Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association between gene expression and CES total editing rate We analyzed association between CES total editing rate and gene expression for 14,961 human genes. (a) Gene set enrichment analysis by hypergeometric test on GO-BP categories and REACTOME pathways revealed that associated genes are mainly involved in immune system response mediated by interferon I and alpha / beta. (b) When we analyze distribution of CES total editing rate and ADAR gene expression, ADAR expression levels explains ∼ 13% of observed variability. No significant effect is observed for ADARB1 expression. ADAR and ADARB1 expression levels are reported as residuals of ridge regression with technical covariates (see description of data in methods section). The graphs report adjusted p-value and R2 value from robust regression analysis. (c) The 1,122 genes associated to CES total editing rate after removing ADAR expression effect were enriched for genes mainly involved in ribonucleoprotein and RNA processing.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Genes associated with CES total editing rate are enriched for ADAR interactors (a) Reconstructed PPI network including ADARs and proteins encoded by best genes significantly associated with global editing levels (FDR < 0.01). Among these proteins, we observed 285 potential ADARs interactors, including 9 direct partners of ADARs proteins. (b) Boxplot of number of ADARs interacting genes observed in 1M random simulations. The observed number of interactions (285) resulted in empirical p-value < 1e-6. (c) ADARs interactors are strongly enriched for RNA binding proteins in GO-MF categories. (d) Distribution of degree and betweenness centrality values among network nodes are represented by violin plots. ADAR1 protein has a major role (higher values) among ADAR proteins. Among ADARs direct partners, ELAVL1, RPA1 and IFI16 showed high values of degree and betweenness centrality, suggesting a central role in the network. (e) ADAR1 interaction with RPA70 (coded by RPA1) and IFI16 determined by co-immunoprecipitation. After immunoprecipitation with ADAR1 antibody, western blot for IFI16 and RPA70 are reported. For a better discrimination two times of exposure are reported in the figure.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: RNA Binding Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed strong associations with CES total editing rate for 4 cell type variables (a), representing proportion of neutrophils, monocytes, dendritic cells (DC) and T helper (Th). Specific cell variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level (p) and correlation coefficient (r) are reported in each plot based on Pearson’s product-moment. Only non-zero observations are plotted.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of biological / pharmacological factors on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed significant associations with CES total editing rate for blood pressure medication, BMI current, Age and Sex (a). Specific biological variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level of association after correction for cell composition is reported (p (cell)) is reported in each plot based on Mann-Whitney-Wilcoxon or Pearson’s product-moment correlation test for binary and continuous variables, respectively. For continuous variables the Pearson correlation coefficient (r) is also reported.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition, biological and pharmacological factors on PCs of editing levels The heathmap represents strength of association between the first 5 principal components of CESs (PCs) and ADAR / ADARB1 expression (upper panel), 7 cell composition variables (middle panel) and 11 biological / pharmacological variables (lower panel). Only factors showing significant association with at least one of the first 5 PCs are represented. Significant p values (< 0.05) are colored in yellow-red scale, while p value > 0.05 are represented in grey scale. Age, BMI, blood pressure medications, smoke and alcohol all associate with PC1. Also time of blood draw seems to have a small, but consistent effect, on different PCs. For each PC, variance explained is represented by the bar plot in the upper side.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association study for SNPs and CES total editing rate (a) Manhattan plot representing the association between 573,801 SNPs and CES total editing rate, where black line represents threshold for the top 100 SNPs (p value ∼ 10e-4). (b) Detailed view of genotyped SNPs located in the region at chromosome 7 that showed significant association with CES total editing rate. Known GWAS associations for human phenotypes from GRASP database are reported in the lower panel. (c) The top associated SNP (rs856554) showed a significant effect on global editing level, while no significant correlation was observed with ADAR and ADARB1 expression. (d) Real-time expression analysis of ADAR and ADARB1 mRNA after B-EBV transfection of LOC730338. Not transfected cells were used as control samples. Data are reported as 2 -ΔΔ ct (expression level of control sample is equal to 1) and represent mean values and standard errors obtained from at least 3 independent evaluations. Unpaired t test was used for statistical analysis (*p< 0.05).

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: bioRxiv

Article Title: Resolvin D2/GPR18 signaling enhances monocytic myeloid-derived suppressor cell function to mitigate abdominal aortic aneurysm formation

doi: 10.1101/2024.02.23.581672

Figure Lengend Snippet: RvD2 administration decreases aortic aneurysm phenotype and preserves morphology to mitigate AAA formation. A , Schematic description of murine topical elastase model with RvD2 treatments. Mice were divided into three groups and treated with either heat-inactivated elastase or elastase on day 0. Mice were then administered either vehicle or RvD2. Aortic diameter was measured on day 14 and tissue harvested for additional analysis. B , RvD2 treated mice demonstrated a significant decrease in aortic diameter compared to vehicle treated mice; *p<0.001 vs. other groups; n=10-20 per group). C , Representative images of aortic phenotype in the respective groups. D , Comparative histology performed on day 14 indicates that elastase-treated mice administered with RvD2 have a marked increase in smooth muscle cell α-actin (SM-α actin) expression, decrease in elastic fiber disruption (Verhoeff-Van Gieson staining for elastin) as well as neutrophil (PMN) and macrophage (Mac-2) infiltration, compared to elastase-treated WT mice alone (n=5 per group). Representative histological images in the respective groups with arrows indicate areas of immunostaining. E , Quantification of histological staining in respective groups; n=5 per group; *p<0.02 vs. other groups.

Article Snippet: Slides of aortic cross-sections were prepared and stained for elastin (Van Gieson’s Solution, catalog no. s289; Poly Scientific R&D Systems, Bay Shore, NY) smooth muscle actin (monoclonal anti-actin α-smooth muscle, Sigma Aldrich, St. Louis, MO), macrophages (purified anti-mouse Mac-2, Cedarlane Laboratories, Burlington, Canada), and neutrophils (rat anti-mouse neutrophils, AbD Serotec, Oxford, UK).

Techniques: Expressing, Disruption, Staining, Immunostaining

Journal: bioRxiv

Article Title: Resolvin D2/GPR18 signaling enhances monocytic myeloid-derived suppressor cell function to mitigate abdominal aortic aneurysm formation

doi: 10.1101/2024.02.23.581672

Figure Lengend Snippet: In vivo GPR18 knockdown reduces the protective effect of RvD2. A , GPR18-siRNA treatment of WT mice demonstrated a significant increase in aortic diameter as compared to control (c)-siRNA treated mice after administration of RvD2 in respective groups (*p=0.004, n=12-13 per group). B , Representative images of aortic phenotype in respective groups. C, Expression of smooth muscle-α actin is significantly increased, and elastin fragmentation as well as macrophage and neutrophil infiltration in aortic tissue are significantly decreased in mice treated with c-siRNA+RvD2 compared to mice treated with GPR18 siRNA+RvD2 (n=5 per group). Arrows indicate areas of immunostaining. D, Quantification of histological staining in respective groups; n=5 per group; *p<0.01 vs. other groups.

Article Snippet: Slides of aortic cross-sections were prepared and stained for elastin (Van Gieson’s Solution, catalog no. s289; Poly Scientific R&D Systems, Bay Shore, NY) smooth muscle actin (monoclonal anti-actin α-smooth muscle, Sigma Aldrich, St. Louis, MO), macrophages (purified anti-mouse Mac-2, Cedarlane Laboratories, Burlington, Canada), and neutrophils (rat anti-mouse neutrophils, AbD Serotec, Oxford, UK).

Techniques: In Vivo, Knockdown, Control, Expressing, Immunostaining, Staining

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