Figure 4: IM30 disturbs the membrane structure and thereby triggers membrane fusion.

(a–e) Cryo- transmission electron microscope images of pure MGDG/PG liposomes (a), of liposomes mixed with solely IM30 (b) or Mg²⁺ (c), as well as of liposomes mixed with both, IM30 and Mg²⁺ (d,e). Notably, at high Mg²⁺ concentrations, vesicles were essentially no longer found inside the holes of the holey carbon film, but mostly on the carbon itself. In contrast, with both IM30 and Mg²⁺ present, the vesicles were found in large amounts inside the holes (d,e), suggesting that attachment of IM30 affects the surface properties of the liposomes. Furthermore, note the large compartmentalized liposomes in d and e, as well as the extended connection between two liposomes in d. Arrows indicate extra electron density found at the liposome surface. The area boxed in d is enlarged in Fig. 5. Scale bar, 200 nm. The magnification was identical in a–e. EM micrographs were taken after 1 min incubation with Mg²⁺ and/or IM30. (f) Sodium dithionite was added to NBD-labelled MGDG/PG liposomes (1), resulting in immediate quenching of the NBD fluorescence of the outer membrane leaflet. (2) When nothing further (light grey), solely 0.94 μM IM30 (light blue) or solely 11 mM MgCl₂ (dark blue) was added to the liposomes, the fluorescence did not considerably decrease further. After addition of increasing IM30 concentrations at a constant MgCl₂ concentration of 7.5 mM (red curves), the NBD-fluorescence decreased further in an IM30 concentration-dependent manner. Addition of Triton X-100, which dissolves the liposomal membranes, results in an immediate quenching of the entire NBD-fluorescence (black). The experiment was repeated two times.