Investigation of SNARE-mediated membrane fusion mechanism using atomic force microscope spectroscopy.

摘要

Membrane fusion is essential for survival in eukaryotic cells. Many physiological processes such as endocytosis and exocytosis are mediated by membrane fusion, which is driven by highly specialized and conserved family of proteins. Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane during neurotransmitter release; however, the mechanism for SNARE-mediated membrane fusion remains to be established. In the current work, we aimed at investigating this mechanism using atomic force microscope (AFM) spectroscopy.;We established an AFM lipid bilayer system, which proved effective in detecting fusion of bilayers and measuring compression forces required to generate fusion. It also revealed that SNARE-mediated membrane fusion proceeds through an intermediate hemifused state. Using this system, we revealed the energy landscape for membrane fusion using a dynamic force approach. We carried out compression force measurements at different compression rates and a significant reduction in the force was observed when SNAREs were present in the bilayers. The results also indicated that a single energy barrier governed membrane fusion in our experimental system. The energy barrier is characterized by its width and height, which determine the slope of the activation potential. With SNAREs in the opposing (trans) bilayers, the width of the barrier increased > 2 fold, which is interpreted as an increase in the compressibility of the membranes and subsequently a greater ease in their deformation and fusion under compression. Moreover, specific perturbations to the SNARE interaction interfered with the observed facilitation of membrane fusion, which indicated the involvement of SNAREs in the observed fusion facilitation and increase in the fusion rate. Furthermore, dissociation kinetics analysis of the SNARE interaction revealed a strong binding force during trans SNARE-complex formation, and a correlation between the strength of the SNARE interaction and the degree of fusion facilitation was established.;In conclusion, the present findings provide support for a mechanism for SNARE-mediated membrane fusion, where trans-interaction between SNAREs provides close apposition of the membranes and reduces fusion energy requirements by locally destabilizing the bilayers, in which the SNAREs are anchored, through pulling on or tilting of their transmembrane segments.