SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion

Abstract

Synaptic vesicles fuse with the plasma membrane in response to Ca(2+) influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large-scale, automated cryo-electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca(2)(+)-triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high-energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.

Figure 1

Observation of docked vesicles on the target membrane.

1. A schematic representation of the in vitro system employed in this study.

2. Kinetics of fusion reactions monitored by a plate reader assay for lipid mixing (see Supplementary Methods). Upon the addition of Ca²⁺, docked and primed vesicles fuse almost instantaneously as evident by a sudden increase in fluorescence signal indicating fast lipid mixing. Reaction mixtures contained or lacked the indicated components. All: all components (syntaxin1/SNAP-25, VAMP2, synaptotagmin 1, complexin, and Munc18) were present in the fusion assay; empty SUVs: protein-free SUVs lacking VAMP2 and synaptotagmin 1 were used; +CD-VAMP2: the cytoplasmic domain of VAMP2 was added to the reaction containing all components.

3. Slice through a representative cryo-electron tomogram showing large populations of SUVs docked onto the target membrane in a reaction containing SNARE proteins, synaptotagmin 1, complexin and Munc18-1. Large populations of docked vesicles were not observed in a control reaction with empty SUVs. See also Supplementary Figs S1, S2A and Movie S1.

Figure 2

Large-scale automated cryoET and blind quantification of major membrane morphologies observed.

1. A–C Cartoons showing the three major types of membrane morphologies observed where SUVs were proximal to GUVs.

2. D–F Tomographic slices showing examples of (A-C). See also Supplementary Fig S2B for a gallery of the protruding target membrane intermediate.

3. G  Blind quantification (see Supplementary Methods) of the instances of each morphology observed in three independent biological replicates of each sample. Raw data are shown in Table 1. Error bars represent the 95% confidence intervals for each observed proportion (see Supplementary Methods and Supplementary Table S1–S2 for details of statistical analysis). The ratio of undocked (gray bar) to docked (red, blue and orange bars) vesicles was quantified using low-magnification images and the ratio of “protrusion”, “contact” and “extended” contact vesicles was quantified using high-magnification cryoET data (see also Supplementary Figs S1, S2 and S3).

Figure 3

Protrusion formation correlates with priming and membrane fusion. Reactions containing VAMP2/synaptotagmin 1 SUVs were mixed on ice with t-SNARE GUVs in the presence of complexin, but in the absence of Munc18.

1. Rate of docking assayed by a liposome sedimentation assay described in [23] at different time points. Docking was found to increase as a function of time. Error bars indicate s.e.m. (n = 3).

2. Kinetics of the fusion reaction at 37°C monitored by a plate reader assay for lipid mixing. Error bars indicate s.e.m. (n = 3).

3. Change in fluorescence signal 10 s after induction with Ca²⁺ for the same samples. The extent of priming was seen to increase as a function of time. Error bars indicate s.e.m. (n = 3).

4. Samples were prepared on ice and plunge-frozen for cryoEM at different time points at 4°C. Representative tomographic slices from these experiments are shown (n = 1 for each time point).

5. Quantification of randomized data from one sample at each time point (see Supplementary Methods) shows that both docking and protrusion formation increase as a function of time. Undocked vesicles (gray), contact (blue), extended contacts (orange), protrusions (red).