Lipid membranes membrane fusion

The adliesion and fiision mechanisms between bilayers have also been studied with the SEA [M, 100]. Kuhl et al [17] found that solutions of short-chained polymers (PEG) could produce a short-range depletion attraction between lipid bilayers, which clearly depends on the polymer concentration (fignre Bl.20.1 It. This depletion attraction was found to mduce a membrane fusion widiin 10 minutes that was observed, in real-time, using PECO fringes. There has been considerable progress in the preparation of fluid membranes to mimic natural conditions in the SEA [ ], which promises even more exciting discoveries in biologically relevant areas. 

While recent attention has been largely on proteins, it should be borne in mind that membrane fusion ultimately involves the merger of phospholipid bilayers. However, little is known about the specific membrane lipid requirements. When membranes fuse, energetically unfavorable transition states are generated that may require specific lipids and lipid domains for stabilization. Although there is some evidence for a specific influence of lipids on exocytosis, it is still unclear whether specific lipid metabolites are needed or even generated at the site of membrane merger. 

Q Studies using v- and t-SNARE ptoteins reconsti-mted into separate lipid bilayer vesicles have indicated that they form SNAREpins, ie, SNARE complexes that hnk two membranes (vesicles). SNAPs and NSF are required for formation of SNAREpins, but once they have formed they can apparently lead to spontaneous fusion of membranes at physiologic temperamre, suggesting that they are the minimal machinery required for membrane fusion. 

Several enveloped viruses, and some physical gene transfer techniques such as electroporation, deliver the nucleic acid into the cell by direct crossing of the cell membrane. Lipid-based, enveloped systems can do this by a physiological, selfsealing membrane fusion process, avoiding physical damage of the cell membrane. For cationic lipid-mediated delivery of siRNA, most material is taken up by endo-cytotic processes. Recently, direct transfer into the cytosol has been demonstrated to be the bioactive delivery principle for certain siRNA lipid formulations [151]. 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

Fig. 9 Utility of de-PEGylation technology in liposomes, (a) PEG derivative possessing a lipid moiety. The covalent bond between PEG and the lipid moiety can be cleaved by stimuli such as those within the acid environment of cancer and inflammation, (b) After binding the target cell via specific recognition of the receptor by the ligand, PEG molecules on the surface of the liposome are cleaved. The release of PEG facilitates membrane fusion of the liposome and liposome decomposition, resulting in efficient drug delivery...

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

Living cells visualization of membranes, lipids, proteins, DNA, RNA, surface antigens, surface glycoconjugates membrane dynamics membrane permeability membrane potential intracellular pH cytoplasmic calcium, sodium, chloride, proton concentration redox state enzyme activities cell-cell and cell-virus interactions membrane fusion endocytosis viability, cell cycle cytotoxic activity... 

Membranes and models membrane organization (e.g. membrane domains, lipid distribution, peptide association, lipid order in vesicles, membrane fusion assays, etc.)... 

Bailey AL, Cullis PR. Modulation of membrane fusion by asymmetric transbilayer distributions of amino lipids. Biochemistry 1994 33 12573. 

Grafting of PEG on the liposome surface interferes with the ability of the liposome to undergo membrane fusion and destabilization in the endosome. Meyer et al. observed this point (33). The stabilization of the lipopiexes into a lamellar phase would be a possible reason for this transfection inhibition, by lack of destabilization into the endosome (34). Thus, cleavable PEG-lipid has been designed to limit the nonspecific interaction with proteins, although restoring the ability of the particles to interact with the endosomal cellular membranes and to release their therapeutic payload. 

An emergent field dealing with the physical/chemical processes that underlie the changes of lipid phase state during such cellular events as membrane fusion, vesicle trafficking, and cell disjunction. 

Transporters, particularly those carrying nonlipophilic species across biomembranes or model membranes, can be regarded as vectorial catalysts (and are also called carriers, translocators, permeases, pumps, and ports [e.g., symports and antiports]). Many specialized approaches and techniques have been developed to characterize such systems. This is reflected by the fact that there are currently twenty-three volumes in the Methods in Enzymology series (vols. 21,22,52-56,81,88,96-98,125-127,156-157, 171-174, and 191-192) devoted to biomembranes and their constituent proteins. Chapters in each of these volumes will be of interest to those investigating transport kinetics. Other volumes are devoted to ion channels (207), membrane fusion techniques (220 and 221), lipids (14, 35, 71, and 72), plant cell membranes (148), and a volume on the reconstitution of intracellular transport (219). See Ion Pumps... 

Kasson, P.M., Pande, V.S. Control of membrane fusion mechanism by lipid composition predictions from ensemble molecular dynamics. PLoS Comput. Biol. 2007, 3, e220. 

As just mentioned, there are a large number of unsolved problems in membrane biophysics, including the questions of local anisotropic diffusion, hysteresis, protein-lipid phase separations, the role of fluctuations in membrane fusion, and the mathematical problems of diffusion in two dimensions Stokes paradox). 

In this chapter we first describe the composition of cellular membranes and their chemical architecture— the molecular structures that underlie their biological functions. Next, we consider the remarkable dynamic features of membranes, in which lipids and proteins move relative to each other. Cell adhesion, endocytosis, and the membrane fusion accompanying neurotransmitter secretion illustrate the dynamic role of membrane proteins. We then turn to the protein-mediated passage of solutes across membranes via transporters and ion channels. In later chapters we discuss the role of membranes in signal transduction (Chapters 12 and 23), energy transduction (Chapter 19), lipid synthesis (Chapter 21), and protein synthesis (Chapter 27). 

During the fusion process the relative surface area decreases with increasing volume indicating a loss of membrane material (about 22% in Fig. 51). In analogy to the fusion process of protoplasts it can be assumed that the excess lipid is removed in form of small, submicroscopic vesicles (Fig. 52). The electric breakdown in the membrane contact zone leads to the formation of several pores in which lipid molecules are randomly oriented (Fig. 52 b). The molecules reorient forming submicroscopic vesicles and the new membrane of the fused vesicle (Fig. 52c). Thus, fused giant liposomes should contain small, submicroscopic vesicles. This could possibly be proven by using fluorescence-labelled lipids for liposome fusion. 

Fig. 52a-c. Scheme of the fusion process of giant liposomes and the formation of small unilamellar vesicles (SUV) at the interface, a) lipid bilayers in contact b) pores generated by electric breakdown and lipid reorientation forming SUVs c) reconstitution of lipid membranes formation of a fused giant liposome and SUVs . 

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