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.
CITATION STYLE
Dudzinski, N. R., Wu, Z., & Karatekin, E. (2019). A nanodisc-cell fusion assay with single-pore sensitivity and sub-millisecond time resolution. In Methods in Molecular Biology (Vol. 1860, pp. 263–275). Humana Press Inc. https://doi.org/10.1007/978-1-4939-8760-3_17
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