Unlike in a whole cell with multiple proteins and protein complexes, this pure phospholipid membrane, alone or in association with SNAREs, is much simpler to study. All AFM images of such lipid membrane and membrane-associated SNARE arrangements were obtained using different AFM imaging forces and cantilevers, confirming similar size, structure, and arrangement of SNAREs. Purified recombinant t- and v-SNARE proteins, when applied to a lipid membrane, form globular complexes (Fig. 1, A-D) ranging in size from 30 to 100 nm in diameter and 3 to 15 nm in height when examined using AFM. Section analysis of the t-SNARE complexes (Fig. 1 D) in a lipid membrane, before (Fig. 1 B) and after (Fig. 1 C) addition of v-SNARE, demonstrate changes in both shape and size of the complexes. A 5% increase in diameter and 40% increase in height were seen after addition of v-SNARE to the t-SNARE complexes in the lipid membrane. Studies of conductance changes in the bilayer following reconstitution of SNAREs into phospholipid membranes supported the AFM observations. Addition of t-SNAREs to v-SNARE reconstituted lipid membranes did not alter membrane current (Fig. 1 E). Likewise, when t-SNAREs were added to the lipid membrane before addition of v-SNARE, (t-SNAREs were brushed onto the lipid bilayer in the chamber followed by addition of v-SNARE), no change in the baseline current of the bilayer membrane was observed (Fig. 1 F).
In contrast to the SNARE complex formed when t-/v-SNAREs were added to the same bilayer, t-SNAREs and v-SNARE in opposing bilayers interact and arrange in circular arrays, forming pore-like structures (Fig. 2, A-D). These pores are conducting, because some vesicles have discharged their contents and appear flattened (Fig. 2 B), measuring 10-15 nm in height as compared to the 40-60 nm size of intact vesicles (Fig. 2 A). Because the t-/v-SNARE complex lies between the opposing bilayers, the discharged vesicles clearly reveal t-/v-SNAREs forming a rosette pattern with a dimple or pore-like depression at the center (Fig. 2, B, C, and D). On the contrary, unfused v-SNARE vesicles associated with the t-SNARE reconstituted lipid membrane reveal only the vesicle profile (Fig. 2 A). These studies demonstrate that the t-/v-SNARE arrangement is in a circular array, with a pore-like structure formed at the center of the complex.
To determine whether the pore-like structures were capable of fusing the opposing bilayers, changes in current across the bilayer were examined. T-SNARE vesicles containing the antifungal agent nystatin, and the cholesterol homolog ergosterol, where added to the cis side of the chamber containing v-SNARE in the bilayer membrane. Nystatin, in the presence of ergosterol, forms a cation-conducting channel in lipid membranes (Woodbury and Miller, 1990
; Cohen and Niles, 1993; Kelly and Woodbury, 1996; Woodbury, 1999). When vesicles containing nystatin and ergosterol incorporate into an ergosterol-free membrane, a current spike can be observed because the nystatin channel collapses as ergosterol diffuses into the lipid membrane (Cohen and Niles, 1993; Kelly and Woodbury, 1996; Woodbury, 1999). As a positive control, a KCl gradient was established to test the ability of vesicles to fuse at the lipid membrane (410 mM cis: 150 mM trans). The KCl gradient provided a driving force for vesicle incorporation that was independent of the presence of SNARE proteins (Kelly and Woodbury, 1996). When t-SNARE vesicles were exposed to v-SNARE reconstituted bilayers, vesicles fused (Fig. 2 E). Fusions of t-SNARE-containing vesicles with the membrane were observed as current spikes as described.
To verify whether the pore-like structures were continuous across the membrane, capacitance and conductance measurements of the membrane were performed (Fig. 3 A). Phospholipid vesicles that come in contact with the bilayer membrane do not readily fuse with the membrane. When phospholipid vesicles were added to the cis side of the bilayer chamber containing v-SNARE in the membrane, a small increase in capacitance was observed with little or no further increase. Simultaneously, an increase in conductance was also observed with little or no further increase over a 5-min period. The increase and no further change in conductance or capacitance is consistent with vesicles making contact with the membrane but not fusing (Fig. 3 B). These vesicles were fusogenic because of a salt (KCl) gradient across the bilayer membrane, inducing fusion of vesicles with the lipid membrane. When t-SNARE vesicles containing nystatin and ergosterol were added to the cis side of the bilayer chamber, an initial increase in capacitance and conductance occurred followed by a stepwise increase in both membrane capacitance and conductance (Fig. 3 C), along with several fusion events observed as current spikes in separate recordings. (Fig. 2 E). The stepwise increase in capacitance suggests that the t-SNARE vesicles dock and are continuous with the bilayer membrane. The simultaneous increase in membrane conductance is most likely a reflection of the increase in membrane charge associated with the docked vesicles and only secondarily due to open vesicle-associated nystatin channels that are conducting through SNARE-induced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane. SNARE-induced fusion occurred at an average rate of four t-SNARE vesicle incorporations every 5 min into the v-SNARE reconstituted bilayer without osmotic pressure, compared to six vesicles using a KCl gradient (n = 7).
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