Direct Visualization of Large and Protein-Free Hemifusion Diaphragms

Abstract

Membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. As supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (HD) and subsequently open to a fusion pore. Direct experimental verification of the HD is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. Time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the GUV-GUV contact region being of several Âμm in size. A detailed analysis showed that this structure is consistent with the formation of an HD. A quantitative model of the hemifusion equilibrium and kinetics of the growing HD was developed. Bilayer tension could be shown to drive HD expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon HD growth. The model and its predictions fit nicely with observations above. In addition we are currently investigating the influence of membrane tension on the fusion pathway directly using the GUV system within a micromanipulation approach.