The role of the vesicular Ca 2+ -sensor Synaptotagmin-1 in synaptic vesicle docking

Next, I studied the function of Synaptotagmin-1 in synaptic vesicle docking.

Synaptotagmin-1, the exocytotic Ca²⁺-sensor, has also been implicated in secretory and synaptic vesicle docking, and in the regulation of total synaptic vesicle numbers, which are reduced in Synaptotagmin-1 KO animals (Jorgensen et al., 1995; Liu et al., 2009; de Wit et al., 2009). In-vitro data from liposome fusion assays indicated a role for Synaptotagmin-1 as a distance-regulator for synaptic vesicle fusion, keeping plasma membrane and vesicle membrane apart to prevent SNARE-complex formation and fusion prior to Ca²⁺ -influx. Ca²⁺-binding to the C₂ domains of Synaptotagmin-1 would then trigger SNARE-nucleation and membrane fusion in a post-docking step (van den Bogaart et al., 2011). In vivo, the role of Synaptotagmin-1 in synaptic vesicle docking in neurons remains a point of heavy discussion, a confounding factor being the variety of model systems, fixation

protocols (chemical vs. cryo-fixation), data acquisition methods (two-dimensional versus three-dimensional EM analyses), and above all, differences in “docking” definitions used to study membrane-attachment by EM (discussed in: Verhage and Sørensen 2008).

3.1.3.6.1. 2D-EM analysis of synaptic morphology in Synaptotagmin-1 KO neurons

For the two-dimensional analysis of basic ultrastructural features in the absence of Synaptotagmin-1, samples from four Synaptotagmin-1 KO animals (N=4) were compared with samples from five control animals (N=5), that were either wildtype or heterozygous for the Synaptotagmin-1 KO allele. No major morphological changes between control and Synaptotagmin-1 KO synaptic profiles were observed in electron micrographs (Fig. 3.14 A, B). First, no changes in the overall number of synaptic vesicles per synaptic profile were detected between genotypes (control: 52.8 ± 2.573, n = 114; Synaptotagmin-1 KO:

50.3 ± 2.608, n = 97 / P = 0.4952, n.s.; Fig. 3.14 C). However, when I normalized the number of synaptic vesicles to the area of the synaptic terminal I could unmask a small but significant reduction in the terminal vesicle density in Synaptotagmin-1 KO synapses (control: 1.595 ± 0.05415, n = 114; Synaptotagmin-1 KO: 1.363 ± 0.055, n = 97 / P = 0.0045, **; Fig. 3.14 D), which was also detected as a reduction of the synaptic vesicle cluster density (control: 3.946 ± 0.063, n = 114; Synaptotagmin-1 KO: 3.658 ± 0.062, n = 97 / P = 0.0034, **; Fig. 3.14 E). No differences in the PSD length (control: 333.4 ± 10.03, n = 114; Synaptotagmin-1 KO: 345.0 ± 11.67, n = 97 / P = 0.3661, n.s.; Fig. 3.14 F) in the number of endosomes per synaptic profile (control: 0.211 ± 0.046, n = 114;

Synaptotagmin-1 KO: 0.175 ± 0.046, n = 97 / P = 0.046; Fig. 3.14 G), or in the number of LDCVs per synaptic profile were detected (control: 0.211 ± 0.046, n = 114;

Synaptotagmin-1 KO: 0.175 ± 0.046, n = 97 / P = 0.5481, n.s.; Fig. 3.14 H).

3.1.3.6.2. 3D-ET analysis of synaptic vesicle docking in Synaptotagmin-1 KO neurons

For the three-dimensional electron tomographic analysis of synaptic vesicle docking in the absence of Synaptotagmin-1, samples from three Synaptotagmin-1 KO animals (N=3) were compared with samples from four control animals (N= 4), that were either wildtype or

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nm; control: 1.494 ± 0.203, n = 23; Synaptotagmin-1 KO: 0.906 ± 0.156, n = 20 / P = 0.0302, *; Fig. 3.15 I) and in the number of closely tethered synaptic vesicles (0-4 nm;

control: 2.062 ± 0.185, n = 23; Synaptotagmin-1 KO: 1.433 ± 0.217, n = 20 / P = 0.0319, *;

Fig. 3.15 J). In contrast to all previously analyzed mouse mutants, I detected a significant decrease in the number of membrane-proximal synaptic vesicles (0-40 nm) in synapses lacking Synaptotagmin-1 in comparison to control synaptic profiles (control: 3.630 ± 0.243, n = 23; Synaptotagmin-1 KO: 2.802 ± 0.289, n = 20 / P = 0.0329, *; Fig. 3.15 H), whereas the number of vesicles within 100 nm distance of the active zone was only slightly, but not significantly, reduced (control: 8.997 ± 0.644, n = 23; Synaptotagmin-1 KO: 7.628 ± 0.500, n = 20 / P = 0.1080, n.s.; Fig. 3.15 G). Moreover, the synaptic vesicle distribution within 100 nm distance of the active zone revealed a reduction in the number of membrane-attached synaptic vesicles, but did not show an accumulation of vesicles close to the active zone membrane as was observed for the KOs of the t-SNARE SNAP25 and of Munc13s (Fig. 3.15 M).

Figure 3.14. Two-dimensional ultrastructural analysis of synaptic morphology in Synaptotagmin-1 KO neurons

Electron micrographs of control (A) and Synaptotagmin-1 KO (B) synaptic profiles acquired from 60 nm-thick ultrathin sections. Mean number of synaptic vesicles (SVs) per synaptic profile (C). Mean number of SVs normalized to synaptic terminal area (SV terminal density; D). Mean number of SVs normalized to SV cluster area (SV cluster density; E). Mean postsynaptic density (PSD) length (F).

Mean number of endosomes per synaptic profile (G). Mean number of large dense-core vesicles (LDCVs) per synaptic profile (H). C-H: Control: N=5, n=114; Synaptotagmin-1 KO: N=4, n=97 (Mean + SEM), P<0.001: ***; P<0.01: **; P<0.05: *. Scale bar: B, 500 nm.

Figure 3.15. Three-dimensional electron tomographic analysis of synaptic vesicle docking in Synaptotagmin-1 KO neurons

Tomographically reconstructed subvolumes of control (A) and Synaptotagmin-1 KO (B) synapses

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Orthogonal views of control (E) and Synaptotagmin-1 KO (F) tomographic models displaying the spatial arrangement of membrane-attached synaptic vesicles within the reconstructed active zone area. Mean number of SVs within 100 nm of the AZ normalized to AZ area (G). Mean number of SVs within 40 nm of the AZ normalized to AZ area (H). Mean number of membrane-attached SVs (within 0-2 nm of the AZ) normalized to AZ area (I). Mean number of membrane-attached and closely-tethered SVs (within 0-4 nm of the AZ) normalized to AZ area (J). Mean outer SV diameter (K). Mean SV volume including the membrane bilayer (L). Spatial distribution of SVs within a 100 nm distance of the AZ membrane (M). Distribution of synaptic vesicle diameters of vesicles within 100 nm of the AZ (N). G-L: Control: N=4, n=23; Synaptotagmin-1 KO: N=3, n=20 (Mean + SEM), P<0.001: ***; P<0.01: **; P<0.05: *. M, N: (807 SVs in control and 650 SVs in Synaptotagmin-1 KO synaptic profiles). Scale bar: B, 100 nm.

In comparison to control synaptic profiles, synapses devoid of Synaptotagmin-1 exhibited reductions in the number of membrane-attached and closely tethered vesicles to 61% and 70% of controls, respectively. The number of membrane-proximal vesicles within 40 nm of the active zone is reduced to 77% compared to control synaptic profiles. However, taking the reduced density of membrane proximal synaptic vesicles into consideration, the number of membrane-attached (0-2 nm) and closely tethered (0-4 nm) vesicles normalized to the total number of vesicles within 40 nm of the active zone show reductions to only 74% and 83% of controls, respectively. In addition to these findings, I did not observe any changes in the mean outer vesicle diameter (control: 46.81 ± 0.548, n

= 23; Synaptotagmin-1 KO: 45.41 ± 0.582, n = 20 / P = 0.0873, n.s.; Fig. 3.15 K, N) or in the mean vesicle volume between the two groups (control: 54578 ± 2011, n = 23;

Synaptotagmin-1 KO: 49958 ± 1941, n = 20 / P = 0.1086, n.s.; Fig. 3.15 L).

In summary, Synaptotagmin-1 KO neurons exhibit a decrease in the number of membrane-attached synaptic vesicles, which points towards a regulatory function of Synaptotagmin-1 in synaptic vesicle docking.