Zhenyong Wu, Ph.D.
Ph.D., Zhejiang University School of Medicine (2012)
Fusion pores play a critical role in cellular activities. Successful release of secretory vesicle contents (neurotransmitters, enzymes, or hormones) is dependent on the formation of a fusion pore (the initial, nanometer-sized connection) between the vesicle and the plasma membrane. The nature of single fusion pores has been the subject of very limited biophysical, biochemical and biological investigations, mainly because of a lack of suitable methods.
To address these questions, we established a novel single fusion pore real time recording assay to directly observe SNARE-meditated fusion pores. SNAREs mediate most intracellular fusion reactions, including synaptic or secretory vesicle exocytosis which underlie neurotransmitter and hormone release. We isolate single flickering pores connecting v-SNARE-reconstituted nanodiscs (vNDs) to a cell ectopically expressing cognate, “flipped” t-SNAREs (tCell), which is voltage-clamped in the cell-attached configuration. For this, we first incorporate the neuronal v-SNARE VAMP2 proteins into soluble lipid discs (~16 nm in diameter). The v-SNARE reconstituted nanodiscs (vNDs) are then added into the patch pipette while a flipped t-SNARE cell is patch-clamped. vNDs slowly diffuse to the patched cell surface where they interact with the outward-facing t-SNAREs present on the cell surface. Fusion of a vND with the cell surface generates a pore that connects the cytosol to the pipette solution, just like ion channels do. Currents that flow through the pore report pore dynamics with sub-millisecond time resoltuion.
Using this assay, single fusion pore dynamics are easily and directly detected. Collaborating with the Rothman lab, we studied the role of v- and t-SNARE transmembrane interactions in pore nucleation and lifetimes (Z. Wu, S. M. Auclair, O. Bello, W. Vennekate, S. Krishnakumar, and E. Karatekin, submitted). We are currently studying how SNAREs cooperate at different stages of pore nucleation and dilation.
Detection of single fusion pores between v-SNARE nanodiscs (vNDs) and flipped t-SNARE cells (tCells).
- Schematic of the setup and vND-tCell fusion.
- A pipette is gigaohm sealed onto the tCell to isolate a patch of membrane in the cell-attached configuration.
- Representative fusion pore currents