Tolerance to changes in membrane lipid composition as a selected trait of membrane proteins

Membrane lipid composition can vary dramatically across the three domains of life and even within single organisms. Here we review evidence that the lipid-exposed surfaces of membrane proteins have generally evolved to maintain correct structure and function in the face of major changes in lipid composition. Such tolerance has allowed evolution to extensively remodel membrane lipid compositions during the emergence of new species without having to extensively remodel the associated membrane proteins. The tolerance of membrane proteins also permits single-cell organisms to vary their membrane lipid composition in response to their changing environments and allows dynamic and organelle-specific variations in the lipid compositions of eukaryotic cells. Membrane protein structural biology has greatly benefited from this seemingly intrinsic property of membrane proteins: the majority of structures determined to date have been characterized under model membrane conditions that little resemble those of native membranes. Nevertheless, with a few notable exceptions, most experimentally determined membrane protein structures appear, to a good approximation, to faithfully report on native structure.     

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.     

Lipid spirals in Bacillus subtilis and their role in cell division

The fluid mosaic model of membrane structure has been revised in recent years as it has become evident that domains of different lipid composition are present in eukaryotic and prokaryotic cells. Using membrane binding fluorescent dyes, we demonstrate the presence of lipid spirals extending along the long axis of cells of the rod-shaped bacterium Bacillus subtilis. These spiral structures are absent from cells in which the synthesis of phosphatidylglycerol is disrupted, suggesting an enrichment in anionic phospholipids. Green fluorescent protein fusions of the cell division protein MinD also form spiral structures and these were shown by fluorescence resonance energy transfer to be coincident with the lipid spirals. These data indicate a higher level of membrane lipid organization than previously observed and a primary role for lipid spirals in determining the site of cell division in bacterial cells.     

Changes in membrane lipid composition cause alterations in epithelial cell–cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell–cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell–cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell–cell adhesion structures in renal papillary collecting duct cells.     

The Fluid—Mosaic Model of Membrane Structure: Still relevant to understanding the structure,function and dynamics of biological membranes after more than 40 years

In 1972 the Fluid—Mosaic Membrane Model of membrane structure was proposed based on thermodynamic principals of organization of membrane lipids and proteins and available evidence of asymmetry and lateral mobility within the membrane matrix [S. J. Singer and G. L. Nicolson, Science 175 (1972) 720–731]. After over 40 years, this basic model of the cell membrane remains relevant for describing the basic nano-structures of a variety of intracellular and cellular membranes of plant and animal cells and lower forms of life. In the intervening years, however, new information has documented the importance and roles of specialized membrane domains, such as lipid rafts and protein/glycoprotein complexes, in describing the macrostructure, dynamics and functions of cellular membranes as well as the roles of membrane-associated cytoskeletal fences and extracellular matrix structures in limiting the lateral diffusion and range of motion of membrane components. These newer data build on the foundation of the original model and add new layers of complexity and hierarchy, but the concepts described in the original model are still applicable today. In updated versions of the model more emphasis has been placed on the mosaic nature of the macrostructure of cellular membranes where many protein and lipid components are limited in their rotational and lateral motilities in the membrane plane, especially in their natural states where lipid–lipid, protein–protein and lipid–protein interactions as well as cell–matrix, cell–cell and intracellular membrane-associated protein and cytoskeletal interactions are important in restraining the lateral motility and range of motion of particular membrane components. The formation of specialized membrane domains and the presence of tightly packed integral membrane protein complexes due to membrane-associated fences, fenceposts and other structures are considered very important in describing membrane dynamics and architecture. These structures along with membrane-associated cytoskeletal and extracellular structures maintain the long-range, non-random mosaic macro-organization of membranes, while smaller membrane nano- and submicro-sized domains, such as lipid rafts and protein complexes, are important in maintaining specialized membrane structures that are in cooperative dynamic flux in a crowded membrane plane. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

生物膜结构研究的一些进展

膜蛋白三维结构的解析存在很多困难.最近几年由于一些通道(如K⁺通道,Cl^-通道,水通道Aquaporin 1等)和泵（如Ca^2＋泵）的结晶获得成功,这些膜蛋白具有原子分辨率三维结构的解析才得以完成,从而基本阐明一些极性分子和离子选择性通过生物膜的分子机理.在膜脂结构方面,动物细胞质膜膜脂的分布是不均匀的.近年来已多方面证明,质膜具有一些被命名为“脂筏（lipid rafts）”和“质膜微囊（Caveolae）”的微区.它们富含鞘脂和胆固醇。简单介绍了这些脂质微区的大小、组分以及动态变化.根据研究结果,这类脂质微区含有大量信号分子,很可能具有信号传递中心的作用.此外,对脂筏在膜运送过程中的作用也进行一些评述.     

An update on plant membrane rafts

The dynamic segregation of membrane components within microdomains, such as the sterol-enriched and sphingolipid-enriched membrane rafts, emerges as a central regulatory mechanism governing physiological responses in various organisms. Over the past five years, plasma membrane located raft-like domains have been described in several plant species. The protein and lipid compositions of detergent-insoluble membranes, supposed to contain these domains, have been extensively characterised. Imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level at the plant plasma membrane, correlating detergent insolubility and membrane-domain localisation of presumptive raft proteins. Finally, the dynamic association of specific proteins with detergent-insoluble membranes upon environmental stress has been reported, confirming a possible role for plant rafts as signal transduction platforms, particularly during biotic interactions.     

Tracking microdomain dynamics in cell membranes

Studies of the diffusion of proteins and lipids in the plasma membrane of cells have long pointed to the presence of membrane domains. A major challenge in the field of membrane biology has been to characterize the various cellular structures and mechanisms that impede free diffusion in cell membranes and determine the consequences that membrane compartmentalization has on cellular biology. In this review, we will provide a brief summary of the classes of domains that have been characterized to date, focusing on recent efforts to identify the properties of lipid rafts in cells through measurements of protein and lipid diffusion.     

Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.     

Lipids as targeting signals: lipid rafts and intracellular trafficking

Our view of biological membranes has evolved dramatically over the last few decades. In the bilayer model from Singer & Nicholson (Science 1972;175:720-731), both proteins and lipids freely diffuse within the plane of the membrane. Currently, however, membranes are viewed as a mosaic of different compartments or domains maintained by an active cytoskeleton network (Ritchie et al. Mol Membr Biol 2003; 20:13-18). Due to interactions between membrane components, several types of subdomains can form with different characteristics and functions. Lipids are likely to play an important role in the formation of so-called lipid-enriched microdomains or lipid rafts, adding another order of complexity to the membrane model. Rafts represent a type of domain wherein lipids of specific chemistry may dynamically associate with each other, to form platforms important for membrane protein sorting and construction of signaling complexes (Simons & Toomre. Nat Rev Mol Cell Biol 2000;1:31-39). Currently, there are several hypotheses concerning the nature of rafts (reviewed in (Edidin. Annu Rev Biophys Biomol Struct 2003;32: 257-283; Zurzolo et al. EMBO Rep 2003;4:1117-1121)). The most commonly cited one, proposed by Kai Simons (Simons & Ikonen. Nature 1997;387:569-572; Pralle et al. J Cell Biol 2000;148:997-1008), suggests that rafts are relatively small structures ( approximately 50 nm) enriched in cholesterol and sphingolipids within which associated proteins are likely to be concentrated. Another proposal (Anderson & Jacobson. Science 2002;296:1821-1825) suggests that rafts are constructed of lipid shells. These are small dynamic assemblies wherein 'raft' proteins are preferentially associated with certain types of lipids. These 'shells' are thermodynamically stable mobile entities in the plane of the membrane that are able to target the protein they encase to preexisting rafts/caveolae domains. In this review we summarize the data suggesting a specific role for lipid domains in intracellular trafficking and sorting and present a modification of the raft model that may help explain the observed phenomena.     

Glycolipid-Enriched Caveolae and Caveolae-Like Domains in the Nervous System

Abstract: Recent years have been characterized by a booming interest in research on caveolae and caveolae-like membrane domains. The interest in this subject grew further, when their involvement in fundamental membrane-associated events, such as signal transmission and lipid/protein sorting, was postulated. Substantial progress has been reached in understanding the biological role of membrane domains in eukaryotic cells. The neuron, however, which perhaps represents one of the greatest challenges to research on membrane traffic and function, has only been partially investigated. The purpose of the present review is to survey this issue in the nervous system. We confine ourselves to the presence of membrane domains in the nervous system and discuss this in the context of three facts: first, glycolipids are peculiarly enriched in both caveolae and caveolae-like domains and are particularly abundant in the nervous system; second, the neuron is characterized by a basic dual polarity, similar in this respect to other polarized cells, where the role of glycolipid-enriched domains for lipid/protein sorting has been better ascertained; and third, neurons evolved from, and are related to, simpler eukaryotic cells, allowing us to find analogies with more investigated nonneuronal cells.     

The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function

Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to - and degradation of - the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott-Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.     

Diversity and versatility of lipid-protein interactions revealed by molecular genetic approaches

The diversity in structures and physical properties of lipids provides a wide variety of possible interactions with proteins that affect their assembly, organization, and function either at the surface of or within membranes. Because lipids have no catalytic activity, it has been challenging to define many of their precise functions in vivo in molecular terms. Those processes responsive to lipids are attuned to the native lipid environment for optimal function, but evidence that lipids with similar properties or even detergents can sometimes partially replace the natural lipid environment has led to uncertainty as to the requirement for specific lipids. The development of strains of microorganisms in which membrane lipid composition can be genetically manipulated in viable cells has provided a set of reagents to probe lipid functions. These mutants have uncovered previously unrecognized roles for lipids and provided in vivo verification for putative functions described in vitro. In this review, we summarize how these reagent strains have provided new insight into the function of lipids. The role of specific lipids in membrane protein folding and topological organization is reviewed. The evidence is summarized for the involvement of anionic lipid-enriched domains in the organization of amphitropic proteins on the membrane surface into molecular machines involved in DNA replication and cell division.     

Structural Interactions between Lipids, Water and S1-S4 Voltage-Sensing Domains

Membrane proteins serve crucial signaling and transport functions, yet relatively little is known about their structures in membrane environments or how lipids interact with these proteins. For voltage-activated ion channels, X-ray structures suggest that the mobile voltage-sensing S4 helix would be exposed to the membrane, and functional studies reveal that lipid modification can profoundly alter channel activity. Here, we use solid-state NMR to investigate structural interactions of lipids and water with S1-S4 voltage-sensing domains and to explore whether lipids influence the structure of the protein. Our results demonstrate that S1-S4 domains exhibit extensive interactions with lipids and that these domains are heavily hydrated when embedded in a membrane. We also find evidence for preferential interactions of anionic lipids with S1-S4 domains and that these interactions have lifetimes on the timescale of ≤ 10^− 3 s. Arg residues within S1-S4 domains are well hydrated and are positioned in close proximity to lipids, exhibiting local interactions with both lipid headgroups and acyl chains. Comparative studies with a positively charged lipid lacking a phosphodiester group reveal that this lipid modification has only modest effects on the structure and hydration of S1-S4 domains. Taken together, our results demonstrate that Arg residues in S1-S4 voltage-sensing domains reside in close proximity to the hydrophobic interior of the membrane yet are well hydrated, a requirement for carrying charge and driving protein motions in response to changes in membrane voltage.     

FSCS Reveals the Complexity of Lipid Domain Dynamics in the Plasma Membrane of Live Cells

The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane.     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Quantitative characterization of the lateral distribution of membrane proteins within the lipid bilayer.

The dependence of the lateral distribution of membrane proteins on the size, protein/lipoid molar ratio, and the magnitude of the interaction potentials has been investigated by computer modeling protein-lipid distributions with Monte Carlo calculations. These results have allowed us to develop a quantitative characterization of the distribution of membrane proteins and to correlate these distributions with experimental observables. The topological arrangement of protein domains, protein plus annular lipid domains, and free lipid domains is described in terms of radial distribution, pair connectedness, and cluster distribution functions. The radial distribution functions are used to measure the distribution of intermolecular distances between protein molecules, whereas the pair connectedness functions are used to estimate the physical extension of compositional domains. It is shown that, at characteristic protein/lipid molar ratios, previously isolated domains become connected, forming domain networks that extend over the entire membrane surface. These changes in the lateral connectivity of compositional domains are paralleled by changes in the calculated lateral diffusion coefficients and might have important implications for the regulation of diffusion controlled processes within the membrane.     

Transit of hormonal and EGF receptor-dependent signals through cholesterol-rich membranes

The functional consequences of changes in membrane lipid composition that coincide with malignant growth are poorly understood. Sufficient data have been acquired from studies of lipid binding proteins, post-translational modifications of signaling proteins, and biochemical inhibition of lipidogenic pathways to indicate that growth and survival pathways might be substantially re-directed by alterations in the lipid content of membranes. Cholesterol and glycosphingolipids segregate into membrane patches that exhibit a liquid-ordered state in comparison to membrane domains containing relatively lower amounts of these classes of lipids. These "lipid raft" structures, which may vary in size and stability in different cell types, both accumulate and exclude signaling proteins and have been implicated in signal transduction through a number of cancer-relevant pathways. In prostate cancer cells, signaling from epidermal growth factor receptor (EGFR) to the serine-threonine kinase Akt1, as well as from IL-6 to STAT3, have been demonstrated to be influenced by experimental interventions that target cholesterol homeostasis. The recent finding that classical steroid hormone receptors also reside in these microdomains, and thus may function within these structures in a signaling capacity independent of their role as nuclear factors, suggests a novel means of cross-talk between receptor tyrosine kinase-derived and steroidogenic signals. Potential points of intersection between components of the EGFR family of receptor tyrosine kinases and androgen receptor signaling pathways, which may be sensitive to disruptions in cholesterol metabolism, are discussed. Understanding the manner in which these pathways converge within cholesterol-rich membranes may present new avenues for therapeutic intervention in hormone-dependent cancers.     

Thermodynamics of membrane domains

The concept of lipid rafts and the intense work toward their characterization in biological membranes has spurred a renewed interest in the understanding of domain formation, particularly in the case of cholesterol-containing membranes. The thermodynamic principles underlying formation of domains, rafts, or cholesterol/phospholipid complexes are reviewed here, along with recent work in model and biological membranes. A major motivation for this review was to present those concepts in a way appropriate for the broad readership that has been drawn to the field. Evidence from a number of different techniques points to the conclusion that lipid-lipid interactions are generally weak; therefore, in most cases, massive phase separations are not to be expected in membranes. On the contrary, small, dynamic lipid domains, possibly stabilized by proteins are the most likely outcome. The results on mixed lipid bilayers are used to discuss recent experiments in biological membranes. The clear indication is that proteins partition preferentially into fluid, disordered lipid domains, which is contrary to their localization in ordered, cholesterol/sphingomyelin rafts inferred from detergent extraction experiments on cell membranes. Globally, the evidence appears most consistent with a membrane model in which the majority of the lipid is in a liquid-ordered phase, with dispersed, small, liquid-disordered domains, where most proteins reside. Co-clustering of proteins and their concentration in some membrane areas may occur because of similar preferences for a particular domain but also because of simultaneous exclusion from other lipid phases. Specialized structures, such as caveolae, which contain high concentrations of cholesterol and caveolin are not necessarily similar to bulk liquid-ordered phase.