(A) Munc18–1 is concentrated in microdomains in inside-out plasma membrane sheets from PC12 cells. Membrane sheets were fixed with paraformaldehyde immediately after preparation, followed by immunostaining for Munc18–1 (right panel). Integrity of the membrane was confirmed using counterstaining by the lipophilic dye TMA-DPH (left panel).
(B) and (C) Time-dependent dissociation of Munc18–1 from the membrane sheets. The experiments were carried out as in (A), but between preparation and fixation, the sheets were washed with buffer for varying time periods (as indicated), resulting in a gradual decrease of Munc18–1 immunostaining intensity over time (C). Values were related to the immediately fixed condition which was set to 100% (n = 3–6 independent experiments for each data point, values are given as mean ± standard error of the mean (SEM); for details see Materials and Methods).
(D) Sensitivity of the Munc18–1 acceptor to SNARE-cleaving light chains of clostridial neurotoxins. Membrane sheets were incubated for 10 min (see also arrow in C) in the absence (value used for normalization) or presence of 10 μM light chains as indicated, followed by fixation and immunostaining for Munc18–1. BoNT/C1 cleaves syntaxin 1A (BotNT/C1-mut, a cleavage-inactive BoNT/C1-point mutant was used as a control), BoNT/E cleaves SNAP-25, and synaptobrevin 2 is sensitive to TeTx. Values are given as mean ± SEM. n = 8, paired t-test analysis none:BoNT/C1 p < 0.0005.
(E) Cleavage efficiency of BoNT/C1. Membrane sheets were incubated for 10 min in the absence (set to 100%) or presence of 10 μM light chains of BoNT/C1 or BoNT/C1-mut, followed by fixation and immunostaining for syntaxin 1. Values are given as mean ± SEM. n = 3.

(A) Plasma membrane sheet generated from a PC12 cell expressing the secretory granule marker NPY-GFP. Left, immunostaining for Munc18–1 (red channel); right, plasma membrane–docked, GFP-filled secretory granules (green channel). Circle indicates a fluorescent bead visible in all channels acting as a spatial reference for vertical shifts occurring during filter change.
(B) Left, overlay from images shown in (A); right, magnified view from overlay. Linescans were placed through the centers of individual secretory granules (174 granules from ten membrane sheets were analyzed; for example, see dotted line), and granules were rated to be associated with a Munc18–1–rich domain when both signals had a maximum to within two pixels. Random co-localization was determined on mirrored images and subtracted (for details see Materials and Methods), resulting in 70% specific co-localization of granules with Munc18–1 domains.

Co-overexpression of myc-tagged Munc18–1 together with GFP-tagged syntaxin 1 (A) and (B) or an open mutant of syntaxin 1 (C) and (D). Membrane-recruited overexpressd Munc18–1 was selectively visualized on the background of endogenous Munc18–1 by immunostaining for the myc-tag. For each condition, the level of overexpressed syntaxin and of overexpressed, recruited Munc18–1 were determined for individual membrane sheets, plotted against each other, and linearly fitted (B) and (D). The ratio of the slopes wild-type (wt) syntaxin:open syntaxin was determined to be 2.1. The graphs contain data points from 411 membrane sheets for wt syntaxin 1/Munc18–1 and 417 for open syntaxin 1/Munc18–1 obtained from ten independent experiments.

(A) Membrane sheets were incubated for 10 min in the absence (set to 100%) or presence of 10 μM of recombinant SNARE proteins as indicated, and Munc18–1 was quantified by immunofluorescence. Each experiment was performed six to seven times, values are given as mean ± standard deviation of the mean (SDM). Paired t-test analysis none:synaptobrevin 2 p < 0.0005 (n = 7); t-test synaptobrevin:SNAP-25 p < 0.005 (n = 6).
(B) Incubation of membrane sheets with or without 10 μM synaptobrevin 2 for varying times, as indicated, followed by immunostaining for Munc18–1. Immunostaining intensity of immediately fixed membrane sheets was set to 100%. For each data point, four to six independent experiments were performed. Values are given as mean ± SEM.
(C) Experiment as in (A), R-SNAREs were added during incubation as indicated. Values are given as mean ± standard deviation of the mean (SDM). Paired t-test synaptobrevin 2:none p < 0.00005 (n = 10), synaptobrevin 2:endobrevin p < 0.0005 (n = 10), and non-paired t-test synaptobrevin:VAMP4 p < 0.005 (n = 10 and 6).

Membrane sheets were prepared from mouse embryonal chromaffin cells (E16–E18), isolated from wild-type or snap25 knockout animals. Membrane sheets were incubated for 10 min in the absence or presence of 10 μM synaptobrevin 2, fixed, and immunostained for Munc18–1. Syntaxin was also visualized by immunostaining in order to make sure that membrane sheets were generated from chromaffin cells and not from fibroblast cells, which are also present in the culture (for details see Materials and Methods).
(A) and (B) Membrane sheets generated from cells isolated from wild-type (WT) (A) or knockout (B) animals. Left panels, syntaxin staining; right panels, Munc18–1 staining. Upper and lower panels in (A) and (B), absence or presence of synaptobrevin during incubation, respectively.
(C) Quantification of Munc18–1 immunostaining intensities. For clarity, values are expressed as percentage relating to values obtained in the absence of synaptobrevin. For each condition, seven independent experiments were performed. Values are given as mean ± SEM. Statistical test. n = 7, paired t-test knockout:wt p < 0.005.

Munc18–1 binds to a partially closed conformation of syntaxin that is organized in clusters and that may (bottom branch) or may not (top branch) be associated with SNAP-25 at a 1:1 stoichiometry. After vesicle docking, synaptobrevin interacts with this complex, thereby displacing Munc18–1. Alternatively, SNAP-25 is not yet associated with the complex, and synaptobrevin binding is associated with the simultaneous recruitment of SNAP-25. As result, SNARE trans-complexes form, leading to exocytosis. It is possible that some syntaxin molecules that are freely diffusing in the membrane are capable of binding Munc18–1 with high affinity in a closed conformation (left) similar to that observed in solution, thereby preventing it from entering SNARE complexes. Such a pool could be reflected by Munc18–1 remaining membrane associated even after extensive washing periods.