…………………………………………..…………………………………………..
FIGURE 1 AFM micrographs and force plots of mica and lipid surface and of SNAREs on lipid membrane. (A) AFM performed on freshly cleaved mica (left) and on lipid membrane formed on the same mica surface (right), demonstrating differences in the force-versus-distant curves. Note the curvilinear shape exhibited in the force-versus-distant curves of the lipid surface in contrast to mica. Three dimensional AFM micrographs of neuronal t-SNAREs deposited on the lipid membrane (B), and after the addition of v-SNARE (C). Section analysis of the SNARE complex in (B) and (C) is depicted in (D). Note that the smaller curve belonging to the t-SNARE complex in (B) is markedly enlarged after addition of v-SNARE. Artificial bilayer lipid membranes are nonconducting either in the presence or absence of SNAREs (E, F). Current verses time traces of bilayer membranes containing proteins involved in docking and fusion of synaptic vesicles while the membranes are held at 
60 mV (current/reference voltage). (E) When t-SNAREs are added to the planar lipid bilayer containing the synaptic vesicle protein, VAMP-2, no occurrence of current spike for fusion event at the bilayer membrane is observed (n = 7). (F) Similarly, no current spike is observed when t-SNAREs (syntaxin 1A-1 and SNAP25) are added to the cis side of a bilayer chamber, following with VAMP-2. Increasing the concentration of t-SNAREs and VAMP-2 protein.
…………………………………………..…………………………………………..
FIGURE 2 Pore-like structures are formed when t-SNAREs and v-SNARE in opposing bilayers interact. (A) Unfused v-SNARE vesicles on t-SNARE reconstituted lipid membrane. (B) Dislodgement or fusion of v-SNARE-reconstituted vesicles with a t-SNARE-reconstituted lipid membrane, exhibit formation of pore-like structures due to the interaction of v- and t-SNAREs in a circular array. The size of the pores range between 50 and 150 nm (B-D). Several 3D AFM amplitude images of SNAREs arranged in a circular array (C) and some at higher resolution (D), illustrating a pore-like structure at the center is depicted. Scale bar is 100 nm. Recombinant t-SNAREs and v-SNARE in opposing bilayers drive membrane fusion. (E) When t-SNARE vesicles were exposed to v-SNARE reconstituted bilayers, vesicles fused. Vesicles containing nystatin/ergosterol and t-SNAREs were added to the cis side of the bilayer chamber. Fusion of t-SNARE containing vesicles with the membrane observed as current spikes that collapse as the nystatin spreads into the bilayer membrane. To determine membrane stability, the transmembrane gradient of KCl was increased, allowing gradient driven fusion of nystatin-associated vesicles.
…………………………………………..…………………………………………..
FIGURE 3 Opposing bilayers containing t- and v-SNAREs respectively, interact in a circular array to form conducting pores. (A) Schematic diagram of the bilayer-electrophysiology setup. (B) Lipid vesicle containing nystatin channels (red) and both vesicles and membrane bilayer without SNAREs, demonstrate no significant changes in capacitance and conductance. Initial increase in conductance and capacitance may be due to vesicle-membrane attachment. To demonstrate membrane stability (both bilayer membrane and vesicles), the transmembrane gradient of KCl was increased to allow gradient-driven fusion and a concomitance increase of conductance and capacitance. (C) When t-SNARE vesicles were added to a v-SNARE membrane support, the SNAREs in opposing bilayers arranged in a ring pattern, forming pores (as seen in the AFM micrograph on the extreme right) and there were seen stepwise increases in capacitance and conductance (60 mV holding potential). Docking and fusion of the vesicle at the bilayer membrane, opens vesicle-associated nystatin channels and SNARE-induced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane. Then further addition of KCl to induce gradient-driven fusion resulted in little or no further increase in conductance and capacitance, demonstrating that docked vesicles have already fused.
…………………………………………..…………………………………………..