Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Abstract

We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 AE 0.5 Å for the investigated lipid compositions.curvature energy | hydration force | lipid bilayer | E xocytosis, intracellular transport, neurotransmission, fertilization, or viral entry, require that two membranes merge into one. This event, membrane fusion, involves a complex interplay of different membrane lipids, proteins, and water molecules on length scales of few nm. Following the "lipidic pore hypothesis", it is now well accepted that membrane fusion involves a sequence of lipidic nonbilayer intermediates, whose formation is catalyzed and guided by a specialized protein machinery (1, 2). A stage termed "hemifusion" in which the lipids of the outer leaflets of two membrane-enclosed compartments about to fuse can mix, whereas the inner leaflets and the enclosed content remain unaltered (3), has been confirmed by a variety of methods including conical electron tomography (4) and, more indirectly; e.g., by electron spin resonance (5) and fluorescence recovery after photobleaching (6). Studying the fusion of protein-free bilayers of well defined lipid composition can contribute useful insights into the physical principles governing the merger of their more complex biological counterparts and clarify the effect of individual lipid species.The first connection between two lipid bilayers is the so-called stalk sketched in Fig. 1A (7). The proximal lipid monolayers have merged into one strongly curved monolayer, whereas the distal ones are still separated and intact. A persistent problem in membrane biophysics is to determine the precise structure of stalks and the free energy barrier for stalk formation. In a long series of papers (8-16), this determination has been attempted within the framework of the continuum theory of membrane elasticity (17, 18). More recently, with the advent of sufficient computational power, simulations including molecular details have become feasible [e.g. (19, 20) and references therein]. While stalk formation between lipid bilayers in close contact is generally accepted as the initial step in possibly all membrane fusion reactions (7), the subsequent stages from stalk to complete fusion are still debated and different pathways may exist (21)....

Non-equilibrium dynamics of lipid bilayers

“…inelastic neutron scattering and revealed a rich spectrum of dynamics from the 0.1ps to the µs time scale [26,27,28,29]. Structural properties of lipid bilayers under varying ambient conditions can well be resolved by specular and diuse x-ray scattering experiments [30,31,32,3,33]. However, an experimental technique capable of probing fast, especially eld and light induced, transient non-equilibrium structural dynamics is necessarily accompanied by two major experimental challenges: (i) Ultrafast temporal resolution on the order of picoseconds is needed in order to probe non equilibrium eects (ii) The outlined eects involve structural changes in the sub-nm length scale.…”

“…The initially unknown relative phase factors ν n ∈ [−1, +1] can be reconstructed by e.g. the swelling method [96], reference data for a broad range of lipid model systems is readily available [30,31]. Details on the Fourier synthesis approach as well as the application of the swelling method to lamellar and non-lamellar lipid phases are extensively reviewed in [53,52,97].…”

Advanced x-ray multilayer waveguide optics

“…The multilayer sample was mounted upright in xz plane by fixing it by double-sided tape on a home-built Aluminum holder. More details on the instrument can be found in [73]. [29].…”

“…X-ray macroscopic reflectivity (XMR) XMR was performed for the multi-guide RBCs, using an in-house Cu K α source (λ = 0.154 nm), equipped with collimating multilayer mirrors and a fully motorized reflectometer [73]. The x-ray reflectivity was recorded with 0.1 mm beam as defined by the entrance slits.…”

Revisit the Correlation between the Elastic Mechanics and Fusion of Lipid Membranes

“…The first energy barrier arises from the need to bring two fusion-destined membranes into close proximity to initiate fusion 6,7 . The barrier, an inter-membrane interaction known as hydration repulsion, results from the resistance to removing inter-membrane water needed for shortening the inter-membrane distance 8 .…”