Lipid Rafts and Caveolae: From Membrane Biophysics to Cell Biology - Hardcover

Hardcover

ISBN 10: 3527312617 ISBN 13: 9783527312610

Publisher: Wiley-VCH, 2006

Synopsis

This keenly awaited first overview of the field represents a complete guide to the structure and function of the most important mammalian cell membrane organelles. Filling a huge gap in the primary literature, this book is the first to cover the subject in detail.
Following an introduction by Kai Simons, the discoverer of lipid rafts and the most prominent scientist in the field, chapters include:
Historical background
Distinct structures and functions
Structural basis
Signaling
Viral entry and virion budding
Cholesterol transport
Caveolins
Lipid shells
Cell polarity and intracellular trafficking
Cancer cells
Of prime importance to molecular and cell biologists, biochemists, membrane scientists, cancer researchers, and virologists.

About this title

About the Author

Christopher Fielding is Neider Professor of Cardiovascular Physiology at the University of California at San Francisco (UCSF). He graduated from University College in London (UK) where he also received his PhD. After appointments at Oxford University and at the University of Chicago, he joined the faculty at UCSF in 1971, being appointed full professor in 1985.
Professor Fielding's main research interest is in the trafficking of cholesterol, its regulation and its role in signal transduction.

From the Back Cover

This keenly awaited first overview of the field represents a complete guide to the structure and function of the most important mammalian cell membrane organelles. Filling a huge gap in the primary literature, this book is the first to cover the subject in detail.
Following an introduction by Kai Simons, the discoverer of lipid rafts and the most prominent scientist in the field, chapters include:

- Historical Background
- Distinct Structures and Functions
- Structural Basis
- Signaling
- Viral Entry and Virion Budding
- Cholesterol Transport
- Caveolins
- Lipid Shells
- Cell Polarity and Intracellular Trafficking
- Cancer Cells

The book is of prime importance to molecular and cell biologists, biochemists, membrane scientists, cancer researchers, and virologists.

Excerpt. © Reprinted by permission. All rights reserved.

Lipid Rafts and Caveolae

From Membrane Biophysics to Cell Biology

John Wiley & Sons

Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All right reserved.
ISBN: 978-3-527-31261-0

Chapter One

Lipid Rafts, Caveolae, and Membrane Traffic Doris Meder and Kai Simons

1.1 Introduction

Cell membranes are dynamic assemblies of a variety of lipids and proteins. They form a protective layer around the cell, but also mediate the communication with the outside world - that is, neighboring cells in a tissue, hormones and growth factors arriving with the blood supply, or pathogens trying to enter the system. The unique feature of cell membranes is that their lipid and protein constituents can self-assemble into 5 nm-thin, two-dimensional fluids composed of two apposing lipid monolayers that form a hydrophobic interior and two polar interfacial regions oriented towards the aqueous medium. This organizing principle - the lipid bilayer - is the oldest, still valid molecular model of biological structures. The first model that incorporated proteins was proposed by Danielli and Davson, and assumed that the bilayer was made up entirely of lipids and that proteins covered the two polar surfaces. Some 40 years later, the fluid mosaic model of the cell membrane proposed by Singer and Nicolson was a conceptual breakthrough. Amphipathic membrane proteins were recognized to reside within, and even span, the whole bilayer that was depicted as a dynamic structure, the components of which are laterally mobile. However, the view that the lipids in the bilayer mainly serve as a homogeneous solvent for proteins has been proven to be too simplistic. Lipids are not only distributed asymmetrically between the two leaflets of the bilayer, but also within the leaflet they are heterogeneously arranged. This chapter will recapitulate the history and recent advances in membrane biology including the lipid raft concept, and then summarize current views on the functions of rafts and caveolae in membrane traffic.

1.2 Basic Organization Principles of a Cell Membrane

The lipid bilayer is a two-dimensional fluid, where lipid molecules exchange slowly between leaflets but are mobile within the leaflet. This mobility consists of two parts:

the "translational freedom" of a molecule - that is, its lateral mobility; and

the "configurational freedom" that is, the ability to flex parts of the molecule and to rotate bonds in its carbon backbone.

Synthetic bilayers change from a liquid state with high translational and configurational freedom into a rigid gel state at a characteristic freezing point. Cell membranes at physiological temperatures are almost always in the liquid state, but can contain regions with high configurational order, as will be described later. Importantly, the lipid bilayer of cell membranes is asymmetric, with a different lipid composition in the two leaflets. The main lipid components of cellular membranes are glycerophospholipids, with the most abundant species being phosphatidylcholine (PC) in the exoplasmic leaflet and phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the inner leaflet, as well as sphingolipids with glycosphingolipids and sphingomyelin (SM) mostly localized to the exoplasmic leaflet. Sterols make up the third lipid class, and are present in both leaflets. Mammalian cell membranes contain only one sterol, namely cholesterol, but probably more than thousand different glyco- and sphingolipid species, emerging from the combinatorial propensity to assemble lipids from different backbones linked in different ways with two varying hydrocarbon chains and a vast number of headgroups. A large number of flippases and translocators tightly control the asymmetric distribution of all these lipids across the bilayer.

Lipids are differentially distributed between cellular organelles. The endoplasmic reticulum and the Golgi-complex contain mainly glycerophospholipids and only small amounts of sphingolipids, whereas the plasma membrane is relatively enriched in SM and glycosphingolipids. Also within the membrane plane of one organelle, lipids are believed to be heterogeneously arranged. Caveolae - small invaginations of the plasma membrane - are enriched in glycosphingolipids, and phosphatidylinositol-3'-phosphate (PI(3)P) is concentrated in subdomains of early endosome membranes. Recently, vacuole-fusion in yeast has been shown to be controlled by microdomains of ergosterol, diacylglycerol and phosphoinositide-3-and-4-phosphate. Furthermore, membranes are differentially susceptible to extraction by detergents such as Triton X-100 or CHAPS at 4 C, with some proteins and lipids being completely solubilized and others forming so-called "detergent-resistant membranes" (DRM; for a review, see). These findings suggested that cell membranes contained microdomains in which lipids were more tightly packed and thus not accessible to the detergent, although it is widely accepted that DRMs do not have an exact in-vivo correlate but are defined by being formed during the detergent treatment. These microdomains were later termed "rafts" and were described as sphingolipid-cholesterol assemblies containing a subset of membrane proteins. Currently, the raft hypothesis is heavily debated, with the main discussion points being the methodologies to study rafts and the size of the domains (see below). The core of the raft concept is that cell membranes phase-separate into different domains and that this is a lipid-driven process. In light of the ongoing discussion in the field, the following sections will provide an overview about what is known about phase separation, first discussing the studies conducted in model membrane systems and later in cell membranes.

1.3 Evidence for Phase Separation in Model Membrane Systems: Liquid-Ordered and Liquid-Disordered Phases

Various model membrane systems have been used by physicists and chemists to study phase separation in lipid mixtures. They are either monolayers or bilayers. Monolayers are either assembled at an air-water interface with the packing density of the lipids being adjusted by applying lateral pressure, or on a supporting lipid monolayer that is fixed to a solid support. Bilayers are used in the supported version as described above, or in the form of vesicles. The most commonly used vesicles are large or giant unilamellar vesicles (LUV or GUV, respectively) composed of only a single bilayer, but also multilamellar vesicles (MLV) are used. The basic principles were first established in simple binary lipid mixtures, but recently ternary mixtures which more closely mimic the composition of the cell plasma membrane have been used. The mixtures usually contain one lipid with a high melting temperature ([T.sub.m]), one with a low [T.sub.m], and cholesterol. GUVs are probably the system closest to a cell membrane, because artifacts from a support are excluded. Still, cell membranes are asymmetric with different lipid compositions of the outer versus the inner leaflet, while the GUVs used so far were all symmetric. Since maintaining an asymmetric lipid distribution is energy-consuming, perhaps by reconstituting lipid translocators into liposomes this drawback can be overcome in the future. Although model membrane systems produce very simplified pictures of cell membranes, there are many examples of a close correlation with experimental data obtained in living cells.

Ipsen et al. were the first to describe the formation of a liquid-ordered phase by cholesterol and saturated phospholipids. This phase can coexist with other lipid phases, and its characteristics are described as follows: the translational order of lipid molecules within the liquid-ordered phase is similar to that in a fluid bilayer state, whereas the configurational order of the hydrocarbon chains compares more to that in a gel state. The formation of the liquid-ordered phase was attributed to the unique chemical nature of cholesterol (for a review, see), but later it was shown that all natural sterols promote domain formation and that also small amounts of ceramide (3 %) can stabilize domains formed in vesicles. Leventis and Silvius showed that the interaction of cholesterol with different lipid species is dependent on the nature of their hydrocarbon chains and, to a lesser extent, also on their headgroup. The interaction preference decreases with SM > PS > PC > PE and with increasing unsaturation of the acyl chains. Whereas the kink in unsaturated hydrocarbon chains is likely to hinder tight packing with the flat sterol ring of cholesterol, the reason for the preferential interaction of cholesterol with SM is still a debated issue.

The first visualization of "raft-like domains" in model membranes was achieved by Dietrich et al. They visualized liquid ordered domains in supported bilayers and GUVs composed not only of synthetic lipid mixtures but also of lipid extracts from brush border membrane, the apical membrane of intestinal cells. Domain formation was cholesterol-dependent, since domains disappeared after treatment with the cholesterol-extracting drug methyl--cyclodextrin. Another big step forward was the establishment of a ternary phase diagram of SM/PC/cholesterol at the physiological temperature of 37 C. This predicts the coexistence of liquid-ordered and liquid-disordered phases for a wide range of compositions mimicking those occurring in the plasma membrane of cells. Most domains observed in model membranes are rather large (i. e., several micrometer in diameter) or they start small when they are being formed and then grow continuously by collision and fusion as the system reaches equilibrium. Contrary to this, raft domains in cells are believed to be small, most likely because the cell membrane is not at equilibrium (see below). Interestingly, fluorescence resonance energy transfer (FRET) measurements on vesicles composed of a ternary lipid mixture mimicking the outer leaflet of the plasma membrane revealed heterogeneities (i. e., domains) of sizes in the tens of nanometer range at 37 C. Large domains were observed with the same lipid mixture only below 20 C.

A slightly different interpretation of liquid-liquid immiscibility observed in model membranes was proposed by McConnell and colleagues. These authors argue for the formation of "condensed complexes" between cholesterol and SM rather than a liquid-ordered phase or domain. The name originates from the observation that cholesterol and SM occupy less surface area when mixed together compared to the sum of the areas occupied by each component alone before mixing. Such a complex is supposed to contain 15-30 molecules with a fixed stoichiometry of 2:1 (SM: cholesterol). These complexes could exist in quite high concentration without necessarily leading to a phase separation (for a review, see). However, the condensed complex theory was developed on monolayer membranes and has not yet been validated for bilayers.

Taken together, there is clear evidence for lipid-driven domain formation in model membrane systems mimicking the outer leaflet of the plasma membrane. On the contrary, domain formation could not be observed in lipid mixtures mimicking the inner leaflet of the plasma membrane. The intermolecular forces leading to phase separation are van der Waals interactions between saturated acyl chains and cholesterol, as well as forces such as hydrophobic shielding or the "umbrella effect", described for cholesterol filling the holes left between the acyl chains of glycosphingolipids with large headgroups. However, none of the systems described so far has included proteins in their analysis, and the question remains whether proteins choose the domain they partition into, or whether they organize a domain around them.

Partitioning experiments have been performed, in which proteins were reconstituted into model membranes, and their phase distribution was analyzed. In this way, glycosyl-phosphatidyl-inositol (GPI)-anchored placental alkaline phosphatase (PLAP; and Thy-1 were shown to partition into the liquid-ordered phase, and the chain length of the GPI-anchor was shown to be important for partitioning of the protein. Similarly, peptides modified with prenyl groups were excluded from liquid-ordered domains, while peptides modified with cholesterol or palmityl chains partitioned significantly into the ordered phase. Partitioning studies with synthetic transmembrane peptides revealed that longer transmembrane domains are incorporated better into liquid-ordered domains than shorter versions. Another important determinant for the partitioning of a molecule is the size and orientation of its dipole moment. The membrane dipole moment is stronger in ordered phases where the dipoles are better aligned. Only molecules displaying a dipole moment with the same orientation as the dipolar potential of the membrane, are predicted to be able to enter the ordered phase. Nevertheless, our knowledge about lipid-transmembrane protein interactions is still scarce and this area of research is a major challenge.

1.4 Evidence for Phase Separation in Cell Membranes: The "Raft Concept"

There are several indications for cell membranes being inhomogeneous fluids and for the existence of lipid-driven phase separation. One key finding was the selective co-clustering of certain membrane components and segregation from others upon application of antibodies to living cells. Co-clustering of lipids was first observed in lymphocytes, where one ganglioside species was capped with antibodies and another species was found to redistribute into the cap. It was then shown that simultaneous addition of two antibodies against apparently homogeneously distributed surface antigens could, in selected cases, lead to their co-clustering and in other cases to their segregation. These findings were explained by certain proteins residing in small raft domains that are below the light microscopic resolution in size, and others residing outside the raft domains. Upon cross-linking by antibodies the small raft domains coalesce into visible, stable clusters that contain several different raft proteins. The antigens that were previously in the non-raft environment are excluded from the coalescing domains and thus form separate clusters upon cross-linking. How these large-scale domains containing multiple raft components could be formed in a homogeneous membrane without the occurrence of phase separation is not obvious, and an alternative explanation for this phenomenon has not been put forward. Since then, two techniques have been used to directly assess liquid order in living cells. Gidwani et al. measured the steady-state anisotropy of the lipid-probe DPH-PC, which is sensitive to cholesterol-induced liquid order. With this approach, they found that approximately 40% of the plasma membrane of mast cells is in a liquid-ordered state. More recently, Gaus et al. were able to directly visualize liquid-ordered domains in living macrophages on the light microscopic level. They applied two-photon imaging of the amphiphilic dye LAURDAN, which changes its emission peak depending on the state of its lipid environment.

Other techniques have also been employed for assessing raft domains in living cells, most of them analyzing the distribution and dynamics of membrane proteins rather than lipids. Pralle et al. measured the local diffusion of a bead attached to a single protein molecule in the plasma membrane of fibroblasts within an area smaller than 100 nm in diameter. In this way, diffusion was not hindered by cytoskeletal constraints but was supposed to be free. Proteins previously shown to be resistant to detergent extraction diffused three times slower than detergent-soluble proteins. After cholesterol depletion, the former diffused as fast as the latter. The first group of proteins was thus assumed to reside in a raft environment and to diffuse together with the whole raft entity. After destruction of this entity by cholesterol extraction the proteins behaved as if they were diffusing in a non-raft environment. From the viscous drag and from the diffusion coefficient, the size of the raft entities was calculated to be approximately 50 nm in diameter. Extrapolated from average protein and lipid densities in cell membranes, one raft entity was calculated to contain roughly 3000 lipid molecules and 10-20 proteins.

(Continues...)

Excerpted from Lipid Rafts and Caveolae Copyright © 2006 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Excerpted by permission.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

Edition details

• Publisher: Wiley-VCH
• Publication date: 2006
• Language: English
• ISBN 10: 3527312617
• ISBN 13: 9783527312610
• Binding: Hardcover
• Edition number: 1
• Number of pages: 294
• Editor: Fielding Christopher J.