(613j) Line Tension and Lipid Sorting Modulate Dynamics of Hemifusion Diaphragm Dissipation

Lipid bilayer fusion is commonly believed to proceed through the stalk-pore mechanism. This hypothesis includes an intermediate in which two distal monolayers of merging bilayers come together to form a new bilayer, called the hemifusion diaphragm (HD). Several factors aid in the formation and stabilization of an HD, including the presence of non-bilayer lipids. Lipids with negative intrinsic curvature (NIC) have been shown to enhance the probability of HD formation and effect the stability and formation of the resulting HD structure. Despite a theoretical basis for the necessity of NIC lipids to the HD, lipid contributions to the dynamics of HD formation are still not fully understood. We studied the effects of intrinsic curvature and lipid concentration on protein-free HD dynamics through the use of molecular dynamics (MD) and a coarse-grained lipid model. Large HD’s (r_HD ∈ 10 nm, 20 nm) were artificially produced. Binary lipid mixtures were added by randomly seeding the HD’s with lipids of varying strengths of NIC (low, moderate, and high) at various compositions ( ̄ x ∈ 0.05, 0.10, 0.17, 0.25, 0.50, 0.75, 1.00). Systems were run for a total of 2 μs in an NP_xxP_yyL_zT ensemble.

HD’s are found to decay to three different metastable end states dependent on intrinsic curvature and composition of the constituent lipids. These end states include a non-fusogenic double bilayer and fusogenic fusion pore and stable HD. Systems decay to a double bilayer at low compositions of NIC lipids or when NIC lipids are weak. Moderate NIC lipids or moderate compositions of strong NIC lipids decay to a metastable fusion pore while high compositions of strong NIC lipids stabilize the HD. Systems which decay to a double bilayer are found to have similar dynamics regardless of lipid composition while in fusogenic systems, lipid compositions are correlated to the rate of HD dissipation. In addition, the method of dissipation appears different between fusogenic and non-fusogenic systems. Non-fusogenic systems formed a network of interconnected bicelles in the stalk region with large pores in the region. In some of these systems, the network of bicelles aligned in such a way that a single flat bilayer formed and the previously joined bilayer was left with a large pore (r_pore ≈ 8 nm) in the center. The fusogenic systems maintained a connected stalk region throughout the dissipation process with only small pores present around the three-junction. The difference in decay rates and method of dissipation alludes to a fundamentally different driving force in fusogenic compositions and non-fusogenic compositions.

We conclude that the metastable end state is dependent on the mode of HD dissipation. When systems do not have sufficient NIC lipids to mediate the line tension of the three-junction, the stalk region forms a network of free edges. In this case, the stalk region harbors a line tension and a compressive force on the interior HD bilayer exists. This compressive force can be measured through a progressively decreasing apparent area per lipid in the HD bilayer as the HD dissipates. In the fusogenic system, no such compression exists. The fusogenic systems are found to sort NIC lipids towards the three-junction to effectively minimize the line tension in the stalk region. In these systems, though no significant compressive force on the HD bilayer exists, dissipation is driven by lipid movement into the three-junction from the HD bilayer. As the HD dissipates, the secondary radius dictated by the HD size decreases which effectively increases the necessary composition of NIC lipid required to minimize the line tension. NIC lipids respond by flipping out of the HD and outer bilayer and into the three-junction. The movement of NIC lipids out of the HD bilayer simultaneously decreases the NIC lipid concentration in the HD bilayer and the radius of curvature of the three-junction. These two factors drive the HD to shed more NIC lipids to mediate the increased line tension in the three-junction and increased effective area per lipid in the HD bilayer. When the NIC lipids have stabilized the three-junction and sorting towards the junction halts, the driving force vanishes and the system assumes a stable HD state.

This work aims to elucidate the effect of NIC lipids in fusion events and further understand the dynamics of the fusion process. In the two proposed dissipation methods, the HD is dependent on lipid flip-flop to shrink the HD. Flippases have previously been indicated as a functional component of the fusion process. Our work implicates flip-flop as a mechanism by which the bilayer may control the fusion process and sheds light on the role of flip-flop in HD formation and dissipation. Though this work uses a highly coarse-grained lipid model, it is believed that these findings may have implications in biological fusion events.