Figure 2.

Diagram summarizing the overall approach followed in the lipid and content mixing assays described here. The color coding is the same as in Figure 1a. PhycoE-Biotin trapped in the T-liposomes is shown as a black square with a yellow circle that represents the fluorescent probe. Cy5-Streptavidin trapped in the VSyt1-liposomes is shown in gray with a red circle that represents the fluorescent probe. Note that the T-liposomes are first incubated at 37 °C for 25 min with Munc18–1 and NSF-αSNAP to disassemble the syntaxin-1-SNAP-25 complexes and form the closed syntaxin-1-Munc18–1 complex. These liposomes are then mixed at 30 °C with the VSyt1-liposomes in the presence of other soluble components such as Munc13–1 C₁C₂BMUNC₂C and excess SNAP-25 (step A), and Ca²⁺ is added after 5 min (step B). Unlabeled streptavidin (gray) is included outside the vesicles to ensure that the content mixing signal does not arise from leakiness.