Putative intermediates of membrane fusion and their transitions. Transitions that are less relevant or speculative are indicated by dashed arrows. Protein elements are colored green. (1) A vesicle approaches the plasma membrane. (2) Proteins hold the vesicle and plasma membrane together, either through separate contacts (a) or through one central contact (b). (3) A proteinaceous fusion pore could form from a central contact as in 2b. (4) Lipid mixing of the outer (proximal) leaflets can begin, first through the formation of a stalk (4a) and then through the formation of an extended hemifusion diaphragm (4b) in which the fused proximal leaflets retract and leave a bilayer formed by the two distal leaflets. (5) A fusion pore formed by a contiguous lipid bilayer curved into an hourglass-like shape. (6) A greatly expanded lipid fusion pore on the way to complete merger of the plasma and vesicle membranes.
A structural model of a synaptophysin–synaptobrevin complex. The model was based on a study by Adams et al. (2015) and generated with PyMOL using a pdb file provided by M. Stowell. (A) The complete complex viewed from the vesicle lumen shows 6 synaptophysins (green) and 12 synaptobrevins (red) in a hexagonal formation. 12 synaptophysin TMDs and 12 synaptobrevin TMDs face inward and could line a fusion pore. (B) The TMDs of two synaptophysin and two synaptobrevin molecules are viewed from within the plane of the membrane. Residues highlighted in yellow were implicated as pore liners by amperometry experiments with synaptobrevin TMD mutants (Chang et al., 2015); synaptobrevin residues 99 and 103 are highlighted on one chain, and residues 101 and 105 are highlighted on the other (compare with Fig. 4 B).
Impedance and amperometry measurements of fusion pores can be interpreted in terms of three successive stages of membrane fusion. (1) Contact (top left), (2) fusion pore opening (top middle), and (3) fusion pore expansion (top right). (left) Impedance recording reveals fusion pore openings as a change in the complex impedance of a patch of membrane to which a vesicle fuses. The imaginary component of the impedance (blue) and real component (red) are used to calculate the fusion pore conductance, γ (green; Lollike et al., 1995). The opening of a fusion pore produces an initial conductance increase, and fusion pore expansion increases the conductance further. (right) Amperometry recording reveals a fusion pore opening as a pre-spike foot (shaded), which represents the flux of catecholamine out of the vesicle at a limited rate. Fusion pore expansion allows content release much more rapidly to produce an amperometric spike.
SNARE TMD residues lining the fusion pore. (A) Residues in the SNARE TMDs identified by various fusion pore measurements are highlighted in green. The first TMD residue of the wheel is labeled with a red arrow. Amperometry identified some residues (Han et al., 2004; Chang et al., 2015). Glutamate flux and labeling by MTSES identified some of the same residues as well as some different residues (Bao et al., 2016). Many of the residues identified in a given assay fell along one face of a helical wheel (wheels generated at http://kael.net/helical.htm). (B) Fusion pore model based on TMD mutagenesis and amperometric pre-spike feet and conductance in endocrine release (Chang et al., 2015).
Ease of formation of a lipidic fusion pore increases with decreasing vesicle size. (A) Smaller vesicles have a larger contact angle, φ, which corresponds to a smaller area of highly curved lipid bilayer (brown). (B) Amperometry traces illustrate that larger vesicles, which contain more catecholamine, have longer duration pre-spike feet (horizontal line segments below the corresponding traces). The integrated amperometric charge in fC quantifies vesicle size. A theoretical model based on A predicts that the logarithm of the inverse pre-spike foot lifetime, τ, is a linear function of the inverse vesicle radius, Rv (proportional to the cube root of the vesicle content), and that the slope depends on the spontaneous curvature of the lipid bilayer (Zhang and Jackson, 2010). (C) Plots from rat chromaffin cells confirm this prediction and show that altering spontaneous curvature with lysophosphatidylcholine (LPC) or oleic acid (OA) changes the slope. (D) Plots from mouse chromaffin cells show that tryptophan mutations in the synaptobrevin TMD alter the slope of these plots (Chang and Jackson, 2015). The effects of both lipids (C) and mutants (D) can be interpreted in terms of changes in spontaneous curvature of the lipid bilayer.
Minimum energy fusion pores. (A) The fusion pore is a surface of revolution around the z axis formed by a lipid bilayer. This shape was obtained by minimizing the mean curvature of two parallel lipid monolayers subject to the constraint of a pore radius Rp = 3.3 nm and a bilayer separation of twice Rb = 5.45 nm (note that these are distances to the center of the bilayer; further note that this minimization omitted the possibility of an inflection that can introduce a bowing shape that reduces the energy further; Yoo et al., 2013). (B) Minimum energies determined for Rb = 3 nm and varied Rp. This plot shows a stable minimum at Rp = ∼2.5 nm. The spontaneous curvature of the lipid (C0) has a major influence on the energy but not on the position of the minimum. (C) Varying both Rp and Rb reveals a global minimum at the location indicated by the circle, at Rp = 2.75 nm and Rb = 4.2 nm (modified from Jackson  with permission from Springer).
A model for transition 3 → 5. (A) The joining of the helical segments of the SNARE motif with the helical TMD is a potential driving force for this transition. In the initial state (top), the SNARE motifs are perpendicular to the TMDs (high energy) and the membranes are flat (low energy). The final state (bottom) has straight SNAREs (low energy) and curved membrane (high energy). (B) As the transition progresses, the hydrocarbon interior becomes transiently exposed to water, creating a barrier to the transition. The angle θ between the TMD and the membrane normal serves as a reaction coordinate for the transition (Jackson, 2010).
The time course of concentration within the synaptic cleft. The concentration directly under the release site was obtained by integrating Eq. 5 at z = 0. α was calculated from Eq. 2 for the indicated values of conductance. N0 = 1,600 (Edwards, 1995) and D = 0.33 µm2 ms−1 (Nielsen et al., 2004). To convert to N to concentration a thickness of the synaptic cleft was taken as 20 nm.
Composite lipid–protein fusion pores. SNAREs are green; synaptophysin TMDs are pink. Models 1–3 illustrate continuity of the proximal monolayer of the vesicle and plasma membrane outside a proteinaceous fusion pore. These models have no contact between phospholipids and the aqueous pore lumen. Models 4–5 illustrate lipid headgroups of a bilayer that forms a pore in which lipid and protein alternate along the walls. Model 6 illustrates protein TMDs lodged among the headgroups of a lipidic fusion pore.