The Fusion Pore Lifetime during SNARE Mediated Fusion of Dense Core Vesicles with t-SNARE Containing Supported Membranes can be Modulated by Asymmetric Lipid Distributions

Abstract

The two leaflets of the plasma membrane contain different lipid compositions. Phosphatidylethanolamine (PE) is predominantly located in the inner cytosolic leaflet and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. Docking and fusion of neuroendocrine dense core vesicles (DCVs) purified from PC12 cells with supported membranes that contain the neuronal SNARE proteins syntaxin 1a and SNAP-25A can be observed by recording the fluorescence of mRuby-tagged neuropeptide Y in a total internal reflection microscope (TIRFM). Single DCV fusion events can be identified by distinct characteristic time-dependent fluorescence line shapes. These line shapes can be simulated by a mathematical model that includes the opening of a stable fusion pore followed by the collapse of the vesicle into the plane of the supported membrane. We prepared supported t-SNARE membranes with different asymmetric PE distributions between the two leaflets and analyzed the life-time of the fusion pore and the fusion probabilities. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.