Direct visualization of large and protein-free hemifusion diaphragms

Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several mum in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.