Complexin arrests a pool of docked vesicles for fast Ca2+-dependent release

Abstract

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger--a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.

Figure 1

Fast Ca²⁺-dependent liposome fusion requires the combined function of CpxII and Syt1. (A) Effect of Syt1 and CpxII on liposome fusion kinetics. v-SNARE or v-SNARE/Syt1-SUVs (2.5 nmol lipid, 12.5 pmol VAMP2, 3.1 pmol Syt1), labelled with rhodamine and NBD lipids were mixed with unlabelled t-SNARE-GUVs (14 nmol lipid, 14 pmol syntaxin 1/SNAP-25) in the absence or presence of 600 pmol CpxII in a final volume of 100 μl and the increase in NBD fluorescence was monitored. After 5 min at 37°C, Ca²⁺ was added to a final concentration of 100 μM and the measurement continued for another 20 min. The results were normalized to the maximum NBD fluorescence signal after detergent lysis as described in Materials and methods. (B) Comparison of the initial Ca²⁺-dependent fusion stimulation in the presence and absence of Syt1 and CpxII. The % increase of the fluorescent signal was determined for each reaction by subtracting the value before the addition of the Ca²⁺ trigger (minute 5) from the value reached 1 min later. Error bars indicate s.e.m. (n=3).

Figure 2

CpxII affects membrane fusion of v-SNARE/Syt1-SUVs with t-SNARE-GUVs. (A) In the presence of CpxII, v-SNARE/Syt1-SUVs accumulate on the surface of t-SNARE-GUVs. Liposomes were mixed in the presence of CpxII and incubated for 1 h on ice to maximize docking. A representative cryoEM picture shows docked v-SNARE/Syt1-SUVs. Scale bar indicates 100 nm. (B) CpxII blocks membrane fusion of v-SNARE/Syt1-SUVs with t-SNARE-GUVs. t-SNARE-GUVs were incubated with v-SNARE-Syt1-SUVs in the absence or presence of CpxII for 5 min at 37°C, and analysed by cryoEM. Total numbers of SUVs visible in five different images collected from three different cryoEM grids were counted. Statistics were performed ‘blind’. (C) Accumulation of v-SNARE/Syt1-SUVs on the surface of t-SNARE-GUVs caused by the presence of CpxII. The bar diagram shows the quantification of the numbers of SUVs docked to the surface of GUVs in the absence or presence of CpxII subsequent to the 5-min incubation at 37°C (five different images collected from three different cryoEM grids). (D) Ca²⁺ triggers efficient fusion of the docked v-SNARE/Syt1-SUVs. Liposomes were incubated in the presence of CpxII under the following conditions and subsequently plunge frozen: (i) 1 min at 4°C; (ii) 5 min at 37°C; (iii) 5 min at 37°C, followed by the addition of 100 μM Ca²⁺ for 1 min at 37°C. The number of SUVs docked to GUV membranes in each sample was counted and the data were normalized to the total number of SUVs present in SUV-GUV incubations under non-fusogenic conditions (1 min at 4°C). Error bars indicate s.e.m. (n=5).

Figure 3

A CpxI construct lacking the inhibitory accessory α-helix does not inhibit Ca²⁺-independent lipid mixing. t-SNARE-GUVs were incubated with v-SNARE-Syt1-SUVs in the absence or the presence of wild-type CpxI (CpxI wt) or a CpxI construct (CpxI aa 41–134), lacking 40 amino acids at the amino terminus. After 5 min at 37°C, Ca²⁺ was added at a final concentration 100 μM. Lipid mixing was analysed as described in Materials and methods.

Figure 4

Ca²⁺-dependent stimulation of lipid mixing by Syt1 requires an intact Ca²⁺-binding site in the C2B domain of Syt1. t-SNARE-GUVs were incubated with CpxII and v-SNARE-SUVs, containing either Syt1 wt (A) or a Syt1 construct containing the double mutation D303/309N (B). After 5 min at 37°C, Ca²⁺ was added at the indicated concentrations. Lipid mixing was monitored and analysed as described in Materials and methods.

Figure 5

Ca²⁺-independent stimulation of lipid mixing by Syt1 requires an intact polybasic motif in the C2B domain of Syt1. t-SNARE-GUVs were incubated with v-SNARE-SUVs, containing either Syt1 wt or a Syt1 construct containing the triple mutation K326, 327, 331Q in the presence or absence of CpxII. Fusion reactions were monitored in the presence (A) or absence (B) of PI(4,5)P2 and analysed as described in Materials and methods.

Figure 6

Docking of SUVs to t-SNARE-GUVs requires an intact polybasic motif in the C2B domain of Syt1. ³H-DPPC labelled SUVs (7.5 nmol lipid) containing the indicated proteins (9.4 pmol Syt1 and/or 38 pmol VAMP2) were mixed with t-SNARE-GUVs (42 nmol lipid, 42 pmol syntaxin 1/SNAP-25) in a final volume of 200 μl in the absence or presence of 20 μM Ca²⁺ and incubated for 5 min at 4°C to allow docking. Subsequently, GUVs were isolated by centrifugation for 5 min at 5000 g and the fraction of bound ³H-labelled SUVs in the pellet was quantified. Counts obtained from control incubations containing protein-free SUVs were subtracted from individual measurements. (A) Illustration of the experimental set-up. (B) Quantification of bound SUVs normalized to 100% SUV input. Error bars indicate s.e.m. (n=3).

Figure 7

Effect of soluble Syt1 C2 domains on lipid mixing. (A) Comparison of lipid mixing reactions containing various soluble Syt1 C2 domains with a reaction containing membrane-anchored Syt1 (Syt1 TMD). t-SNARE-GUVs and v-SNARE-SUVs were incubated with CpxII in the presence of 6 μM soluble Syt1 C2A and C2B domains, which were added separately, in combination, or joined by their native linker. In the reaction, membrane-anchored Syt1 (Syt TMD) is present at 31 nM. (B) The soluble Syt1 C2B domain inhibits lipid mixing of v-SNARE/Syt1-SUVs with t-SNARE-GUVs. t-SNARE-GUVs and v-SNARE/Syt1-SUVs were incubated with 6 μM of the indicated Syt1 C2 constructs. After 5 min at 37°C, Ca²⁺ was added to a final concentration of 100 μM. Lipid mixing was monitored and analysed as described in Materials and methods.