Atomic force microscope spectroscopy reveals a hemifusion intermediate during soluble N-ethylmaleimide-sensitive...

This study investigated the effect of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptors (SNAREs) on the fusion of egg L-alpha-phosphatidylcholine bilayers using atomic force microscope (AFM) spectroscopy. AFM measurements of the fusion force under compression were acquired to reveal the energy landscape of the fusion process. A single main energy barrier governing the fusion process was identified in the absence and presence of SNAREs in the bilayers. Under compression, a significant downward shift in the fusion dynamic force spectrum was observed when cognate v- and t-SNAREs were present in the opposite bilayers. The presence of vesicle-associated membrane protein (VAMP) and binary syntaxin and SNAP 25 in the apposed bilayers resulted in a reduction in the height of the activation potential by approximately 1.3 k(B)T and a >2-fold increase in the width of the energy barrier. The widening of the energy barrier in the presence SNAREs is interpreted as an increase in the compressibility of the membranes, which translates to a greater ease in the bilayer deformation and subsequently the fusion of the membranes under compression. Facilitation of membrane fusion was observed only when SNAREs were present in both bilayers. Moreover, addition of the soluble cytoplasmic domain of VAMP, which interferes with the interaction between opposing v- and t-SNAREs, prevented such facilitation. These observations implicated the interaction between the cytoplasmic domains of opposing SNAREs in the observed fusion facilitation, possibly by destabilizing the bilayers through pulling on their transmembrane segments. Our AFM compression measurements revealed that SNARE-mediated membrane fusion proceeded through a sequence of two approximately 5 nm collapses of the membrane, an observation that is consistent with the existence of a hemifused state during the fusion process.