Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains.
The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle -the fusion pore- can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, "flipped" t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.
Department of Cellular and Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.|Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, CT, 06516, USA.
Zhenyong
Z
Wu
Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, CT, 06516, USA.|Department of Cell Biology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.
Sarah M
SM
Auclair
Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, CT, 06516, USA.|Department of Cell Biology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.
Oscar
O
Bello
Department of Cellular and Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.|Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, CT, 06516, USA.
Wensi
W
Vennekate
Department of Cellular and Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.|Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, CT, 06516, USA.
Natasha R
NR
Dudzinski
Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, CT, 06516, USA.|Department of Cell Biology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.
Shyam S
SS
Krishnakumar
Department of Cellular and Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.|Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, CT, 06516, USA.|Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, CT 06520, USA.|Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS) UMR8250, 45, rue des Saints Pères, 75270 Paris Cedex 06, France.
Erdem
E
Karatekin
D002118:Calcium / Q000378:metabolism; D002459:Cell Fusion / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism*; D005089:Exocytosis; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion; D018377:Neurotransmitter Agents; D011485:Protein Binding; D000072417:Protein Domains; D050600:SNARE Proteins / Q000737:chemistry* / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism
D018377:Neurotransmitter Agents; D050600:SNARE Proteins; D002118:Calcium