Symmetrical arrangement of proteins under release-ready vesicles in presynaptic terminals

Abstract

Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca²⁺ influx. This Ca²⁺-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially "tethered" to the PM by a variable number of protein densities (∼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the "primed" vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca²⁺ influx.

Keywords: SNARE protein; cryoelecton tomography; synaptic vesicles; vesicle priming.

Fig. 1.

Cryoelectron tomography analysis of synaptic vesicles in primary hippocampal neurons. (A) Low magnification (220×) cryo-EM micrograph of the hippocampal neurons cultured directly on Au grids revealed the presence of neuronal networks of varying densities. (B) Cryo-EM micrograph at 3,600× magnification of the highlighted area (dashed white box in A) revealed the presence of neuronal boutons. (C) High magnification cryo-EM micrograph of the synaptic bouton (highlighted by the dashed white box in B) was collected at 26,000× using Titan Krios equipped with a Volta phase plate and energy filter. (D) Representative tomographic slice and corresponding 3D segmentation rendering (E) shows the distribution of synaptic vesicles in the neuronal boutons. Both the tomographic slice and the 3D segmentation revealed the presence of synaptic vesicles (SVs) at different stages of docking and the neuronal ultrastructure such presynaptic membrane (PrM), postsynaptic membrane (PoM), dense core vesicles (DCVs), endosomal vesicles (EVs), microtubules (MTs), mitochondria (Mito), and postsynaptic mitochondria (PostMito).

Fig. 2.

Classification of side-view docked vesicles as a function of interbilayer distance. (A) Representative tomographic slices of SVs at various stages of docking and (B) quantification of the observed distribution of docked synaptic vesicle (SV) based on the SV to presynaptic membrane interbilayer distance (Bottom) are shown. Side-view vesicles (∼2,500 bin4 subtomograms with 1 pixel = 2.16 nm) were used for the analysis.

Fig. 3.

Cryo-ET 3D reconstruction of protein organization under docked vesicles. (A) Representative tomographic slices of primed SVs that have a SV–PrM interbilayer distance of ≤6 nm (Top). Pronounced protein density at the docking site is marked by orange arrowheads. The 3D reconstruction of the observed protein density, following several rounds of alignment and classification, revealed a hexameric arrangement of protein densities at the docking interface either with or without a sixfold rotational symmetry imposed (Bottom). (B) Representative tomographic slices of the tethered SVs that have a SV–PrM interbilayer distance of ≥8 nm (Top). These SVs were connected to PrM by varying numbers of long protein tethers (red arrowheads), but averaging failed to reveal any organized structure at the SV–PrM docking interface (Bottom). The yellow arrowheads represent the vertical position in the SV–PrM interface (in the XZ slice or side view) at which the XY plane (or top view) is rendered.

Fig. 4.

Symmetrical arrangement of protein density under docked synaptic vesicles. (A) Subtomogram class averages of SVs that are docked to the PrM. All the eight 3D class averages revealed a symmetrical organization of proteins at the SV–PrM interface. Side-view vesicles, with SV–PrM interbilayer distance of less than ∼6 nm and top/bottom-view vesicles, with a diameter of 43.88 ± 5.32 nm were pooled together for the subtomogram analysis. Top row: Slice through the center of the tomogram along the z axis. Bottom row: Corresponding slices through the volume in the XY plane at the vertical position highlighted by yellow arrowheads on the Top. (B) The cryo-ET 3D reconstruction of the SV–PrM interface reveals a symmetrical organization of six protein densities at the SV–PrM interface. (Left) Slice through the reconstructed volume along the z axis of the subtomogram averaged structure (XZ slice, side view) revealed protein densities connecting the SV to the PrM. (Right) Slice through the volume in XY plane (top view) at the vertical position highlighted by yellow arrowheads in the XZ slice revealed the symmetrical arrangement of protein densities at the SV–PrM interface. (C) Surface representation of the cryo-ET 3D reconstruction of the protein organization at the SV–PrM interface at different orientations. The cryo-ET 3D map at threshold level σ = 0.85 was segmented using UCSF Chimera for the surface representation. The top, side, and bottom views of the 3D reconstructions reveal six symmetrically organized protein densities (orange) connecting the SV (light blue) to the PrM (light gray).