A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution

Abstract

During exocytosis, vesicles fuse with the plasma membrane and release their contents. The fusion pore is the initial, nanometer-sized connection between the plasma membrane and the cargo-laden vesicle. A growing body of evidence points toward the fusion pore being a regulator of exocytosis, but the shortcomings of current experimental techniques to investigate single-fusion pores make it difficult to study factors governing pore behavior. Here we describe an assay that fuses v-SNARE-reconstituted nanodiscs with cells ectopically expressing "flipped" t-SNAREs to monitor dynamics of single fusion pores in a biochemically defined system using electrical recordings. We also describe a fluorescence microscopy-based approach to monitor nanodisc-cell fusion that is much simpler to employ, but cannot resolve single pores.

Keywords: Electrophysiology; Exocytosis; Fusion pore; Membrane fusion; Nanodisc; SNAREs.

Figure 1.

The fusion pore is a key intermediate during exocytosis. Left: complex formation between v-SNARE proteins on the vesicle and t-SNARE proteins on the plasma membrane drive fusion between the membranes. This results in the formation of a fusion pore (middle). The initial pore is only 1–3 nm wide and can fluctuate in size, flicker open-closed multiple times, then either reseal or dilate irreversibly (right).

Figure 2.

Fusion of a nanodisc with a cell creates a pore that connects the cytosol to the extracellular medium. Nanodiscs containing v-SNAREs fuse with the membrane of a cell containing flipped t-SNAREs facing outward. A fusion pore is formed that connects the nanodisc membrane to that of the cell. Figure modified from.

Figure 3.

Nanodisc-cell single fusion pore assay. a) A pipette containing v-SNARE nanodiscs is used to establish an on-cell patch on a flipped t-SNARE cell. Discs that are initially layered on top of a disc-free solution in the pipette diffuse to the cell surface. When a disc that reaches the cell surface fuses with it, a fusion pore is formed. (Figure from). b) An example of a fusion pore current burst (using 16 v-SNARE copies per disc). The threshold for detecting open-pore sub-periods is indicated as the dashed red line just below the baseline. Current must be <−0.25 pA for 60 ms or longer to be considered due to an open pore. Burst open sub-states are indicated by colored lines above the pore. c) a second example of a fusion pore current burst, as in b.