Cryo-EM tomography and automatic segmentation delineate modular structures in the postsynaptic density

Abstract

Postsynaptic densities (PSDs) are large protein complexes associated with the postsynaptic membrane of excitatory synapses important for synaptic function including plasticity. Conventional electron microscopy (EM) typically depicts PSDs as compact disk-like structures of hundreds of nanometers in size. Biochemically isolated PSDs were also similar in dimension revealing a predominance of proteins with the ability to polymerize into an extensive scaffold; several EM studies noted their irregular contours with often small granular structures (<30 nm) and holes. Super-resolution light microscopy studies observed clusters of PSD elements and their activity-induced lateral movement. Furthermore, our recent EM study on PSD fractions after sonication observed PSD fragments (40-90 nm in size) separate from intact PSDs; however, such structures within PSDs remained unidentified. Here we examined isolated PSDs by cryo-EM tomography with our new approach of automatic segmentation that enables delineation of substructures and their quantitative analysis. The delineated substructures broadly varied in size, falling behind 30 nm or exceeding 100 nm and showed that a considerable portion of the substructures (>38%) in isolated PSDs was in the same size range as those fragments. Furthermore, substructures spanning the entire thickness of the PSD were found, large enough to contain both membrane-associated and cytoplasmic proteins of the PSD; interestingly, they were similar to nanodomains in frequency. The structures detected here appear to constitute the isolated PSD as modules of various compositions, and this modular nature may facilitate remodeling of the PSD for proper synaptic function and plasticity.

Keywords: automatic segmentation; cryo-EM tomography; modular organization; nanodomain; postsynaptic density.

FIGURE 1

Isolated PSDs with and without sonication, imaged by cryo-EM tomography. (A) A 1.4-nm-thick virtual slice through a reconstructed volume of a sonicated PSD. The slice shows electron-dense structures apparently completely isolated from other structures (vertical black arrowhead), those apparently incompletely disconnected from other structures showing a few connections with adjacent ones though they were noticeably away from each other in most of their parts (horizontal black arrowhead), other apparently condense structures (black asterisk), and empty spaces devoid of such structures (white asterisk) including filamentous structures (black arrow). (B) A 1.3-nm-thick virtual slice through a reconstructed volume of an isolated PSD without sonication (control PSD). The slice also shows electron-dense structures apparently completely isolated from other structures (horizontal black arrowhead), those incompletely isolated from other structures (vertical black arrowhead), other apparently condense structures (black asterisk), and empty spaces devoid of such structures (white asterisk) including filamentous structures (black arrow). Scale bar = 100 nm.

FIGURE 2

Cryo-EM tomography and subsequent segmentation of two sonicated PSDs. (A,H) A virtual slice (2.8 nm thick) of each of two different PSDs through their reconstructed volumes. Black arrows point to clearly delineated structures within the PSDs likely to be loosened upon sonication. Scale bar = 100 nm. (B,I) Virtual slices with regions of interest containing PSDs marked in red on the two-dimensional slices, segmented by hand without any repetitive manual refinement. (C,J) A surface model of the volume of interest (VOI) containing each of the PSDs by manual segmentation. Here, outlines of segmented regions on slices form lateral surface of the VOI; the outlines of the first and last slices form the top and bottom bases of the VOI, showing that modification of the VOI is required to obtain the properly segmented PSD. (D,K) A surface model of each of the segmented PSDs after lowering the isodensity level to reduce noise. Note that the models are still highly noise-embedded. (E,L) A surface model after application of ASOM onto each of the VOIs markedly reducing the noisy structures automatically. Signal-to-noise ratios increased from 0.93 to 1.9 for panel (D) and from 0.10 to 0.51 for panel (K). (F,M) Surface models of differently color-coded substructures or modules within each of the PSDs, obtained by 3D watershed segmentation. White arrows point to the automatically segmented modular structures corresponding to those visually noted in panels (A,H) by black arrows. (G,N) Histograms showing size distribution of modules within the PSDs. White dotted lines indicate the range from 40 to 90 nm. The spheres of 50 nm in diameter in panels (E,L) were included as a guidance of the scale.

FIGURE 3

Surface models of unsonicated PSDs and their modules viewed at four different angles after segmentation. (A) Surface models of six different PSDs in gray after application of ASOM, visualized en face. The surfaces of the PSDs were irregular and lumpy. Holes or gaps were present in some of the PSDs (see the fourth and fifth PSDs). The PSDs separated into modules by the 3D watershed segmentation and color-coded were viewed (B) en face, (C) after ∼45-degree rotation, (D) after ∼135-degree rotation, and (E) from behind. (F) Histograms of the size of PSD modules. The distributions were asymmetric and skewed to the left except for one distribution (sixth distribution). Most modules were less than 125 nm in size, and the average size was ∼50 nm. A sphere of 50 nm in diameter in the first panel (A) was included as a guidance of the scale.

FIGURE 4

Surface models of modules spanning the whole thickness of the PSDs (Trans-PSD modules) viewed at four different angles. The Trans-PSD modules of four different PSDs were viewed (A) en face, (B) after ∼45-degree rotation, (C) ∼135-degree rotation, and (D) from behind. The PSDs contained one to eight Trans-PSD modules. Each Trans-PSD module fell into a range from 6 × 10⁴ nm³ to 5 × 10⁵ nm³ in volume taking up 0.5–6% of the volume of its PSD for control PSDs (four rows from the top) and from 1 × 10⁵ nm³ to 5 × 10⁵ nm³ in volume taking up 1.4–7% for sonicated PSDs (two rows from the bottom).