Protein-Free Membrane Fusion Probed by Single Giant Unilamellar Vesicle Imaging - the Role of Membrane Charge

Abstract

Membrane fusion is a ubiquitous process in biology. The outcome of fusion is followed by successive steps of fusion intermediates, including (i) protein tethering in apposing bilayers, (ii) membrane adhesion, (ii) hemifusion and eventually (iv) full fusion, the latter leading to complete mixing of membranes and aqueous contents. Studying membrane fusion in vivo is, however, very challenging. By a combination of microscopic approaches, we investigate the role of membrane charges on the interaction and fusion of cationic large (DOTAP:DOPE:DPPE-Rh - 1:1:0.1 mol) and individual giant unilamellar vesicles (LUVs-GUVs) in a pure lipid system. Fluorescencence Resonance Energy Transfer (FRET) efficiency, in which the donor is reconstituted in the GUVs and the acceptor incorporated upon membrane merging, is measured and directly related to fusion efficiency. LUVs stably adhere and diffuse onto the surface of neutral (POPC) GUVs with minimal membrane merging (FRET efficiency 0.1). In sharp contrast, very efficient fusion occurs with the negative GUV membranes (POPC:POPG - 1:1) - FRET 0.6-1. An increase in membrane area after contact with the fusogenic LUVs was clearly detected for this composition. Excess of negative lipids on GUVs (pure POPG) relative to positive charges decrease fusion efficiency (FRET 0.3) - a charge-neutralization process. Transferred lipids upon fusion undergo similar diffusion to those on GUV membranes (∼ 1 μm2/s) With the developed LUV-GUV system, we studied the charge-dependent membrane adhesion and fusion separately and individually for the very same interacting partners, not possible with traditional in vitro systems. The system could also be easily adapted to protein-reconstituted studies.Financial support: FAPESP.