Dynamics of asymmetric membranes and interleaflet coupling as intermediates in membrane fusion

Abstract Membrane fusion is a tool to increase the complexity of model membrane systems. Here, we use silica nanoparticles to fuse liquid-disordered DOPC giant GUVs and liquid-ordered DPPC:Cholesterol (7:3) GUVs. After fusion, GUVs display large membrane domains as confirmed by fluorescence confocal microscopy. Laurdan spectral imaging of the membrane phases in the fused GUVs shows differences compared to the initial vesicles indicating some lipid redistribution between phase domains as dictated by the tie lines of the phase diagram. Remarkably, using real-time confocal microscopy we were able to record the dynamics of formation of asymmetric membrane domains in hemifused GUVs and detected interleaflet coupling phenomena by which the DOPC-rich liquid-disordered domains in outer monolayer modulates the phase state of the DPPC:Cholesterol inner membrane leaflet which transitions from liquid-ordered to liquid-disordered phase.. We find that internal membrane stresses generated by membrane asymmetry enhance the efficiency of full fusion compared to our previous studies on symmetric vesicle fusion. Furthermore, under these conditions, the liquid disordered monolayer dictates the bilayer phase state of asymmetric membrane domains in >90% of observed cases. By comparison to the findings of previous literature, we suggest that which monolayer phase dominates the bilayer properties could be a mechanoresponsive signalling mechanism sensitive to the local membrane environment. Statement of Significance Natural biomembranes are highly asymmetric in lipid composition between the two leaflets of its bilayer structure. While the majority of membrane biophysics studies are conducted on symmetric lipid bilayers, it has become increasingly apparent that these asymmetric lipid compositions have a strong impact on the overall properties and behaviours of the membrane. However, achieving controlled membrane asymmetry in artificial model membranes is non-trivial, making the study of related biophysical mechanisms challenging. Here, we present an experimental framework to image the dynamics of asymmetric membrane domains formed as intermediates during membrane fusion of giant unilamellar vesicles (GUVs) triggered by silica nanoparticles. We directly observe interleaflet coupling of the phases in asymmetric membranes, where the liquid disordered domains most commonly dominate. By comparison with other literature on asymmetric membranes, this may indicate that the phase state of asymmetric membranes can be mechanoresponsive to its local environment. Furthermore, enhanced efficiency of full fusion of these GUV membranes indicates that stresses generated by membrane asymmetry can promote the formation of the full fusion pore during vesicle fusion processes.