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?-Actinin Anchors PSD-95 at Postsynaptic Sites.

ABSTRACT: Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein ?-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of ?-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of ?-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to ?-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify ?-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.

SUBMITTER: Matt L

PROVIDER: S-EPMC5963734 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Publications

α-Actinin Anchors PSD-95 at Postsynaptic Sites.

Matt Lucas L Kim Karam K Hergarden Anne C AC Patriarchi Tommaso T Malik Zulfiqar A ZA Park Deborah K DK Chowdhury Dhrubajyoti D Buonarati Olivia R OR Henderson Peter B PB Gökçek Saraç Çiğdem Ç Zhang Yonghong Y Mohapatra Durga D Horne Mary C MC Ames James B JB Hell Johannes W JW

Neuron 20180208 5

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impa ...[more]

PMID: 29429936

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Project description:Combining tomography with electron microscopy (EM) produces images at definition sufficient to visualize individual protein molecules or molecular complexes in intact neurons. When freeze-substituted hippocampal cultures in plastic sections are imaged by EM tomography, detailed structures emerging from 3D reconstructions reveal putative glutamate receptors and membrane-associated filaments containing scaffolding proteins such as postsynaptic density (PSD)-95 family proteins based on their size, shape, and known distributions. In limited instances, structures can be identified with enhanced immuno-Nanogold labeling after light fixation and subsequent freeze-substitution. Molecular identification of structure can be corroborated in their absence after acute protein knockdown or gene knockout. However, additional labeling methods linking EM level structure to molecules in tomograms are needed. A recent development for labeling structures for TEM employs expression of endogenous proteins carrying a green fluorescent tag, miniSOG, to photoconvert diaminobenzidine (DAB) into osmiophilic polymers. This approach requires initial mild chemical fixation but many of structural features in neurons can still be discerned in EM tomograms. The photoreaction product, which appears as electron-dense, fine precipitates decorating protein structures in neurons, may diffuse to fill cytoplasm of spines, thus obscuring specific localization of proteins tagged with miniSOG. Here we develop an approach to minimize molecular diffusion of the DAB photoreaction product in neurons, which allows miniSOG tagged molecule/complexes to be identified in tomograms. The examples reveal electron-dense clusters of reaction product labeling membrane-associated vertical filaments, corresponding to the site of miniSOG fused at the C-terminal end of PSD-95-miniSOG, allowing identification of PSD-95 vertical filaments at the PSD. This approach, which results in considerable improvement in the precision of labeling PSD-95 in tomograms without complications due to the presence of antibody complexes in immunogold labeling, may be applicable for identifying other synaptic proteins in intact neurons.

Dataset Information

?-Actinin Anchors PSD-95 at Postsynaptic Sites.

Publications

α-Actinin Anchors PSD-95 at Postsynaptic Sites.

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