Journal: bioRxiv

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

doi: 10.1101/2024.06.17.599435

Figure Lengend Snippet: (A), Close-up view of region II of the primary interface. Proteins are represented by ribbon diagrams and stick models with nitrogen atoms in dark blue, oxygen in red, sulfur in yellow orange and carbon in salmon color (SNARE complex) or green (Syt1 C 2 B domain). Selected residues are labeled. ( B) Schematics of SNAP25 mutations utilized. ( C) Example traces and ( D) quantification of the EPSC amplitude recorded for autaptic hippocampal neurons obtained from rescue experiments with SNAP25-WT, SNAP25-D51N, E52Q, E55Q, or D51N/E52Q. (E) Example traces and ( F) quantification of the readily releasable pool (RRP) charge induced by 500 mM sucrose application obtained from the same neurons as in (C). ( G) Quantification of the vesicle release probability (Pvr). ( H) Example traces and ( I) quantification of the miniature EPSC frequency (mEPSC) obtained from the same neurons as in (C). ( J) Schematics of Stx1A mutations utilized. ( K) Example traces and (L) quantification of the EPSC amplitude recorded for autaptic hippocampal neurons obtained from rescue experiments with Stx1A-WT, Stx1A-E228Q/D231N and Stx1A-D231N/E234Q/E238Q. (M) Example traces and (N) quantification of the readily releasable pool (RRP) induced by 500 mM sucrose application obtained from the same neurons as in (K). (O) quantification of the vesicle release probability (Pvr). (P) Example traces and (Q) quantification of the miniature EPSC frequency (mEPSC) obtained from the same neurons as in (K). Each data point represents a single recorded neuron. Between 35 and 39 neurons per group from 3 independent cultures were recorded and are shown as mean +/- SEM. Normalization in this and subsequent experiments was computed by dividing response from each neuron against mean values of the WT rescue group for each individual culture. ns: not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: After blocking with 5 % milk powder (Carl Roth GmbH) for 1 hour at room temperature, membranes were incubated with mouse monoclonal anti-syntaxin-1A (1:10,000; Synaptic Systems), mouse anti-Syt1 (1:1,000; Synaptic Systems), mouse anti-SNAP25 (1:10,000; Synaptic Systems) and mouse monoclonal anti-betaTubulinIII (1:10,000; Sigma) antibodies for 1 hour at room temperature.

Techniques: Labeling

Journal: bioRxiv

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

doi: 10.1101/2024.06.17.599435

Figure Lengend Snippet: (A) Close-up view of Region II of the primary interface. Proteins are represented by ribbon diagrams and stick models with nitrogen atoms in dark blue, oxygen in red, sulfur in yellow orange and carbon in salmon color (SNARE complex) or green (Syt1 C 2 B domain). Selected residues are labeled. (B) Schematics of Synaptotagmin1 (Syt1) mutations utilized. (C) Example traces and (D) quantification of the EPSC amplitude recorded for Syt1/7 DKO autaptic hippocampal neurons rescued with Syt1 WT, Syt1 R398Q, Syt1 R399Q, or Syt1 R398Q/R399Q (E) Example traces and (F) quantification of the readily releasable pool (RRP) charge induced by 500 mM sucrose application obtained from the same neurons as in (C). (G) Quantification of the vesicle release probability (Pvr). (H) Example traces and (I) quantification of the miniature EPSC frequency obtained from the same neurons as in (C). Each data point represents a single recorded neuron. Between 39 and 42 neurons per group from 3 independent cultures were recorded and are shown as mean +/- SEM. ns: not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: After blocking with 5 % milk powder (Carl Roth GmbH) for 1 hour at room temperature, membranes were incubated with mouse monoclonal anti-syntaxin-1A (1:10,000; Synaptic Systems), mouse anti-Syt1 (1:1,000; Synaptic Systems), mouse anti-SNAP25 (1:10,000; Synaptic Systems) and mouse monoclonal anti-betaTubulinIII (1:10,000; Sigma) antibodies for 1 hour at room temperature.

Techniques: Labeling

Journal: bioRxiv

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

doi: 10.1101/2024.06.17.599435

Figure Lengend Snippet: (A) Close-up view of Region II of the primary interface. Proteins are represented by ribbon diagrams and stick models with nitrogen atoms in dark blue, oxygen in red, sulfur in yellow orange and carbon in salmon color (SNARE complex) or green (Syt1 C 2 B domain). Selected residues are labeled. (B) Schematics of Synaptotagmin1 (Syt1) mutations utilized. (C) Example traces and (D) quantification of the EPSC amplitude recorded for Syt1/7 DKO autaptic hippocampal neurons rescued with Syt1 WT, Syt1 R281A, Syt1 K288A, or Syt1 R281A/K288A. (E) Example traces and (F) quantification of the readily releasable pool (RRP) charge induced by 500 mM sucrose application obtained from the same neurons as in (C). (G) Quantification of the vesicle release probability (Pvr). (H) Example traces and (I) quantification of the miniature EPSC frequency obtained from the same neurons as in (C). Each data point represents a single recorded neuron. Between 44 and 45 neurons per group from 3 independent cultures were recorded and are shown as mean +/- SEM. ns: not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: After blocking with 5 % milk powder (Carl Roth GmbH) for 1 hour at room temperature, membranes were incubated with mouse monoclonal anti-syntaxin-1A (1:10,000; Synaptic Systems), mouse anti-Syt1 (1:1,000; Synaptic Systems), mouse anti-SNAP25 (1:10,000; Synaptic Systems) and mouse monoclonal anti-betaTubulinIII (1:10,000; Sigma) antibodies for 1 hour at room temperature.

Techniques: Labeling

Journal: bioRxiv

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

doi: 10.1101/2024.06.17.599435

Figure Lengend Snippet: (A) Close-up view of Region I and Region II of the primary interface. Proteins are represented by ribbon diagrams and stick models with nitrogen atoms in dark blue, oxygen in red, sulfur in yellow orange and carbon in salmon color (SNARE complex) or green (Syt1 C 2 B domain). Selected residues are labeled. (B) Schematics of Synaptotagmin1 (Syt1) mutations utilized. (C) Example traces and (D) quantification of the EPSC amplitude recorded for Syt1/7 DKO autaptic hippocampal neurons rescued with Syt1 WT, Syt1 Y338D, Syt1 A402T and Syt1 Y338D/A402T mutants. (E) Example traces and (F) quantification of the readily releasable pool (RRP) charge induced by 500 mM sucrose application obtained from the same neurons as in (C). (G) Quantification of the vesicle release probability (Pvr). (H) Example traces and (I) quantification of the miniature EPSC frequency obtained from the same neurons as in (C). Each data point represents a single recorded neuron. Between 41 and 45 neurons per group from 3 independent cultures were recorded and are shown as mean +/- SEM. ns: not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: After blocking with 5 % milk powder (Carl Roth GmbH) for 1 hour at room temperature, membranes were incubated with mouse monoclonal anti-syntaxin-1A (1:10,000; Synaptic Systems), mouse anti-Syt1 (1:1,000; Synaptic Systems), mouse anti-SNAP25 (1:10,000; Synaptic Systems) and mouse monoclonal anti-betaTubulinIII (1:10,000; Sigma) antibodies for 1 hour at room temperature.

Techniques: Labeling

Journal: bioRxiv

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

doi: 10.1101/2024.06.17.599435

Figure Lengend Snippet: (A) Close-up view of Region I of the primary interface. Proteins are represented by ribbon diagrams and stick models with nitrogen atoms in dark blue, oxygen in red, sulfur in yellow orange and carbon in salmon color (SNARE complex) or green (Syt1 C 2 B domain). Selected residues are labeled. (B) Schematics of Synaptotagmin1 (Syt1) mutations utilized. (C) Example traces and (D) quantification of the EPSC amplitude recorded for Syt1/7 DKO autaptic hippocampal neurons rescued with Syt1 WT, Syt1 Y338W, Syt1 E295A, and Syt1 E295A/Y338W mutants (E) Example traces and (F) quantification of the readily releasable pool (RRP) charge induced by 500 mM sucrose application obtained from the same neurons as in (C). (G) Quantification of the vesicle release probability (Pvr). (H) Quantification of the number of synaptic vesicles (SV) in the RRP. (I) Average traces and (J) close-up view of synaptic responses induced by an application of 500 mM sucrose for Syt1 WT (n = 65), Syt1/7 DKO (n = 26), Syt1 Y338W (n = 32), Syt1 E295A (n = 35) and Syt1 E295A/Y338W (n = 38) mutants. (K) Quantification of the response onset latency normalized to Syt1 rescue. ( L) Example traces and quantification of the miniature EPSC frequency (M) , amplitude (N) and the spontaneous release rate as a ratio of mEPSC frequency and the number of SV in the RRP (O) obtained from the same neurons as in (C). Each data point represents a single recorded neuron. Between 45 and 46 neurons per group from 3 independent cultures were recorded and are shown as mean +/- SEM. ns: not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: After blocking with 5 % milk powder (Carl Roth GmbH) for 1 hour at room temperature, membranes were incubated with mouse monoclonal anti-syntaxin-1A (1:10,000; Synaptic Systems), mouse anti-Syt1 (1:1,000; Synaptic Systems), mouse anti-SNAP25 (1:10,000; Synaptic Systems) and mouse monoclonal anti-betaTubulinIII (1:10,000; Sigma) antibodies for 1 hour at room temperature.

Techniques: Labeling

Journal: bioRxiv

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

doi: 10.1101/2024.06.17.599435

Figure Lengend Snippet: (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) Ca 2+ -triggered release probability. In A, C and E, molecular graphics of the C2B domain-SNARE complex interaction from PDB accession code 5CCH . 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, grey 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, grey 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 C 2 B 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 Ca 2+ (blue circles) induces reorientation of the C 2 B domain to allow insertion of both Ca 2+ binding loops into the plasma membrane and coordination of the Ca 2+ ions by the C 2 B 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: After blocking with 5 % milk powder (Carl Roth GmbH) for 1 hour at room temperature, membranes were incubated with mouse monoclonal anti-syntaxin-1A (1:10,000; Synaptic Systems), mouse anti-Syt1 (1:1,000; Synaptic Systems), mouse anti-SNAP25 (1:10,000; Synaptic Systems) and mouse monoclonal anti-betaTubulinIII (1:10,000; Sigma) antibodies for 1 hour at room temperature.

Techniques: Labeling, Membrane, Binding Assay