БИОЛОГИЧЕСКИЕ МЕМБРАНЫ, 2012, том 29, № 1-2, с. 65-72
= ОБЗОРЫ
УДК 577.352
MEMBRANE FUSION © 2012 Yu. A. Chizmadzhev
Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Leninsky prosp., 31, Moscow, 119071 Russia; Fax: (495) 952-55-82; e-mail: chiz@elchem.ac.ru Received 28.09.2011
The process of membrane fusion in the case of lipid bilayers, as well as induced by influenza virus is reviewed shortly. The methods of studying fusion kinetics in pure lipid and lipid—protein systems are described. The main theories of molecular fusion machines are presented. Open questions and unsolved problems are discussed in details. In conclusion, possible ways to solve the remaining problems are suggested.
Keywords: fusion, stalk, hemifusion diaphragm, hemagglutinin, fusion pore, dimple.
INTRODUCTION
Neurobiological community celebrates the 90th birthday of Professor B.I. Khodorov, outstanding person and brilliant scientist. His contribution to modern neurobiology cannot be overestimated. I met Boris Izrailevich after the appearance of his famous book Problems of Excitability (1969), which played a crucial role in my scientific fate. Working at Frumkin Institute of Electrochemistry on different subjects far from neu-robiology, under the influence of Boris Izrailevich I decided to switch over to studies of the mystery of nerve impulse. In collaboration with Boris Izrailevich and my colleagues we developed a theory of excitable media, elucidated the mechanisms of ion transport through different channels. In recent years, we focused on membrane fusion, having in mind synaptic transmission. The main results obtained in this field are presented below. I am thankful to my teacher in neurobiology, Professor B.I. Khodorov, who drew me to this remarkable field of knowledge and for 40 happy years has been my guide and friend. Now I take the opportunity to express my appreciation and relay my best wishes to Boris Izrailevich.
Membrane fusion is a key event in a multitude of cellular processes, such as synaptic transmission, intracellular trafficking, muscle development, fertilization, viral infection, etc. In all cases fusion implies the membrane merger and a free exchange of the content between two compartments. In general, biological fusion is a result of well-cooperated interplay between special proteins and lipids operating as transitory molecular machines. After the emergence of the famous book of Erwin Shrodinger What is life? The Physical Aspect of the Living Cell [1], living cell is considered as a factory consisting of many workshops. Phenotypic knowledge about these machines is obtained by non-invasive methods. Real structure of protein-lipid fu-
sion machines as well as physical mechanism of its action can be elucidated on model systems drawing nearer to biological objects. Quantitative description of these models is useful if it gives definite messages stimulating experimental studies.
For a long time it has been thought that in different cases of biological fusion Nature uses particular ideas. However, it is established now that even in such diverse cases as viral infection and synaptic signal transmission fusion proceeds along very similar pathways. Moreover, generic features of biological events are the same as in the case of the lipid bilayer fusion.
The objective of this short review is to present recognized concepts in membrane fusion and to discuss the main unsolved problems. We start from a model of two bilayer lipid membranes (BLM), which provided a significant progress in the field of membrane fusion.
FUSION OF LIPID BILAYERS
Planar lipid bilayers are metastable systems but they do not fuse spontaneously unless they are brought into close contact by an outer force [2]. This force is in a balance with repulsion between two membranes arising from thermally induced undulations and hydration forces. Fusion reaction is dilated in stages. After some waiting time it initiates from a local nanoscopic bridge that forms between the adjacent monolayers. The idea of such intermediate named stalk was first stated by Gingell and Ginsberg [3]. This becomes reasonable if we look at fusion of liquid droplets that initiates from the formation of a narrow neck increasing in time spontaneously. The origin of driving force is an area decrease minimizing surface energy. The only difference between two systems—BLM and liquid droplets—is that the membranes are liquid crystals and possess bending elasticity. Therefore, creation of a neck—stalk—in the case of BLM demands a work to
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Fig. 1. Stages of BLM—BLM fusion [2]. A, Experimental device. B, Molecular geometry (a) of lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) and preferential structures of monolayer (b) and bilayer (c) formed by the lipids. C, Fusion intermediates: initial state (a); prefusion state induced by pressure (b); stalk formation (c); hemi-fusion diaphragm (d); electroporation (e); membrane tube (f).
bend planar monolayers. Stalk may expand in radial direction producing hemifusion diaphragm. As a result, only one bilayer, instead of two, remains at this stage. Direct measurements have shown that two monolayers of the hemifusion diaphragm belong to the different fusing BLMs [2]. It means that stalk operates as a zipper, which takes out inner monolayers into meniscus and brings together outer monolayers (Fig. 1). Driving force for monolayer fusion lies in metastability of planar BLM. Actually, any planar bi-layer has an excess surface energy, which can be thrown down after the membrane rupture. However, BLM is rather stable, in spite of thermal undulations and presence of different kind of structural defects including through pores. It is well known that the membrane rupture is induced by the appearance of supercritical pore, which spontaneously enlarges. Creation of such pore can be stimulated by application of the external electric field. Exactly this way was used in the experiments to stimulate rupture of hemifusion diaphragm that leads to the final state, corresponding to complete fusion [2]. BLM rupture can be induced also by tension increase. Therefore, in the course of fusion, membranes undergo two consecutive stages of topological reconfiguration: stalk and pore formation. As a result, highly curved local structures enriched by elastic energy appear in planar membranes. The geometry of these two structures is similar and can be approximated by toroids, although orientation of lipid mole-
cules relative to neutral surface is opposite. At the first glance it seems puzzling why BLMs choose such a complicated way for fusion. The matter is that lipid bi-layers trade a single high barrier related to direct merger of the membranes for two smaller barriers. The first one corresponds to the pathway through monolayer fusion, while the second one is related to the pore formation in hemifusion diaphragm. The height of the barriers is determined by the features of the prefusion state. In the case of BLM—BLM fusion, a prefusion state arising in the course of undulations is transient. Thermal fluctuations cannot maintain close contact between membranes. As a result, a barrier for a direct merger is rather high. However, it is well known that such chemicals as polyethylene glycol dehydrate contact region and induce fast and direct fusion. It means that formation of the close contact due to some chemicals or external forces can decrease drastically the barrier for a direct membrane fusion. There is an obvious similarity between BLM—BLM and protein-induced fusion. In the first case, pressure difference creates a state of close contact, where in the course of thermal fluctuations a monolayer stalk appears. In the second case, spring-loaded proteins accomplish a significant work to build up prefusion state. Creation of stalk is governed afterwards by thermal fluctuations, as in the previous case.
The main experimental results concerning stalk evolution were described by a theory of membrane fu-
sion developed by Markin and Kozlov [4]. This theory plays a key role in the current progress of this field. Following Helfrich [5], they considered bending deformation as a mode giving the main contribution to the free energy. In the subsequent studies this theory was extended to the case of splay and tilt deformations [6, 7]. This approach allowed a decrease in stalk energy. Refuse of toroidal shape and invention of the new one, which was found by variation procedure, made stalk model even more plausible [8]. Calculations of the energy showed that stalk evolution strongly depends on molecular geometry of lipid molecules (Fig. 1). Thus, lysolipids (e.g., lysophosphatidylcho-line, LPC) have a large polar head group and a small, in the cross-section, hydrophobic tale. LPC is known as a pore-forming agent similar to usual detergents. In macroscopic terms it means that LPC incorporated into a monolayer induces positive spontaneous curvature. On the contrary, phosphatidylethanolamine (PE) has a small polar head group creating negative spontaneous curvature. Monolayer energy tends to a minimum when its geometrical curvature coincides with spontaneous curvature. Such structures as stalk and lipid pore of quasitoroidal geometry are characterized by two curvatures, equatorial and meridional. Calculations show that in the case of stalk negative meridional curvature dominates and minimizes stalk energy in the presence of PE, i.e., for the proximal monolayers with negative spontaneous curvature. On the contrary, presence of LPC in proximal monolayers increases stalk energy. These theoretical predictions, proved experimentally on BLMs, made incorporation of LPC and PE the recognized tools for stalk mechanism testing in numerous studies of BLM and biological fusion. Lipid pore formation and rupt
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