Supplementary Figure 6: MCS R-Pre only detached from the plasma membrane at high ionic strength and after incorporation of the cationic lipid DOTAP.

MCS R-Pre only is MCS⁺ without the hydrophobic membrane attachment unit (MA1 in Fig. 1). (a) Merged donor (green) acceptor (red) intensity images of MCS R-Pre only in membrane lawns before and after solution change from 10 mM to 275 mM. Scale bar = 5 μm. (b) Donor (green) and acceptor (red) and acceptor/donor (R/G) ratio before and after the solution change (black arrow) from 10 mM to 275 mM. 75 ± 10% of the total donor and acceptor intensity of MCS R-Pre only was lost from the membrane sheet after solution change, indicating that the hydrophobic membrane anchor was required to retain MCS at the plasma membrane. Data are representative of n = 5 independent experiments. (c) Donor (green) acceptor (red) intensity images of MCS R-pre only in membrane lawn of COS-7 cell at the indicated time point during fusion of DOTAP-enriched lipid vesicles. (d) Intensity plot of donor (green) acceptor (red) of MCS R-pre only and membrane fused DOTAP lipids (cyan) that were labeled with a far-red fluorescent dye, DiR. The fusion of positively charged DOTAP lipids caused R-pre only to detach from the membrane.