Whether bipolar lipid membranes can undergo fusion or not and how such behavior is related to the conformations (O vs U) of lipids has been a subject of intense investigation. Leriche and coworkers demonstrated that vesicles composed purely of an acyclic bipolar lipid can undergo Ca 2+ -induced fusion without loss of contents. They further probed lipid ordering using 2 H-NMR and concluded that the bipolar lipids adopt both O-and U-shapes in membranes although precise percentages were not determined. H-NMR experiments on model acyclic bipolar lipid membranes suggest that 90% of the lipids exist in spanning or O-shape and 10% in U-shape. Many studies on archaeal lipid mixtures attempted to establish a rational link between the lipid structures and membrane fusion (or the lack thereof) induced by Ca 2+ or PEG. For example, Relini et al. noted that vesicles formed from archaeal lipid extracts which displayed transformations between lamellar and non-lamellar phases underwent fusion implying that lipids that can form non-lamellar phases are necessary to induce membrane curvature during fusion. In previous sections, we showed that under the influence of temperature, salt concentration, pH, or divalent cations, GDGTs show diverse mesophases, especially the formation of inverted non-lamellar phases such as hexagonal and cubic via dehydration of headgroups. Although those lamellar to non-lamellar phase transitions were observed in bulk lipid dispersions, the presence of such lipids in vesicle membranes likely increase the propensity to form local transient non-lamellar structures essential for fusion to take place. Therefore, we asked whether the tendency of GDGT lipids to form non-lamellar phases is related to membrane fusion. To test the fusion behavior of GDGT vesicles, we chose influenza virus as a fusogenic partner as the latter possesses an efficient fusion machinery embedded in a bilayer lipid membrane that triggers a fusion pathway representative of many biological fusion processes. 51 We followed a widely utilized fluorescence microscopy assay to observe fusion of viruses with lipid vesicles on a single particle level. In this method, the vesicles are allowed to fuse with bound influenza virus labeled with selfquenching concentrations of Texas Red-DHPE by acidification of the medium (Figure ). 100 nm vesicles composed overwhelmingly of GDGT lipids (PG2 mix -PG2:GD1a:Biotin-DPPE:Atto 647N-DMPE 98.45:1:0.5:0.05; PG1Glc mix -PG1Glc:GD1a:Biotin-DPPE:Atto 647N-DMPE 98.45:1:0.5:0.05 by molar ratio) were prepared in a pH 7.4 buffer. The vesicles were tethered to a functionalized glass slide through interaction of neutravidin with Biotin-DPPE (Figure ). Next, influenza virus particles labeled with a self-quenching concentration of Texas Red-DHPE were added. The α2,3-silaic acid containing sialoglycolipid GD1a served as the receptor for influenza hemagglutinin binding. The pH was lowered by flowing in an acidic buffer (pH 5.1). Upon hemifusion, the outer leaflets of viral and vesicle membranes merge and the Texas Red-DHPE gets diluted. Consequently, its fluorescence turns on as evident from a sudden spike in fluorescence intensity (Figure ). Interestingly, we observed on a single particle level that many dim fluorescent spots (corresponding to a virion labeled with self-quenched TR-DHPE) turned brighter (Figure ). We plotted the time to the onset of lipid mixing into cumulative distribution function for many vesicle populations and found that the fusion of GDGT vesicles follow a kinetics comparable to that with vesicles composed of monopolar phospholipids and cholesterol ("Phospholipid mix") (Figure ). Next, we asked whether the fusion events led to full fusion or whether they were arrested at the hemifusion stage. To address this question, we prepared "PG2 mix" vesicles encapsulating self-quenching concentration of the water-soluble dye sulforhodamine B (SRB) (Figure ). In the event of a full fusion, the interiors of the virus and vesicle compartments become one and the fluorescence of SRB is enhanced upon dilution. We allowed influenza virus particles (unlabeled) to bind to tethered GDGT vesicles and then lowered the pH. We observed many events where a dim fluorescent spot turned bright indicating transfer of SRB from vesicle to virus through the fusion pore with nearly identical kinetics as compared to vesicles composed of monopolar phospholipids ("Phospholipid mix") (Figure ).