BBA - Molecular Cell Research (v.1746, #3)

Seeing spots: Complex phase behavior in simple membranes by Sarah L. Veatch; Sarah L. Keller (172-185).
Liquid domains in model lipid bilayers are frequently studied as models of raft domains in cell plasma membranes. Micron-scale liquid domains are easily produced in vesicles composed of ternary mixtures of a high melting temperature lipid, a low melting temperature lipid, and cholesterol. Here, we describe the rich phase behavior observed in binary and ternary systems. We then discuss experimental challenges inherent in mapping phase diagrams of even simple lipid systems. For example, miscibility behavior varies with lipid type, lipid ratio, lipid oxidation, and level of impurity. Liquid domains are often circular, but can become noncircular when membranes are near critical points. Finally, we reflect on applications of phase diagrams in model systems to rafts in cell membranes.
Keywords: Lipid; Cholesterol; Phase diagram; Miscibility; Raft; Liquid-ordered; Gel;

Fluorescence methods to detect phase boundaries in lipid bilayer mixtures by Frederick A. Heberle; Jeffrey T. Buboltz; David Stringer; Gerald W. Feigenson (186-192).
Phase diagrams of lipid mixtures can show several different regions of phase coexistence, which include liquid-disordered, liquid-ordered, and gel phases. Some phase regions are small, and some have sharp boundaries. The identity of the phases, their location in composition space, and the nature of the transitions between the phases are important for understanding the behavior of lipid mixtures. High fidelity phase boundary detection requires high compositional resolution, on the order of 2% compositional increments. Sample artifacts, especially the precipitation of crystals of anhydrous cholesterol, can occur at higher cholesterol concentrations unless precautions are taken. Fluorescence resonance energy transfer (FRET) can be used quantitatively to find the phase boundaries and even partition coefficients of the dyes between coexisting phases, but only if data are properly corrected for non-FRET contributions. Self-quenching of the dye fluorescence can be significant, distorting the data at dye concentrations that intuitively might be considered acceptable. Even more simple than FRET experiments, measurements of single-dye fluorescence can be used to find phase boundaries. Both FRET and single-dye fluorescence readily detect the formation of phase domains that are much smaller than the wavelength of light, i.e. “nanoscopic” domains.
Keywords: Ternary mixture; Phase boundary; Fluorescence resonance energy transfer; Single-dye fluorescence; Nanoscopic domain; Cholesterol;

The special physical and functional properties ascribed to lipid rafts in biological membranes reflect their distinctive organization and composition, properties that are hypothesized to rest in part on the differential partitioning of various membrane components between liquid-ordered and liquid-disordered lipid environments. This review describes the principles and findings of recently developed methods to monitor the partitioning of membrane proteins and lipids between liquid-ordered and liquid-disordered domains in model membranes, and how these approaches can aid in elucidating the properties of rafts in biological membranes.
Keywords: Membrane domain; Cholesterol; Cellular membrane; Lipid raft; Fluorescence spectroscopy; Fluorescence microscopy; Electron spin resonance; Sphingolipid;

Sphingolipid and cholesterol-rich liquid ordered lipid domains (lipid rafts) have been studied in both eukaryotic cells and model membranes. However, while the coexistence of ordered and disordered liquid phases can now be easily demonstrated in model membranes, the situation in cell membranes remains ambiguous. Unlike the usual situation in model membranes, under most conditions, cell membranes rich in sphingolipid and cholesterol may have a “granular” organization in which the size of ordered and/or disordered domains is extremely small and domains may be of borderline stability. This review attempts to explain the origin of the divergence between of our understanding of rafts in model membranes and in cells, and how the physical properties of model membranes can help explain many of the ambiguities concerning raft formation and properties in cells. How physical principles of ordered domain formation relate to limitations of detergent insolubility and cholesterol depletion methods used to infer the presence of rafts in cells is also discussed. Possible modifications of these techniques that may increase their reliability are considered. It will be necessary to study model membrane systems more closely approximating cell membranes in order gain a complete understanding of raft properties in cells. Very high concentrations of membrane cholesterol and proteins may explain key physical characteristics of domains in cellular membranes, and are the two of the most obvious factors requiring additional study.
Keywords: Phase separation; Phase diagrams; Triton X-100; DRM; Lipid rafts; Cholesterol; Sphingolipid; Plasma membrane; Liquid ordered state; Liquid disordered state;

Rafts in cell membranes have been a subject of much debate and many models have been proposed for their existence and functional significance. Recent studies using Forster's resonance energy transfer (FRET) microscopy have provided one of the first glimpses into the organization of putative raft components in living cell membranes. Here we discuss how and why FRET microscopy provides an appropriate non-invasive methodology to examine organization of raft components in cell membranes; a combination of homo and hetero-FRET microscopy in conjunction with detailed theoretical analyses are necessary for characterizing structures at nanometre scales. Implications of the physical characteristics of the organization of GPI-anchored proteins in cell membranes suggest new models of lipid-based assemblies in cell membranes based on active principles.
Keywords: Raft; GPI-anchored protein; Homo-FRET; Hetero-FRET; Microscopy; Active organization;

The cell membrane is a 2-dimensional non-ideal liquid containing dynamic structures on various time-space scales, and the raft domain is one of them. Existing literature supports the concept that raft dynamics may be important for its formation and function: the raft function may be supported by stimulation-induced raft association/coalescence and recruitment of various raftophilic molecules to coalesced rafts, and, importantly, they both may happen transiently. Thus, one must always consider the limited association time of a raft or a raftophilic molecule with another raft, even when one interprets the results of static experiments, such as immunofluorescence and pull-down assays. Critical considerations on the chemical fixation mechanism and immunocolocalization data suggest that the temporary nature of raft-based molecular interactions may explain why colocalization results are sensitive to subtle variations in experimental conditions employed in different laboratories.
Keywords: Raft domain; Time scale; Transient recruitment; Chemical fixation; Immunofluorescence colocalization; Temporary molecular interaction;

Lipid segregation and IgE receptor signaling: A decade of progress by David Holowka; Julie A. Gosse; Adam T. Hammond; Xuemei Han; Prabuddha Sengupta; Norah L. Smith; Alice Wagenknecht-Wiesner; Min Wu; Ryan M. Young; Barbara Baird (252-259).
Recent work to characterize the roles of lipid segregation in IgE receptor signaling has revealed a mechanism by which segregation of liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment. Previous work showed that the membrane skeleton coupled to F-actin regulates stimulated receptor phosphorylation and downstream signaling processes, and more recent work implicates cytoskeletal interactions with ordered lipid rafts in this regulation. These and other results provide an emerging view of the complex role of membrane structure in orchestrating signal transduction mediated by immune and other cell surface receptors.
Keywords: Lipid raft; Immunoreceptor signaling; Tyrosine kinase and phosphatase; Cytoskeleton; Membrane domain;

Growth factor receptors have been shown to be localized to lipid rafts and caveolae. Consistent with a role for these cholesterol-enriched membrane domains in growth factor receptor function, the binding and kinase activities of growth factor receptors are susceptible to regulation by changes in cholesterol content. Furthermore, knockouts of caveolin-1, the structural protein of caveolae, have confirmed that this protein, and by implication caveolae, modulate the ability of growth factor receptors to signal. This article reviews the findings pertinent to the relationship between growth factor receptors, lipid rafts and caveolae and presents a model for understanding the disparate observations regarding the role of membrane microdomains in the regulation of growth factor receptor function.
Keywords: Lipid raft; Caveola; Cholesterol; Growth factor; Insulin receptor; EGF receptor;

Ras signaling from plasma membrane and endomembrane microdomains by S.J. Plowman; J.F. Hancock (274-283).
Ras proteins are compartmentalized by dynamic interactions with both plasma membrane microdomains and intracellular membranes. The mechanisms underlying Ras compartmentalization involve a series of protein/lipid, lipid/lipid and cytoskeleton interactions, resulting in the generation of discrete microdomains from which Ras operates. Segregation of Ras proteins to these different platforms regulates the formation of Ras signaling complexes and the generation of discrete signal outputs. This temporal and spatial modulation of Ras signal transduction provides a mechanism for the generation of different biological outcomes from different Ras isoforms, as well as flexibility in the signal output from a single activated isoform.
Keywords: Ras; Microdomain; Lipid raftt; Acylation; Plasma membrane; Endomembrane;

Ceramide-enriched membrane domains by Claudia R. Bollinger; Volker Teichgräber; Erich Gulbins (284-294).
Cellular activation involves the re-organization of receptor molecules and the intracellular signalosom in the cell membrane. Recent studies indicate that specialized domains of the cell membrane, termed rafts, are central for the spatial organization of receptors and signaling molecules. Rafts are converted into larger membrane platforms by activity of the acid sphingomyelinase, which hydrolyses raft-sphingomyelin to ceramide. Ceramide molecules spontaneously associate to form ceramide-enriched microdomains, which fuse to large ceramide-enriched membrane platforms. The acid sphingomyelinase is activated by multiple stimuli including CD95, CD40, DR5/TRAIL, CD20, FcγRII, CD5, LFA-1, CD28, TNF, the Interleukin-1 receptor, the PAF-receptor, CD14, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, treatment with γ-irradiation, UV-light, doxorubicin, cisplatin, disruption of integrin-signaling and under some conditions of developmental death. Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.
Keywords: Ceramide; Acid sphingomyelinase; Membrane domain; Cellular infection;

In recent years, it has been unambiguously shown that caveolae and lipid rafts can internalize cargo upon stimulation by multivalent ligands, demonstrated by the infectious entry routes of certain non-enveloped viruses that bind integrins or glycosphingolipids. We currently understand little about the membrane trafficking principles of this endocytic route, but it is clear that we cannot use paradigms from classical membrane traffic. Recent evidence indicates that caveolae- and lipid raft-mediated endocytosis plays important roles in cell adhesion and anchorage-dependent cell growth, but the underlying mechanisms are not known. In this review, I will introduce new models based on current research that aims at identifying the core machinery, regulatory components and design principles of this endocytic route in order to understand its role in cellular physiology. Again, viruses are proving to be excellent tools to reach that goal.
Keywords: Caveolae; Caveosome; Simian Virus 40; Polyoma Virus; Echovirus 1; Endocytosis; Lipid raft; Integrin; Ganglioside; Membrane trafficking;

The role of lipid rafts in the pathogenesis of bacterial infections by David W. Zaas; Matthew Duncan; Jo Rae Wright; Soman N. Abraham (305-313).
Numerous pathogens have evolved mechanisms of co-opting normal host endocytic machinery as a means of invading host cells. While numerous pathogens have been known to enter cells via traditional clathrin-coated pit endocytosis, a growing number of viral and bacterial pathogens have been recognized to invade host cells via clustered lipid rafts. This review focuses on several bacterial pathogens that have evolved several different mechanisms of co-opting clustered lipid rafts to invade host cells. Although these bacteria have diverse clinical presentations and many differences in their pathogenesis, they each depend on the integrity of clustered lipid rafts for their intracellular survival. Bacterial invasion via clustered lipid rafts has been recognized as an important virulence factor for a growing number of bacterial pathogens in their battle against host defenses.
Keywords: Bacteria; Lipid raft; Caveolae; Caveolin;

Raft trafficking of AB5 subunit bacterial toxins by Wayne I. Lencer; David Saslowsky (314-321).
Cholera and the related AB5-subunit toxins co-opt plasma membrane (PM) glycolipids to move retrograde into the endoplasmic reticulum (ER) of the host cell where a portion of the toxin is retro-translocated to the cytosol to induce disease. Only glycolipids that associate strongly with detergent insoluble membrane microdomains can sort the toxins backwards from PM to ER. The way certain lipids and proteins are clustered in the plane of the membrane to form lipid rafts likely explains how the glycolipids can function as sorting motifs for the toxins.
Keywords: AB5 toxin; GM1; Lipid raft; Retrograde transport; Ceramide; Ganglioside; Sphingomyelin; Cholesterol; Cholera toxin; Shiga toxin;

Getting rid of caveolins: Phenotypes of caveolin-deficient animals by Soazig Le Lay; Teymuras V. Kurzchalia (322-333).
The elucidation of the role of caveolae has been the topic of many investigations which were greatly enhanced after the discovery of caveolin, the protein marker of these flask-shaped plasma membrane invaginations. The generation of mice deficient in the various caveolin genes (cav-1, cav-2 and cav-3) has provided physiological models to unravel the role of caveolins or caveolae at the whole organism level. Remarkably, despite the essential role of caveolins in caveolae biogenesis, all knockout mice are viable and fertile. However, lack of caveolae or caveolins leads to a wide range of phenotypes including muscle, pulmonary or lipid disorders, suggesting their implication in many cellular processes. The aim of this review is to give a broad overview of the phenotypes described for the caveolin-deficient mice and to link them to the numerous functions so far assigned to caveolins/caveolae.
Keywords: Caveolin; Caveolae; Knockout mouse; Metabolic disorder; Signaling;

Structure of caveolae by Radu V. Stan (334-348).
The introduction of the electron microscope to the study of the biological materials in the second half of the last century has dramatically expanded our view and understanding of the inner workings of cells by enabling the discovery and study of subcellular organelles. A population of flask-shaped or spherical invaginations of the plasma membrane were described and named plasmalemmal vesicles or caveolae. Until the discovery of caveolin-1 as their first molecular marker in early 1990s, the study of caveolae was the exclusive domain of electron microscopists that demonstrated caveolae at different surface densities in most mammalian cells with few exceptions. Electron microscopy techniques in combination with other approaches have also revealed the structural features of caveolae as well as some of their protein and lipid residents. This review summarizes the data on the structure and components of caveolae and their stomatal diaphragms.

A number of recent studies have provided new insights into the complexity of the endocytic pathways originating at the plasma membrane of mammalian cells. Many of the molecules involved in clathrin coated pit internalization are now well understood but other pathways are less well defined. Caveolae appear to represent a low capacity but highly regulated pathway in a restricted set of tissues in vivo. A third pathway, which is both clathrin- and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluid. The relationship of this pathway, if any, to macropinocytosis or to the endocytic pathways of lower eukaryotes remains an interesting open question. Our understanding of the regulatory mechanisms and molecular components involved in this pathway are at a relatively primitive stage. In this review, we will consider some of the characteristics of different endocytic pathways in high and lower eukaryotes and consider some of the common themes in endocytosis. One theme which becomes apparent from comparison of these pathways is that apparently different pathways can share common molecular machinery and that pathways considered to be distinct actually represent similar basic pathways to which additional levels of regulatory complexity have been added.
Keywords: Endocytosis; Lipid raft; Caveolae; Cholesterol; Clathrin;

Cumulative Contents (364-365).