A Network of Three Types of Filaments Organizes Synaptic Vesicles for Storage, Mobilization, and Docking

Three types of filaments connect to synaptic vesicles. A, All synaptic vesicles (blue, or red if directly connected to filaments extending from the active zone membrane) link to the active zone membrane or each other by filamentous material, forming a coherent group of vesicles termed a vesicle cloud. Typically, four small docking filaments (teal) from the active zone surround each vesicle that contacts the active zone. Longer cluster filaments are found either extending from the active zone (white) or free throughout the vesicle cloud (gold). Small bridge filaments (purple) are distributed throughout the cloud and pair vesicles. B, Same view and color code as in A, but vesicles are removed to expose the underlying filamentous network. Scale bar, 50 nm.

Distributions of lengths of three types of synaptic filament. A, Histogram of lengths of 70 docking filaments, 301 bridge filaments, and 111 cluster filaments pooled from two experiments. B, Scattergrams of the lengths of the same three types of filaments showing that their distributions in the two experiments are indistinguishable. Crossbars indicate the combined filament type mean. Cluster filaments are the most variable in length.

Small filaments extend from the active zone membrane to surround docked vesicles. A features projection images of each vesicle, with colored arrows to indicate electron-dense material corresponding to similarly colored material in B and C. Filaments associated with each vesicle are arbitrarily colorized for contrast, but the active zone membrane is gray throughout. B features the three-dimensional renderings of small filamentous material, docking filaments, from the active zone as well as the synaptic vesicle (red) to which they connect. C shows the rendering in B from an en face view. The bottom panels of A–C feature a fused vesicle. Scale bars, 35 nm.

Two filament types associate with cloud vesicles and the active zone membrane. A, B, and C show three different views of four vesicles (red) that do not contact the active zone but have filament connections to it. A features projection images of each vesicle. Colored arrows indicate electron-dense material that correspond to filaments rendered in the same color in B and C. B features the three-dimensional renderings of long, modular filamentous cluster filaments (white) or singular docking filaments (teal). Both filament types are shown extending from the active zone. C shows the rendering in B from another view. The kinks (gray arrowhead) that are characteristic of a cluster filament are often accompanied by a side branch (gray arrow). Scale bar, 35 nm.

Each synaptic vesicle associates with many filaments. A, All attached filaments and vesicles associated with the vesicle indicated by the asterisk are rendered. Vesicles are color coded so that blue vesicles contact both cluster (gold) and bridge (purple) filaments, while green vesicles contact only bridge filaments. The plasma membrane of the synapse was situated below, well outside the scope of this figure. B–F show reoriented renderings of the individual filaments in A. B, Single cluster filament. C, Example of a possible isolated subunit of a cluster filament, but necessarily classified as a bridge filament. D–F, Renderings of three typical bridge filaments. Scale bar, 35 nm.

Bridge filament connections and superclusters link all synaptic vesicles into a single cloud. A, Within the first 250 nm from the active zone, all cluster filaments are shown (in gold, or white if they touch the active zone) with the vesicles they connect to (blue). Vesicles can connect to multiple cluster filaments, which produce large knots of vesicles that are referred to as superclusters. B, Bridging filaments (purple) and docking filaments (teal) are added to the rendering in A as well as synaptic vesicles (green) that only connect with bridge or docking filaments. Scale bar, 50 nm.