Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Self-quenching of Alexa 647 attached to the C-terminus of Syntaxin was used to probe oligomerization of Syntaxin in proteoliposomes. (B) Alexa 647 fluorescence intensity normalized to bPC:Chol Syx condition. Kruskal-Wallis test for non-Gaussian distribution was used for statistical analysis. (C) Probing the state of Q-SNAREs by solubilizing Q-SNARE proteoliposomes in SDS. (D) SDS gel electrophoresis of solubilized Q-SNAREs and Western blotting with anti-Syntaxin-1a (left) and anti-SNAP25 (right) antibodies. (E and F) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (E) and Syntaxin/SNAP25 (F) samples. In (F) oligomer populations are marked with asterisks to indicate that they might consist of the mixture of Syntaxin’s homo-oligomers and Syntaxin/SNAP25 hetero-oligomers. Two-way ANOVA was used. Error bars represent standard error.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Fluorescence, SDS-Gel, Electrophoresis, Western Blot

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) TIRF microscopy was used to examine binding of fluorescently labeled Munc18 to planar supported membranes. (B) TIRF microscopy binding curves of 0.5 µM Munc18 to planar supported membranes containing Syntaxin only. (C) Maximum fluorescence intensity values obtained from fits of the binding curves from (B) to 1 st order kinetic functions. (D) TIRF microscopy binding curves of 0.5 µM Munc18 to planar supported membranes containing Syntaxin co-reconstituted with SNAP25. (E) Maximum fluorescence intensity values obtained from fits of the binding curves from (D) to 1 st order kinetic functions. Error bars represent standard error. T-test with Welch’s correction for unequal SDs was used in (C) and (E) . We additionally used t-test with Welch’s correction to compare maximum fluorescence intensity values between Syntaxin only and Syntaxin:SNAP25 membranes of each lipid condition. Differences were statistically significant, with p=0.0001 for PM 1% PIP2 membranes, and p=0.0113 for bPC:Chol membranes.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Microscopy, Binding Assay, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Design of the co-floatation assay in a Nycodenz density gradient. (B) Distribution of lipids probed by NBD-DOPE in PM proteoliposomes in the gradient. (C) Distribution of Syntaxin in PM and bPC:Chol proteoliposomes in the gradient. (D, E) Distribution of Munc18 in the gradient with PM and bPC:Chol proteoliposomes with Syntaxin only (D) and Syntaxin co-reconstituted with SNAP25 (E) . Error bars indicate standard error of the mean.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques:

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Change of fluorescence intensity of Syntaxin-289C-Alexa647 in bPC:Chol membranes after the addition of 0.5 µM Munc18. (B and C) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (B) and Syntaxin/SNAP25 (C) samples in bPC:Chol membranes before and after incubation with 0.5 µM Munc18. (D) Change of fluorescence intensity of Syntaxin-289C-Alexa647 in PM membranes after the addition of 0.5 µM Munc18. (E and F) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (E) and Syntaxin/SNAP25 (F) samples in PM membranes before and after incubation with 0.5 µM Munc18.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Fluorescence, Incubation

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Visual representation of residues of Syntaxin that were site-specifically labeled with Alexa 546 for FLIC microscopy experiments. The N-peptide is marked green, the H abc domain is marked orange, the SNARE motif is marked red, and the 7 labeled residues are indicated. (B, C) Distances of the labeled sites of Syntaxin from the membrane before (solid bars) and after (open bars) incubation with 0.5 µM Munc18 in PM 1% PIP2 supported lipid bilayers with Syntaxin only (B) or Syntaxin co-reconstituted with SNAP25 (C) . (D) Distance of residue 192 of Syntaxin (N-terminal end of SNARE motif) co-reconstituted with SNAP25 in bPC:Chol and PM 1% PIP2 lipid bilayers before and after incubation with 0.4 µM Munc18. Error bars represent standard error. For statistical analysis, one-way ANOVA test was used, asterisks indicate ** p=0.003 and **** p<0.0001.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Labeling, Microscopy, Membrane, Incubation, Residue

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Docking and fusion of insulin granules containing C-peptide-GFP with Q-SNARE-containing planar supported membranes were imaged using TIRF microscopy. (B) Example of a fluorescence intensity trace with marked onsets of docking and fusion, and the docking to fusion delay time dt. (C) Docking of granules to supported PM 1% PIP2 or bPC:Chol Syntaxin/SNAP25 membranes without and with Munc18, normalized to the number of docked granules with PM 1% PIP2 membranes without Munc18. (D) Fusion efficiency measured as a percentage of docked granules that proceeded to fusion under the same conditions as in (C) . (E-J) Analysis of fusion kinetics. (E and F) Comparison of docking to fusion delay times with and without the addition of Munc18 in PM 1% PIP2 membranes (E) and bPC:Chol membranes (F) . (G and H) Cumulative distribution function of the delay times (data points) and the fit with a parallel reaction model N ( t ) = (1 − exp − kt ) m , where k is the rate constant and m is the number of parallel reactions occurring (solid line) in PM 1% PIP2 (G) and bPC:Chol (H) membranes. (I and J) Half-times of the maximum fusion calculated from the fits of the cumulative distribution function of the delay times in PM 1% PIP2 (I) and bPC:Chol membranes (J) . For statistical analysis, Kruskal-Wallis test for non-Gaussian distribution was used in multiple comparison tests. Kolmogorov-Smirnov test for cumulative distributions was used to compare docking to fusion delay times.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Microscopy, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Cryo-EM micrographs of PM 1% PIP2 proteoliposomes reconstituted with Munc18/Syntaxin/SNAP25. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (B) 2D class averages of Munc18/Syntaxin/SNAP25 in proteoliposomes. The scale bar is 120 Å. (C) Cryo-EM reconstruction of Munc18/Syntaxin/SNAP25 complex in proteoliposomes. (D) Cryo-EM micrographs of nanodiscs containing Munc18/Syntaxin complexes. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (E) 2D class averages of Munc18/Syntaxin in nanodiscs. The scale bar is 110 Å. (F) Cryo-EM reconstruction of protein complex from Munc18/Syntaxin in nanodiscs. (G) Cryo-EM micrographs of nanodiscs containing Munc18/Syntaxin/SNAP25 complexes. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (H) 2D class averages of Munc18/Syntaxin/SNAP25 in nanodiscs. The scale bar is 110 Å. (I) Cryo-EM reconstruction of protein complex from Munc18/Syntaxin/SNAP25 in nanodiscs.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Side-by-side comparisons of the two density maps when opened together in ChimeraX. (B) Comparison of the protein density in the two maps when they are fit into each other in ChimeraX. (C) Additional views of the comparison of the two maps when they are fit into each other in ChimeraX. The black and pink arrows point to the main and secondary extra densities, respectively, that are observed in Munc18/Syntaxin/SNAP25 but lacking in Munc18/Syntaxin.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Comparison