A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution
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.
Key wordsMembrane fusion Exocytosis Nanodisc SNAREs Fusion pore Electrophysiology
We thank all members of the Karatekin laboratory for stimulating discussions, D. Zenisek and F. Sigworth (Cellular and Molecular Physiology, Yale University) for expert advice and discussions, and James E. Rothman, Oscar Bello, Shyam Krishnakumar, and other members of the Rothman laboratory (Cell Biology, Yale University) for critical advice and introducing us to the use of nanodiscs. This work was supported by the National Institute of General Medical Sciences (grant R01GM108954), and a Kavli Foundation Neuroscience Scholar Award (to EK). NRD was supported by NIH Training Grant T32 NS41228 funded by the Jointly Sponsored NIH Predoctoral Training Program in the Neurosciences.
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