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. 2019 Jan;593(2):144-153.
doi: 10.1002/1873-3468.13316. Epub 2019 Jan 18.

Symmetrical Organization of Proteins Under Docked Synaptic Vesicles

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Free PMC article

Symmetrical Organization of Proteins Under Docked Synaptic Vesicles

Xia Li et al. FEBS Lett. .
Free PMC article

Abstract

During calcium-regulated exocytosis, the constitutive fusion machinery is 'clamped' in a partially assembled state until synchronously released by calcium. The protein machinery involved in this process is known, but the supra-molecular architecture and underlying mechanisms are unclear. Here, we use cryo-electron tomography analysis in nerve growth factor-differentiated neuro-endocrine (PC12) cells to delineate the organization of the release machinery under the docked vesicles. We find that exactly six exocytosis modules, each likely consisting of a single SNAREpin with its bound Synaptotagmins, Complexin, and Munc18 proteins, are symmetrically arranged at the vesicle-PM interface. Mutational analysis suggests that the symmetrical organization is templated by circular oligomers of Synaptotagmin. The observed arrangement, including its precise radial positioning, is in-line with the recently proposed 'buttressed ring hypothesis'.

Keywords: PC12 cells; SNARE proteins; cryo-electron tomography; regulated exocytosis; synaptotagmin.

Figures

Figure 1
Figure 1
The neurite varicosities of NGF‐differentiated PC12 cell revealed by CLEM and cryo‐EM. (A) Micrographs of PC12 cells grown directly on Au‐grids with or without NGF differentiation, shows the appearance of extended neurite (yellow box in middle plane) with varicosities (yellow circle in the right panel) after NGF differentiation. These neurite varicosities were thin enough to allow cryo‐EM analysis. (B) NGF‐differentiation (3 days or 7 days) increased the relative frequency of small, clear synaptic‐like vesicles as compared to large, dense core vesicles. The vesicle diameters were estimated from tomograms assuming they are spheres. (C) A representative cryo‐CLEM workflow used to identify and image VAMP2‐4X‐pHluorin expressing neurites. Left panel: A fluorescent light microscope and wide bright‐field LM image which were used to identify VAMP2‐4X‐pHluorin‐transfected neurite (yellow arrow). Middle panel: low magnification (220×) cryo‐EM image of the same area. Right panel: high magnification cryo‐EM image of the neurite extension (yellow box in middle panel). The obvious markers (cell body, red arrows) were used to coordinate between the images and yellow box denotes the varicosity along the transfected neurites which was used to collect the tilt images.
Figure 2
Figure 2
Representative tomographic slices and 3D segmentation rendering of NGF‐induced PC12 neurite varicosities shows the distribution of vesicles within the varicosities. Tomographic slices of VAMP2‐4X‐pHluorin‐transfected neurite varicosities both at (A) center of the varicosity and (B) proximal to the PM are shown (C) Segmentation of the neurite varicosity built from the tomographic density volume in (A and B). Only vesicles that locate proximal to the PM (red arrowhead/pink vesicles) were included in our analysis, while vesicles floating inside the varicosity (yellow arrowhead/orange vesicles) were excluded.
Figure 3
Figure 3
Cryo‐ET 3D reconstruction of the protein organization at vesicle‐ PM interface. 3D‐reconstructions both without (A) and with sixfold rotational symmetry imposed (B) reveals six symmetrically organized protein density under a docked vesicle. Left panels show the slices through the reconstructed volume of the averaged subtomograms. Top: slice along z‐axis. Red arrow points to relative position of the PM. Bottom: slice through the volume in XY plane at the vertical position highlighted by yellow dotted line on the top. Right panel shows the surface representation of the 3D map filtered to 36 Å at σ = 2 (magenta) and σ = 1 (transparent gray) threshold levels. (C) Rigid body fitting of crystal structures of constituent proteins into the densities below the vesicle. (A) Given the relatively low resolution, it is not possible to rigorously assign specific domains to features in the 3D map, but each of the pronounced density observed between the vesicle and PM were best fitted with a protein complex, consisting of SNAP (SNAP‐25 – green, Syntaxin‐1A – red, VAMP2 – blue); Cpx (cyan), two Synaptotagmin C2B molecules – primary (magenta) and tripartite (yellow) and Munc18 (light blue). Note: Fitting into the sixfold rotationally symmetrized cryo‐ET map at threshold level σ = 2 is shown and Syt1 C2A domains are omitted for clarity. The following X‐ray structures (PDB code) 5W5C (Syt1‐Cpx‐Syt1); 4JEU (Munc‐18/Syntaxin‐1 Habc domain complex) were manually fitted into the cryo‐ET map using USCF Chimera software.
Figure 4
Figure 4
Protein organization at the vesicle‐membrane interface is templated by Synaptotagmin‐ring like oligomers. (A) Subtomogram class averages of docked vesicles in the PC12 cells expressing Syt1 oligomerization mutant (F349A) shows weak protein density, but lacks organization at the vesicle–PM interface. Top row: Slice through the center of tomogram along z axis. Bottom row is the corresponding slices through the volume in XY plane at the vertical position highlighted by yellow dotted line on the top panel. (B) Modeling shows how Syt1 ring‐like oligomers containing 18 Syt1 molecules are perfectly positioned to template the observed symmetrical organization of the exocytic modules. The six SNAREpins, bound to the regulatory proteins, can be positioned on top of the Syt1 oligomer bound via the ‘primary’ binding site. In addition to the X‐ray structures 5W5C (Syt1‐Cpx‐Syt1); and 4JEU (Munc‐18/Syntaxin‐1 Habc domain complex), the Syt1 oligomer model from Wang et al. 11 was used in the model building process using USCF Chimera software.

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