SNARE-mediated membrane fusion trajectories derived from force-clamp experiments.
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 atomic-force 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.
Institute of Physical Chemistry, University of Göttingen, 37077 Goettingen, Germany.
Marieelen
M
Oelkers
Institute of Physical Chemistry, University of Göttingen, 37077 Goettingen, Germany.
Hannes
H
Witt
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany.
Partho
P
Halder
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany.
Reinhard
0000-0003-1542-3498
R
Jahn
Institute of Physical Chemistry, University of Göttingen, 37077 Goettingen, Germany; ajansho@gwdg.de.
Andreas
A
Janshoff
D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008561:Membrane Fusion; D008853:Microscopy / Q000379:methods; D050600:SNARE Proteins / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry
D008051:Lipid Bilayers; D008081:Liposomes; D050600:SNARE Proteins; D012822:Silicon Dioxide
AFM; SNARE; force spectroscopy; hydration barrier; membrane fusion