Membranes

From LipidomicsWiki

Membrane Microdomains

Classically, the cell membrane was regarded as a rather inert lipid bilayer with interspersed biologically active proteins. This Singer–Nicholson fluid mosaic concept proposed that the lipid bilayer functions as a neutral two-dimensional solvent, having little influence on membrane protein function. However, recently it was discovered, that within model lipid bilayers certain lipids exist in several distinct phases, and may assume states of increasing liquid-disorder, such as gel, liquid-ordered and liquid-disordered states. In the liquid-ordered phase, phospholipids with saturated hydrocarbon chains pack tightly with cholesterol but nevertheless remain mobile in the plane of the membrane. This prompted the discovery of specialized membrane microdomains, which perform a crucial function in membranes by providing a platform for the assembly of receptors to form specialized, both ligand and cellspecific signaling complexes. These specialized membrane domains are characterized by their detergent-insolubility and by a unique lipid-rich composition, which have been functionally assigned the name: detergent-insoluble glycolipid-enriched complexes (DIGs). As opposed to the Singer-Nicholson fluid mosaic concept, regarding the plasma membrane lipid bilayer as an inert hydrophobic barrier including functional proteins, the raft model postulated and in many cases proved the existence of dynamic lipid structures within the plasma membrane (Simons). Rafts are enriched in cholesterol, tightly connected with sphingolipids and phospholipids, and contain proteins with hydrophobic membrane anchors (Harder, Graf). Of these, the glycosylphosphadidylinositol (GPI)-anchored proteins, including the protein-tyrosine kinases, acylated and prenylated proteins, such as the α-subunits of the heterotrimeric G proteins, cholesterol- and fatty acid-linked proteins, including hedgehog, as well as other short chain fatty acid conjugated membrane proteins form the most prominent groups (Figure 61). Membrane microdomains or rafts constitute platforms for the dynamic assembly of both celland stimulus specific receptor clusters, which are of utmost importance for the regulation of cell adhesion, receptor binding, co-activation and signal transduction. One of the major functions of rafts is the formation of an organized platform concentrating individual receptors, ligands and effectors on both sides of the membrane. If receptor activation by ligand binding takes place in a lipid raft, the signaling complex is protected from non-raft enzymes such as membrane phosphatases that otherwise could affect the signaling process. Rafts thus have an important regulatory function in preventing inappropriate activation of inhibition processes. In general, raft binding recruits receptor-proteins, such as growth factor receptors, to a new micro-environment, where the phosphorylation state can be modified by local kinases and phosphatases, resulting in enhanced downstream signaling, which occurs with increased stringency. The specific inclusion or exclusion of particular proteins, allowing a specific concentration of functionally interacting partners is one further major property of membranemicrodomains. In the case of growth factor receptors it may function as a pre-stimulatory biological signal, that the cell is prepared to generate cell membrane lipids necessary for cell division, and allow the growth factor receptor to be activated. These membrane platforms additionally constitute crucial docking sites for adaptor molecules, scaffolds and serve as bridging elements for regulated domain interaction. In fact, distinct types of membrane microdomains, such as rafts or caveolae, are not only defined by the specific composition of glycosphingolipids, but their functional diversity is due to this distinctive distribution of glycolipids. Currently, three structural types of glycoclusters are distinguishable (Figure 62):
1) glycosphingolipids organized with cytoplasmic signal transducers and sphingolipid-bound tetraspanins (Pltsp) with or without growth factor receptors
2) transmembrane mucin-type glycoproteins with clustered O-linked glycoepitopes for cell adhesion and associated signal transducers at the cholesterol-rich membrane domain
3) N-glycosylated transmembrane adhesion receptors complexed with tetraspanins and gangliosides, as typically seen with the integrin-tetraspanin-ganglioside complex (Hakomori).
The structural variety of glycoclusters and the carbohydrate and lipid-dependent cell signaling and cell adhesion processes indicate a potentially great role for the induction of cell
differentiation and maintenance.

Another specialized form of membrane microdomains, the caveolae, further include a unique protein called "caveolin" (Graf, Anderson). Caveolae constitute caveolin-induced Figure 62. Schematic models of types 1, 2, and 3 glycosynapse.
(A) Type 1 glycosynapse with GSL clusters, PL tetraspanin (PLtsp), and growth factor receptor (in this example EGF-R). Clusters of GSLs are organized with signal transducer molecules (TDa, TDb). Stimulation of GSL region ‘‘a’’ causes strong signaling ‘‘x’’ through Tda, whereas stimulation of region ‘‘b’’ causes weaker signaling ‘‘y’’ through TDb because of the presence of the blocking factor PLtsp in that region. When EGF-R is located in a GSL-rich domain, signaling through growth factor (EGF) to activate tyrosine phosphorylation (P-Y) is blocked by the association of EGF-R with GSL (in this example, ganglioside GM3). Binding of GM3 to EGF-R may result from interaction of GM3 with carbohydrate N-linked to EGF-R, as suggested by a previous study, and by our studies with N-glycosylation inhibitors. Note that the majority of Nglycosylation (pink oval chains) is localized at the fourth domain from the top, close to ganglioside clusters (purple ovals), such that interaction with gangliosides at the membrane surface is favored.
B) Type 2 glycosynapse with mucin-type transmembrane glycoprotein at cholesterol-rich membrane domain. Examples are shown for MUC-1 and PSGL-1. In MUC-1, the number of tandem repeats varies from 20 to 120,and each repeat is a 20-aa sequence. In PSGL-1, the number of tandem repeats is 15, and each repeat is a 10-aa sequence. The units,
having multiple O-linked structure with glycosyl adhesion epitope, are organized with various signal transducers (TDa, TDb, TDc). In human and mouse T-cell lines, cSrc, lck56, Lyn, Fyn, and CD45 are detected. Both MUC-1 and PSGL-1 are associated with a membrane domain rich in cholesterol (indicated by yellow rods). Cells expressing type 2 glycosynapse are capable of binding to cells expressing P-selectin, E-selectin, or siglecs. A specific structure with three tyrosine phosphates and O-linked glycan to define P-selectindependent adhesion in PSGL-1 was recently elucidated.
(C) Type 3 glycosynapse with integrin receptor (ITR) and tetraspanin (Tsp.). N-glycosylation (pink oval chains) of ITR is essential for connection and stabilization of thesubunits and also for interaction of ITR with tetraspanin CD82. Interaction of some tetraspanins (e.g., CD9) with ITR requiresGM3ganglioside. The complex is more stable with complete N-glycosylation and is located at a low-density domain showing resistance to cyclodextrin. invaginations of sphingolipid–cholesterol microdomains, which are capable to trap proteins or lipid clusters associating with rafts. Thereafter, the entire caveolae are internalized in a signal-dependent manner.

Caveolae, which are enriched in GPI-anchored proteins and glycosphingolipids, may act as centers from which multiple signaling pathways originate. However, signaling can also occur in the absence of caveolins that determine caveola formation, for example in T-lymphocytes (Fra). In fact, it is conceivable that in T-lymphocytes, the tetraspan as well as the pentaspan family may provide analogous molecules as caveolins to determine similar specialized membrane microdomains. In contrast to caveolins these receptors, characterized by multiple membrane-spanning units, do not form invaginations, but share the affinity for certain lipid classes. Analysis of the cell-type-specific lipid composition of membrane microdomains (caveolae) reveals that major components differ considerably between cells of different lineages. While caveolae in fibroblasts are particularly rich in sphingomyelin, ceramide and diacylglycerol, the cholesterol content is rather low. By contrast in epithelial cells. e. g. the Madin-Darby canine kidney (MDCK) cell line, cholesterol content of caveolae may approach 25%. Furthermore in these cells, gangliosides and sphingomyelin represent major constituents of caveolae. The lipid composition of membrane microdomains (caveolae) for different cell types is summarized in Table 12.Specialized membrane microdomains are believed to be present on all cell types, including transformed cells, while the distribution of caveolae is more restricted to endothelial cells, fibroblasts, adipocytes, muscle cells and neuronal cells. Moreover the specialized membrane microdomains may be functionally divided to liquid-ordered domains on the plasma membrane, which regulate receptor function and signal transduction, and those "signalosomes" which may regulate the crosstalk between the plasma membrane and the intracellular organelles. Notably, lipids involved in signal transduction have also been localized to DIGs. These include phosphoinositides such as phosphatidylinositol-( 4,5)- bisphosphate and sphingomyelin, which have been long known to exert specific functions in growth and differentiation in multiple cellular systems (Hope). The functional properties of the caveolae, which are divergent from that of classical rafts, are summarized in Figure 63. Besides the plasma membrane, cholesterol and fatty acid-rich membrane microdomains arepresent in intracellular organelles, including the ER, the golgi complex, the nuclear membrane pore complexes and the outer mitochondrial membrane. Evidence is accumulated that these rafts play a crucial role in the recruitment and the concentration of specific membrane lipids and proteins.

Lipids as basic units for Biomembranes

Lipids are central to the regulation and control of cellular processes by acting as basic building units for biomembranes, which are the platforms for the vast majority of cellular
functions. Moreover, membrane microdomains defined by their specific lipid composition are increasingly recognized as central stages for cell regulation; degradation products of
membrane lipids frequently act as signaling molecules regulating gene expression and protein function. The wide spectrum of regulatory or “bioactive” lipids includes metabolites
derived from sterol pathways, eicosanoids, saturated and unsaturated fatty acids, neutral lipids, glycolipids as well as the large array of products derived from enzymatic and
nonenzymatic degradation of sphingo- and glycerophospholipids. The recent developments in lipid mass spectrometry set the scene for a completely new way to understand the composition of membranes, cells and tissues in space and time by allowing the precise identification and quantification of alterations of the total lipid profile after specific perturbations. In combination with most recent proteome and genome analysis tools as well as novel imaging techniques using synthesized probes, it will now be possible to
unravel the complex network between lipids, genes and proteins in an integrated approach.