Figure 6. The Munc13-1 C₁C₂BMUNC₂C fragment bridges V-liposomes to T-liposomes.

(A,C,E,G) Intensity autocorrelation curves measured by DLS after 3 min incubations at 20°C on samples containing: (A) V-vesicles alone or in the presence of C₁C₂BMUNC₂C and 100 μM EGTA or 500 μM Ca²⁺; (C) T-vesicles alone or in the presence of C₁C₂BMUNC₂C and 100 μM EGTA or 500 μM Ca²⁺; (E) plain vesicles containing no PS alone or in the presence of C₁C₂BMUNC₂C and 100 μM EGTA or 500 μM Ca²⁺, or a 1:1 mixture of plain vesicles and T-vesicles in the presence of C₁C₂BMUNC₂C and 100 μM EGTA; (G) V-vesicles alone, T-vesicles alone, or 1:1 mixtures of V- and T-vesicles in the presence of C₁C₂BMUNC₂C and 100 μM EGTA or 500 μM Ca²⁺. (B,D,F,H) Bar diagrams showing the particle size distribution in samples containing: (B) V-vesicles in the presence of C₁C₂BMUNC₂C and 100 μM EGTA; (D) T-vesicles alone or in the presence of C₁C₂BMUNC₂C and 100 μM EGTA; (F) a 1:1 mixture of plain vesicles and T-vesicles in the presence of C₁C₂BMUNC₂C and 100 μM EGTA; (H) a 1:1 mixture of V- and T-vesicles in the presence of C₁C₂BMUNC₂C and 100 μM EGTA. These bar diagrams correspond to the autocorrelation curves of selected samples among those shown in (A,C,E,G) and are intended to illustrate that mixtures of clustered and non-clustered vesicles can be readily distinguished (F), and that Ca²⁺-free C₁C₂BMUNC₂C does not cluster isolated V-vesicles (B) but bridges V- to T-vesicles (H).

DOI: http://dx.doi.org/10.7554/eLife.13696.016

Figure 6—figure supplement 1. Concentration dependence of the liposome clustering activity of Munc13-1 C₁C₂BMUN and C₁C₂BMUNC₂C.

The diagrams show intensity autocorrelation curves measured by DLS after 3 min incubations at 30°C for mixtures of V- and T-liposomes at the same concentrations used for lipid and content mixing assays (0.125 and 0.25 mM lipids, respectively) in the presence of 0.1 mM EGTA and different concentrations of C₁C₂BMUN (A) or C₁C₂BMUNC₂C (B).

DOI: http://dx.doi.org/10.7554/eLife.13696.017

Figure 6—figure supplement 2. Lipid binding to distinct Munc13-1 fragments monitored by liposome co-floatation assays.

(A) Analysis of Munc13-1 fragments that co-float with liposomes by SDS-PAGE and coomassie blue staining. The four lanes on the left correspond to the co-floatation assays. The four lanes on the right show loading controls (1 μg of protein) for quantification. (B) Relative binding of Munc13-1 C₁C₂BMUNC₂C with respect to C₁C₂BMUN measured by co-floatation experiments. Band intensities were quantified with ImageJ and normalized with the corresponding control. The calculated values were further normalized with the average value obtained for C₁C₂BMUN. The diagram shows averages and standard deviations from triplicate experiments.

DOI: http://dx.doi.org/10.7554/eLife.13696.018

Figure 6—figure supplement 3. Ca²⁺-free C₁C₂BMUNC₂C does not cluster V-liposomes.

(A,B) Intensity autocorrelation curves measured by DLS after 3 min incubations at 30°C on samples containing: (A) V-vesicles alone or in the presence of C₁C₂BMUNC₂C and 100 μM EGTA or 500 μM Ca²⁺; (B) T-vesicles alone or in the presence of C₁C₂BMUNC₂C and 100 μM EGTA or 500 μM Ca²⁺.

DOI: http://dx.doi.org/10.7554/eLife.13696.019

Figure 6—figure supplement 4. Minimal stimulation of lipid mixing between V- and T-liposomes in the presence of C₁C₂BMUNC₂C at 20°C.

Lipid mixing assays between V- and T-liposomes in the presence of C₁C₂BMUN and 100 μM EGTA were performed as in Figure 1 at 20 or 37°C without addition of Ca²⁺.

DOI: http://dx.doi.org/10.7554/eLife.13696.020