Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neurotransmitter release is triggered by a calcium-induced rearrangement in the Synaptotagmin-1/SNARE complex primary interface.

doi: 10.1073/pnas.2409636121

Figure Lengend Snippet: Fig. 6. Summary of primary interface mutational phenotypes and a putative model of Syt1-mediated Ca2+-triggered SV fusion. (A) Map of residues that (B) modulate priming function. (C) Map of residues that (D) modulate spontaneous fusion clamping function. (E) Map of residues that modulate (F) Ca2+-triggered release probability. In A, C, and E, molecular graphics of the C2B domain–SNARE complex interaction from PDB accession code 5CCH (15). Proteins are represented by ribbon diagrams and sphere models. Carbons are represented in salmon color (SNARE complex) or green (Syt1 C2B domain). Selected residues are labeled in red if they show a significantly increased response compared to WT rescue, gray if they are not significantly different from WT rescue, and blue if they are significantly decreased compared to their respective WT rescue protein. In B, D, and F, residues are labeled in red if they show a significantly increased response compared to WT rescue, gray if they are not significantly different from WT rescue, and blue if they are significantly decreased compared to their respective WT rescue protein. The black dotted line represents WT rescue whereas the dark blue dotted line symbolizes the result for Syt1/Syt7 DKO response. (G) Model of how Syt1 triggers neurotransmitter release. In the primed, fusion-clamped state of the release apparatus, the Syt1 C2B domain (orange) binds to the SNARE complex through the primary interface and to the plasma membrane through a polybasic region. Regions I and II are represented schematically with pink diamond shape forms. Binding of Ca2+ (blue circles) induces reorientation of the C2B domain to allow insertion of both Ca2+ binding loops into the plasma membrane and coordination of the Ca2+ ions by the C2B domain ligands and phospholipid head groups. Because of the reorientation, region I dissociates, but region II remains in contact, which in turn communicates a “rowing force” from the Syt C2B reorientation onto the SNARE complex that facilitates extension of the synaptobrevin and syntaxin-1 helices into the jxt linkers, which leads to fast membrane fusion.

Article Snippet: Primary mouse monoclonal anti- syntaxin- 1A (1:10,000; Synaptic Systems, Cat #110111), anti- Syt1 (1:1,000; Synaptic Systems, Cat #105011), anti- SNAP25 (1:10,000; Synaptic Systems, Cat #111111), and monoclonal anti- betaTubulinIII (1:10,000; Sigma, Cat #T8660) were used.

Techniques: Labeling, Clinical Proteomics, Membrane, Binding Assay

Journal: bioRxiv

Article Title: Inhibitory synaptic vesicles have unique dynamics and exocytosis properties

doi: 10.1101/2020.09.21.289314

Figure Lengend Snippet: (A) Schematic diagram depicting a streptavidin-conjugated quantum dot (QD) conjugated to biotinylated antibodies against the luminal domain of VGAT. (B) Colocalization of VGAT-QD‒loaded inhibitory vesicles (green) and CypHer5E-VGAT‒labeled presynaptic boutons (red) in cultured hippocampal neurons. Scale bar: 1 µm. (C) Three-dimensional trajectory of a VGAT-QD‒loaded inhibitory vesicle overlaid on the x - y plane of a CypHer5E-VGAT‒ labeled presynaptic bouton. The color bar represents elapsed time; electrical stimulation (10 Hz) started at 20 s, and the vesicle underwent exocytosis at 32.0 s. (D) Fluorescence images of the VGAT-QD‒loaded vesicle shown in panel C taken at the indicated times. Scale bar: 0.5 µm. (E) Three-dimensional position, radial distance from the momentary position to the fusion site (R), and fluorescence intensity (F) of the VGAT-QD‒loaded vesicle shown in panel C. Note the photoblinking events (e.g., at approximately 8 s, 13 s and 15 s), confirming the presence of one QD inside the vesicle. Electrical stimuli (10 Hz) were applied for 120 s starting at 20 s (green horizontal bar).

Article Snippet: The biotinylated monoclonal mouse anti-Syt1 antibody (105 311BT, Synaptic Systems) or the biotinylated anti-VGAT antibody (131 103CpH, Synaptic Systems) was conjugated to streptavidin-conjugated quantum dots (cat. A10196, Thermo Fisher Scientific), and vesicles were loaded as described previously ( ).

Techniques: Cell Culture, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Inhibitory synaptic vesicles have unique dynamics and exocytosis properties

doi: 10.1101/2020.09.21.289314

Figure Lengend Snippet: Cumulative distribution of the net displacement between the initial location and fusion site (A) , fusion time (B) , and total length traveled (C) for VGAT-QD‒labeled synaptic vesicles (n = 80 vesicles) and Syt1-QD‒loaded synaptic vesicles (n = 49 vesicles). * p <0.05, ** p <0.01, and NS, not significant (Kolmogorov-Smirnov test (K-S test)).

Article Snippet: The biotinylated monoclonal mouse anti-Syt1 antibody (105 311BT, Synaptic Systems) or the biotinylated anti-VGAT antibody (131 103CpH, Synaptic Systems) was conjugated to streptavidin-conjugated quantum dots (cat. A10196, Thermo Fisher Scientific), and vesicles were loaded as described previously ( ).

Techniques:

Journal: bioRxiv

Article Title: Inhibitory synaptic vesicles have unique dynamics and exocytosis properties

doi: 10.1101/2020.09.21.289314

Figure Lengend Snippet: (A) Fusion time is plotted against the net displacement; each symbol represents an individual VGAT-QD‒loaded inhibitory vesicle, and the solid red line represents the linear regression (Pearson’s r = 0.77). (B) Cumulative distribution of net velocity measured for VGAT-QD‒labeled synaptic vesicles and Syt1-QD‒loaded synaptic vesicles. (C) Fusion time is plotted against the total travel length; each symbol represents an individual VGAT-QD‒loaded inhibitory vesicle, and the solid red line represents the linear regression (Pearson’s r = 0.94). (D) Cumulative distribution of vesicle speed measured for VGAT-QD‒labeled synaptic vesicles and Syt1-QD‒loaded synaptic vesicles. ** p <0.01 and NS, not significant (K-S test).

Article Snippet: The biotinylated monoclonal mouse anti-Syt1 antibody (105 311BT, Synaptic Systems) or the biotinylated anti-VGAT antibody (131 103CpH, Synaptic Systems) was conjugated to streptavidin-conjugated quantum dots (cat. A10196, Thermo Fisher Scientific), and vesicles were loaded as described previously ( ).

Techniques:

Journal: bioRxiv

Article Title: Inhibitory synaptic vesicles have unique dynamics and exocytosis properties

doi: 10.1101/2020.09.21.289314

Figure Lengend Snippet: (A) Fluorescence images taken at the indicated times for an inhibitory vesicle loaded with a VGAT-conjugated QD; electrical stimuli (10 Hz) were applied at 20 s. The intersection of the perpendicular lines marked the position before the first fusion. Scale bar: 0.5 μm. (B) Three-dimensional position, radial distance (R), and fluorescence (F) of a VGAT-QD‒ loaded vesicle that underwent kiss-and-run (K&R) fusion (red arrow) followed by full-collapse fusion (FCF, blue arrow). Electrical stimuli (10 Hz) were applied for 120 s starting at 20 s (green horizontal bar). (C) Average normalized fluorescence intensity traces (with SEM) time-aligned to the first fusion event for vesicles that underwent full-collapse fusion (FCF) (C1), vesicles that underwent kiss-and-run (K&R) fusion followed by FCF (C2), and vesicles that underwent K&R fusion but never underwent FCF (C3). The dotted horizontal line represents a normalized fluorescence intensity value of 0.12, (the expected normalized fluorescence right after full-collapse fusion). (D) Relative distribution of the fusion modes measured in VGAT-QD‒labeled vesicles (D1) and Syt1-QD‒loaded vesicles (D2) that underwent exocytosis, showing that 65% and 27% of vesicles, respectively, underwent K&R fusion. (E-F) Cumulative distribution of total travel length for inhibitory and excitatory vesicles that underwent either FCF (E) or K&R fusion (F). (G-H) Cumulative distribution of fusion time for inhibitory and excitatory vesicles that underwent either FCF (G) or K&R fusion (H). * p <0.05, ** p <0.01, and NS, not significant (K-S test).

Article Snippet: The biotinylated monoclonal mouse anti-Syt1 antibody (105 311BT, Synaptic Systems) or the biotinylated anti-VGAT antibody (131 103CpH, Synaptic Systems) was conjugated to streptavidin-conjugated quantum dots (cat. A10196, Thermo Fisher Scientific), and vesicles were loaded as described previously ( ).

Techniques: Fluorescence

Journal: The Journal of comparative neurology

Article Title: Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

doi: 10.1002/cne.25654

Figure Lengend Snippet: FIGURE 1 Whole mount preparation of the carotid sinus with double immunofluorescence for P2 × 3 (green) and Syt1 (red). (a) Low magnification view of the carotid sinus showing P2 × 3-immunoreactive flower-spray nerve endings. Syt1 immunoreactivity is shown in flower-spray endings and in the network of varicose nerve fibers. (b and c) Three-dimensional view of the basal surface of the terminal part of the flower-spray ending indicated in rectangle in Panel a. Arrows indicate thick parent axon for the ending. Panel b shows flower-spray endings could be distinguished from network of thin varicose nerve fibers with Syn1 immunoreactivity. (d–f) Higher magnification view of the rectangle in Panel a. (d) Punctate P2 × 3 immunoreactivity is shown in the terminal part of the endings. (e) Syt1 immunoreactivity is shown as smaller dots in the terminal parts. (f) The merged figure shows that P2 × 3 and Syt1 immunoreactivities are distinct from each other.

Article Snippet: A mouse monoclonal anti-Syt1 antibody (clone ASV48, MAB4364, R&D Systems, Minneapolis, MN, USA; RRID AB_2199304) was raised against the rat brain synaptic plasmamembrane (Matthew, Tsavaler, and Reichardt 1981).

Techniques: Immunofluorescence

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal antibody anti-Syt1 , Synaptic Systems , Cat#105103; RRID: AB_11042457.

Techniques: Virus, Recombinant, cDNA Synthesis, Western Blot, Stripping, Immunodetection, Plasmid Preparation, Isolation, Sequencing, Software