Lipid Domains (eBook)

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2015 | 1. Auflage
372 Seiten
Elsevier Science (Verlag)
978-0-12-803327-2 (ISBN)

Current Topics in Membranes is targeted toward scientists and researchers in biochemistry and molecular and cellular biology, providing the necessary membrane research to assist them in discovering the current state of a particular field and in learning where that field is heading. This volume offers an up to date presentation of current knowledge in the field of Lipid Domains.

Written by leading experts
Contains original material, both textual and illustrative, that should become a very relevant reference material
The material is presented in a very comprehensive manner
Both researchers in the field and general readers should find relevant and up-to-date information

Current Topics in Membranes is targeted toward scientists and researchers in biochemistry and molecular and cellular biology, providing the necessary membrane research to assist them in discovering the current state of a particular field and in learning where that field is heading. This volume offers an up to date presentation of current knowledge in the field of Lipid Domains. Written by leading experts Contains original material, both textual and illustrative, that should become a very relevant reference material The material is presented in a very comprehensive manner Both researchers in the field and general readers should find relevant and up-to-date information

Chapter One

Supported Lipid Bilayers as Models for Studying Membrane Domains

Volker Kiessling∗, Sung-Tae Yang and Lukas K. Tamm Department of Molecular Physiology and Biological Physics, Center for Membrane Biology, University of Virginia, Charlottesville, VA, USA
∗ Corresponding author: E-mail: vgk3c@virginia.edu

Abstract

Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes. We highlight methods to produce asymmetric lipid bilayers with lipid compositions that mimic those of the extracellular and cytoplasmic leaflets of cell membranes and the functional reconstitution of membrane proteins into such systems. Questions related to interleaflet domain coupling and membrane protein activation have been addressed and answered using advanced reconstitution and imaging procedures in symmetric and asymmetric supported membranes with and without coexisting lipid phase domains. Previously controversial topics regarding anomalous and anisotropic diffusion in membranes have been resolved by using supported membrane approaches showing that the propensity of certain lipid compositions to form “rafts” are important but overlaid with “picket-fence” interactions that are imposed by a subtended cytoskeletal network.

Keywords

Integrin; Lipid asymmetry; Lipid bilayer; Lipid raft; Liquid-disordered; Liquid-ordered; Membrane domain; Membrane protein; Rac; Ras; SNARE; Supported membrane

1. Introduction

A fascinating challenge of membrane biology over the last decade has been the study of lipid domains in cell membranes. Cellular membranes are composed of a bewildering variety of different lipids that form their lipid bilayer matrix into which membrane proteins of various topologies are embedded. The functional reasons for the enormous complexity of lipid compositions in biomembranes are only poorly understood. Some lipids activate enzymes while others have distinct roles in cell signaling. Yet other lipids and combinations of lipids are subject to thermodynamic forces that drive them into small or large assemblies in the plane of the lipid bilayer. A classic example is the combination of sphingomyelins (SMs), cholesterol, and unsaturated phospholipids that phase-separate into large domains in lipid model membranes (Crane & Tamm, 2003; Dietrich, Bagatolli, et al., 2001; Feigenson, 2009; Veatch & Keller, 2005). SMs and cholesterol are enriched in liquid-ordered (Lo) domains, whereas unsaturated phospholipids are enriched in liquid-disordered (Ld) domains. The lipids have more extended, i.e., more “ordered” alkyl chains and diffuse about an order of magnitude more slowly in Lo than in Ld phases. Despite these different properties, the lipids are still laterally mobile in both phases, i.e., they are considered to form a two-dimensional “liquid” as opposed to a gel phase. Gel phases with very slowly diffusing lipids are formed, e.g., by SMs or ceramides, in the absence of cholesterol. Whether Lo-phase lipid domains, often referred to as “lipid rafts,” also occur in cell membranes has been the subject of intense debates over the past 15 years or so (Edidin, 2003; Kusumi et al., 2012; Lingwood & Simons, 2010; Pike, 2006).

Lipid rafts were originally defined by cell biologists based on the observation that the extraction of cell membranes with cold detergent yielded fractions that were enriched in SMs and cholesterol (Brown & London, 1998; Simons & Ikonen, 1997). These fractions also frequently contained proteins that are known to cooperate in cell signaling. Although it was later shown that the aggregation of sphingomyelin and cholesterol can be artifactually enhanced by the action of detergent extraction (Lichtenberg, Goni, & Heerklotz, 2005), higher resolution optical techniques on cell membranes have revealed that these lipids retain special properties in cellular membranes that organize them in nanoscale domains even in the presence of the high densities of membrane proteins that are encountered in cell membranes (Honigmann, Mueller, et al., 2014). These domains are not only very small (10–100 nm), but may also be relatively short-lived and undergoing continuous lipid and protein exchange, which makes them quite difficult to detect in cell membranes. However, lipid domains in pure lipid model systems are much larger (1–10 μm) and long-lived, which renders their study much easier in these systems. Despite these clear differences between lipid domains in cellular and model membranes, we believe that studies of lipid domains and their interactions with membrane proteins in model systems yield highly valuable insight into their nature since the model systems amplify and reveal underlying physical–chemical principles that must also occur in cell membranes, but are much more difficult to uncover in live cells.

There have been two principal model systems to study the coexistence of Lo and Ld phases in lipid bilayers. They are giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs). This article reviews pertinent results on lipid domain formation, lipid asymmetry, and protein insertion in SLBs. Although we focus on SLBs, many of the same phenomena can also be observed in GUVs. Despite the high complementarity between these two model systems, they both have their own advantages and disadvantages. In GUVs, one does not need to worry about potential adverse interactions with the substrate that may occur in supported bilayers, but SLBs are flat and extend over many millimeters and therefore offer much better in-focus imaging capabilities than GUVs.

SLBs have been introduced more than 30 years ago as model systems for mimicking cellular membranes (Tamm, 1984; Tamm & McConnell, 1985). They are particularly well suited to study the lateral mobility and phase behavior of lipids (Groves, Boxer, & McConnell, 1997; McConnell, Watts, Weis, & Brian, 1986; Sackmann, 1996; Tamm, 1988; Wright, Palmer, & Thompson, 1988). It has been recognized early on that integral membrane proteins with significant protein domains on both sides of the membrane cannot be reconstituted into supported bilayers in a laterally mobile form. Therefore, multiple strategies have been devised to lift SLBs from their solid supports by introduction of an intervening polymer cushion. Several such designs improve the lateral mobility of integral membrane proteins (Floyd, Ragains, Skehel, Harrison, & van Oijen, 2008; Goennenwein, Tanaka, Hu, Moroder, & Sackmann, 2003; Naumann et al., 2000; Wagner & Tamm, 2000). As will be reviewed below, the same kinds of polymer cushions are also advantageous to preserve symmetric and asymmetric lipid domain structures in supported membranes.

2. Methods for Supported Lipid Bilayer Preparation

To set the stage, we will first briefly review methods for supported bilayer preparation and their applications for intra- and interleaflet lipid interactions. In particular, we want to emphasize the utility of the different techniques for the study of domain structure, bilayer asymmetry, and reconstitution of integral membrane proteins.

2.1. Direct vesicle fusion

The most common technique to form supported bilayers is the direct vesicle fusion (VF) method (Brian & McConnell, 1984; Kalb, Frey, & Tamm, 1992) (Figure 1(A)), which was developed shortly after the original Langmuir–Blodgett/Langmuir–Schäfer (LB/LS) method (see below). Small unilamellar vesicles (SUVs, diameter <50 nm) or large unilamellar vesicles (LUVs, diameter >50 nm) of a specific lipid composition are prepared and added to a clean hydrophilic substrate. Typical substrates are glass or quartz for fluorescence microscopy applications and mica for atomic force microscopy (AFM) applications. After a 30–60-min incubation time, during which vesicles adhere to the substrate and rupture to form a continuous membrane, the nonadhering vesicles are washed out with buffer.

The simplicity of the procedure makes this a very attractive approach. However, for the study of domains in membranes, this method has several disadvantages. First, the domains formed from phase-separating lipid compositions are too small to be visualized by normal fluorescence microscopy. Yet, this problem can be circumvented by using AFM or optical superresolution detection techniques. Second, integral membrane proteins usually end up mostly immobile in the supported bilayer. The water cleft between the substrate and lipid bilayer is only 1–2 nm wide (Fromherz, Kiessling, Kottig, & Zeck, 1999; Kiessling & Tamm, 2003). Proteins with large cytosolic or extracellular domains that are facing outward in the vesicle get stuck to the substrate upon vesicle adhesion. Protein mobility can be improved by cushioning the bilayer with polymers like cellulose from the solid substrate (Goennenwein et al., 2003). A different protein reconstitution approach that results in highly oriented protein insertion is the so-called direct incorporation method (Milhiet et al., 2006). A small volume of protein stabilized in...

Erscheint lt. Verlag	8.6.2015
Sprache	englisch
Themenwelt	Naturwissenschaften ► Biologie ► Biochemie
	Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
	Naturwissenschaften ► Biologie ► Ökologie / Naturschutz
	Naturwissenschaften ► Biologie ► Zellbiologie
	Technik
ISBN-10	0-12-803327-4 / 0128033274
ISBN-13	978-0-12-803327-2 / 9780128033272

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