Membrane fusion is a ubiquitous process, essential for cellular development, communication, and drug delivery. It consists of consecutive steps including docking of the two bilayers, membrane destabilization, fusion of the outer leaflets (hemifusion), opening and expansion of the fusion neck and finally full fusion. In our study, we investigated the fusion between small cationic liposomes (~100 nm) and giant vesicles (tens of microns in diameter) containing increasing fractions of anionic lipids. The cationic liposomes have been developed as a promising drug carrier system, as they are able to fuse with the plasma membrane and efficiently transport encapsulated material to the cytosol. Giant unilamellar vesicles (GUVs) are well-established membrane models, which can be easily followed with optical microscopy and can therefore unravel key features of membrane processes. We put forward advanced microscopy techniques and mechanical manipulation to assess the fusion efficiency in this system. This allows us not only to quantify the degree of fusion, but also to assess the increase in the area acquired by the GUVs as fusion proceeds. Surprisingly, the resulting vesicles develop external tubes suggesting the generation of positive spontaneous curvature in the membrane.

Our cover art for the January 8 issue of Biophysical Journal shows image sequences of GUVs obtained with phase contrast and confocal microscopy. We chose to represent the sequences obtained with modern digital cameras as old film rolls so as to create an antique atmosphere, as though they had been acquired with old cameras. The GUVs are initially labeled with a green dye and the liposomes contain a red probe. The chosen dyes form a FRET (fluorescence resonance energy transfer) pair, implying that excitation of the donor probe leads to emission of the acceptor once they are close enough, indicating fusion. The first sequence shows different GUVs containing the green dye and increasing fractions of the red one, which allowed us to obtain a FRET calibration curve and thus quantify the fusion efficiency from image sequences of individual GUVs (as shown in the second film roll). Our results show that the liposomes dock to and/or hemifuse with neutral GUVs (last sequence) but are able to extensively fuse with GUVs containing anionic lipids (second, third and fourth sequences). Efficient fusion leads to a massive increase in the GUV area occasionally followed by external tubulation. This can be well seen in the second sequence, in which the GUV, initially spherical and green, turns red as it gains area until tubulation occurs in the last snapshot. The area increase is evident in the phase contrast sequence, which was obtained under an AC field that induces a prolate deformation whose extension correlates with the excess area. The cationic liposomes induce mainly docking and hemifusion with neutral GUVs, resulting in an increase in membrane tension that eventually leads to vesicle rupture and restructuration in tubular morphologies (images in the background). We can conclude that the fusogenic system is very efficient for membranes containing anionic lipids, which can be therapeutically interesting, because cancer cells usually expose anionic lipids in their external leaflet.

- Rafael Lira, Tom Robinson, Rumiana Dimova, Karin Riske