Figure 7.
Promotion and inhibition of complete fusion by altering membrane lipid composition. (A) LPC in the inner monolayer of RBC membrane promotes complete fusion. Fusion was triggered at 22°C, pH 5.1, 10 min. No exogenous lipid was added in the control experiments. Lauroyl LPC was either added to the medium to incorporate into outer membrane monolayers (170 μM), loaded into RBC to partition into inner membrane monolayer, or both loaded into RBC and added to the outer membrane monolayers (170 μM). (B) Loading LPC into RBC ghosts decreases the number of activated HA molecules required for fusion pore formation and expansion, downstream of FIF. Fusion of HAb2 cells with either RBC loaded with LPC or control RBC was triggered at 4°C, pH 4.9, 10 min. Then cells were incubated in neutral pH medium for 10 min, treated by proteinase K (0.1 mg/ ml, still at 4°C) for 45 s, 150 s, or not treated at all. After 20 min more at neutral pH, 4°C, the temperature was raised to 22°C. (C) Complete fusion is inhibited by OA. Fusion was triggered at 22°C, pH 4.9, 10 min in the medium containing different concentrations of OA. In A–C, complete fusion (cell pairs with both PKH26 and CF transferred from RBC to HAb2 cells) and hemifusion (cell pairs with PKH26 but not CF transferred) were assayed with fluorescence microscopy after 20 min of incubation at neutral pH. Each point is mean ± SE, n > 3.