Palmitoylation of A-kinase anchoring protein 79/150 modulates its nanoscale organization, trafficking, and mobility in postsynaptic spines.

A-kinase anchoring protein 79-human/150-rodent (AKAP79/150) organizes signaling proteins to control synaptic plasticity. AKAP79/150 associates with the plasma membrane and endosomes through its N-terminal domain that contains three polybasic regions and two Cys residues that are reversibly palmitoylated. Mutations abolishing palmitoylation (AKAP79/150 CS) reduce its endosomal localization and association with the postsynaptic density (PSD). Here we combined advanced light and electron microscopy (EM) to characterize the effects of AKAP79/150 palmitoylation on its postsynaptic nanoscale organization, trafficking, and mobility in hippocampal neurons. Immunogold EM revealed prominent extrasynaptic membrane AKAP150 labeling with less labeling at the PSD. The label was at greater distances from the spine membrane for AKAP150 CS than WT in the PSD but not in extra-synaptic locations. Immunogold EM of GFP-tagged AKAP79 WT showed that AKAP79 adopts a vertical, extended conformation at the PSD with its N-terminus at the membrane, in contrast to extrasynaptic locations where it adopts a compact or open configurations of its N- and C-termini with parallel orientation to the membrane. In contrast, GFP-tagged AKAP79 CS was displaced from the PSD coincident with disruption of its vertical orientation, while proximity and orientation with respect to the extra-synaptic membrane was less impacted. Single-molecule localization microscopy (SMLM) revealed a heterogeneous distribution of AKAP150 with distinct high-density, nano-scale regions (HDRs) overlapping the PSD but more prominently located in the extrasynaptic membrane for WT and the CS mutant. Thick section scanning transmission electron microscopy (STEM) tomography revealed AKAP150 immunogold clusters similar in size to HDRs seen by SMLM and more AKAP150 labeled endosomes in spines for WT than for CS, consistent with the requirement for AKAP palmitoylation in endosomal trafficking. Hidden Markov modeling of single molecule tracking data revealed a bound/immobile fraction and two mobile fractions for AKAP79 in spines, with the CS mutant having shorter dwell times and faster transition rates between states than WT, suggesting that palmitoylation stabilizes individual AKAP molecules in various spine subpopulations. These data demonstrate that palmitoylation fine tunes the nanoscale localization, mobility, and trafficking of AKAP79/150 in dendritic spines, which might have profound effects on its regulation of synaptic plasticity.

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains.

Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

Identification of PSD-95 in the Postsynaptic Density Using MiniSOG and EM Tomography.

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.

Depolarization of Hippocampal Neurons Induces Formation of Nonsynaptic NMDA Receptor Islands Resembling Nascent Postsynaptic Densities.

Depolarization of neurons in 3-week-old rat hippocampal cultures promotes a rapid increase in the density of surface NMDA receptors (NRs), accompanied by transient formation of nonsynaptic NMDA receptor clusters or NR islands. Islands exhibit cytoplasmic dense material resembling that at postsynaptic densities (PSDs), and contain typical PSD components, including MAGUKS (membrane-associated guanylate kinases), GKAP, Shank, Homer, and CaMKII detected by pre-embedding immunogold electron microscopy. In contrast to mature PSDs, islands contain more NMDA than AMPA receptors, and more SAP102 than PSD-95, features that are shared with nascent PSDs in developing synapses. Islands do not appear to be exocytosed or endocytosed directly as preformed packages because neurons lacked intracellular vacuoles containing island-like structures. Islands form and disassemble upon depolarization of neurons on a time scale of 2-3 min, perhaps representing an initial stage in synaptogenesis.

Trafficking of AMPA receptors at plasma membranes of hippocampal neurons.

The number of AMPA receptors at synapses depends on receptor cycling. Because receptors diffuse rapidly in plasma membranes, their exocytosis and endocytosis need not occur near synapses. Here, pre-embedding immunogold electron microscopy is applied to dissociated rat hippocampal cultures to provide sensitive, high-resolution snapshots of the distribution of surface AMPA receptors in spines, dendrites, and cell bodies that will be informative about trafficking of AMPA receptors. The density of the label for GluR2 varies, but is consistent throughout cell body and dendrites in each individual neuron, except at postsynaptic densities (PSDs), where it is typically higher. Glutamate receptor 2 (GluR2) labels at PSDs significantly increase after synaptic activation by glycine treatment and increase further upon depolarization by high K(+). Islands of densely packed labels have consistent size and density but vary in frequency under different experimental conditions. These patches of label, which occur on plasma membranes of cell bodies and dendrites but not near PSDs, are taken to be the aftermath of exocytosis of AMPA receptors. A subpopulation of clathrin-coated pits in cell bodies and dendrites label for GluR2, and the number and amount of label in individual pits increase after NMDA treatment. Coated pits near synapses typically lack GluR2 label under basal conditions, but ∼40% of peri-PSD pits label for GluR2 after NMDA treatment. Thus, exocytosis and endocytosis of AMPA receptors occur mainly at extrasynaptic locations on cell bodies and dendrites. Receptors are not preferentially exocytosed near PSDs, but may be removed via endocytosis at peri-PSD locations after activation of NMDA receptors.