Fife promotes synaptic vesicle docking. (A) Single, central virtual slice of ∼0.7 nm through an EM tomogram of a wild-type NMJ synapse. (B) 3D model of an extensively segmented wild-type active zone prepared using HPF/FS techniques, illustrating the cytomatrix (blue) and synaptic vesicles (clustered pool in gray, tethered vesicles in gold, release-ready vesicles in contact with the cytomatrix in magenta, and release-ready vesicles distal to the cytomatrix in olive), tethers linking synaptic vesicles to the presynaptic membrane (purple), and connectors linking synaptic vesicles within the clustered pool (green). (C–D) Representative virtual slices with segmented cytomatrices superimposed (top) and 3D models (bottom) of cytomatrices (blue) and membrane-associated synaptic vesicles (magenta) at representative wild-type (C) and Fife^AC/Df (D) active zones. (E–F) Virtual slices of ∼0.7 nm from wild-type tomograms (E and F) and the same virtual slices with 3D models of segmented features superimposed (bottom) of representative tethered synaptic vesicles (gold) linked to the presynaptic membrane by single (E) or multiple (F) long filaments (purple). (G–H) Virtual slices from wild-type tomograms and the same virtual slices with 3D models of segmented features superimposed (bottom) of representative docked synaptic vesicles (olive) with (G) or without (H) numerous short connections (red) between the vesicle and presynaptic membranes. (I) Quantification of the number of synaptic vesicles per active zone tethered by long filaments (gold vesicles) in wild type (4.8 ± 1.3 vesicles per active zone, n = 16 active zones) and Fife^AC/Df (4.7 ± 1.4 vesicles per active zone, n = 13 active zones P = 0.81). (J) Quantification of the number of synaptic vesicles per active zone docked at the presynaptic membrane (magenta and olive vesicles) in wild type (2.7 ± 0.45 vesicles, n = 14 active zones) and Fife^AC/Df (1.3 ± 0.37 vesicles, n = 9 active zones P = 0.04). (K) Histogram of the mean number of docked synaptic vesicles, in 30-nm bins, at indicated distances from the dense projection center in wild type (n = 38 docked vesicles at 14 active zones) and Fife^AC/Df (n = 12 docked vesicles at nine active zones). (L) Quantification of the number of docked synaptic vesicles per active zone in contact with the dense projection (magenta vesicles) in wild type (1.7 ± 0.30 vesicles, n = 14 active zones) and Fife^AC/Df (0.9 ± 0.31 vesicles, n = 9 active zones P = 0.08). Student’s t test. Error bars represent SEM.