SNARE-mediated membrane fusion trajectories derived from force-clamp experiments

Marieelen Oelkers, Hannes Witt, Partho Halder, Reinhard Jahn, Andreas Janshoff*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomicforce microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.

Original languageEnglish
Pages (from-to)13051-13056
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number46
DOIs
Publication statusPublished - 15 Nov 2016
Externally publishedYes

Keywords

  • AFM
  • Force spectroscopy
  • Hydration barrier
  • Membrane fusion
  • SNARE

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