SNARE-Mediated Fusion Pore Dynamics from Quantitative TIRF Microscopy

Abstract

SNARE proteins are involved in most intracellular fusion events. SNAREs drive complete fusion but are also thought to generate dynamic flickering pores and reversible fusion. Live cell amperometry and simultaneous capacitance recordings indicated that the characteristic pre-spike foot may correspond to a flickering pore which later opens completely (Alvarez de Toledo et al., Nature, 1993). Here we study fusion and pore statistics between SNARE-reconstituted vesicles and supported bilayers (SBLs) using quantitative total internal reflection fluorescence microscopy (TIRFM) with single-lipid resolution. Based on the intensities of the fluorescent lipid-labeled vesicles and the course of the time-dependent intensity increase upon fusion as lipids diffuse through the pore into the SBL, we determined vesicle sizes, the delay time to create the initial pore, and the rate of intermembrane lipid mixing through the pore. These measurements required us to develop a quantitative image analysis algorithm which accounts for TIRFM effects including the spatial decay of incident light, polarization effects, fluorescence dequenching and bleaching. In cholesterol-free vesicles, lipid transfer from the vesicle to the SBL was slower than would be expected were the fusion pore fully open, suggesting that the pore flickers between open and closed states. To quantify these effects we developed a mathematical model of the stochastic fusion pore and the passage of lipids from vesicle to SBL through the pore. Combining the model and experimental measurements, we infer the fraction of time for which the pore is open. Without cholesterol, the pore favors the closed state. We find cholesterol has profound effects: it decreases the delay time between docking and fusion and, once formed, the pore remains fully open. Thus, our results suggest that cholesterol favors the open pore state. Additionally, we report measurements of vesicle curvature-dependence of fusion and pore flickering.