5). As MAGUKs, PSD95 and SAP97 share a series of highly homologous protein-interacting domains but diverge at their N-terminal domains, which affects their trafficking into and out of the PSD, as well as interactions with AMPARs and NMDARs (3, 6, 7). The SAP97β-isoform, like almost all SAP97 molecules, contains an N-terminal L27 domain that interacts with other L27 domaincontaining proteins, particularly with a differ...

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Super-Resolution Imaging Reveals That AMPA Receptors Inside Synapses Are Dynamically Organized in Nanodomains Regulated by PSD95

Cited by 512 publications

References 70 publications

Recent applications of superresolution microscopy in neurobiology

Recent applications of superresolution microscopy in neurobiology

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

Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation

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