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Membrane Microdomains (membrane + microdomain)
Kinds of Membrane Microdomains Selected AbstractsMembrane microdomains and proteomics: Lessons from tetraspanin microdomains and comparison with lipid raftsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2006François Le Naour Dr. Abstract Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures. [source] Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transductionGENES TO CELLS, Issue 2 2007A.K.M. Mahbub Hasan A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm,egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm,egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm,egg interaction and signaling during Xenopus fertilization. [source] Initiation of TCR signaling: regulation within CD3 dimersIMMUNOLOGICAL REVIEWS, Issue 1 2003Balbino Alarcón Summary: The number of possible T cell activation outcomes resulting from T cell receptor (TCR) engagement suggests that the TCR is able to differentially activate a myriad of signaling pathways depending on the nature of the stimulus. The complex structural organization of the TCR itself could underlie this diversity of responses. Assembly and stoichiometric studies have helped us to shed some light on the initiation of TCR signaling. The TCR is composed of TCR and CD3 dimers. Changes in the interaction between CD3 subunits within the CD3 dimers and in the interaction of these dimers with the TCR heterodimer could be the triggering mechanism that initiates the first activation events. One of the hallmarks of these early changes in TCR conformation is the induced recruitment of the adapter protein Nck to a proline-rich sequence of the cytoplasmic tail of CD3,, but there may be others. According to our most recent observations, the TCR is organized in pre-existing clusters within plasma membrane microdomains, exhibiting a complexity above and beyond that of dimer composition complexity. How the presence of TCR in clusters influences TCR avidity and propagation of TCR signals is something that has yet to be investigated. [source] Regulation of T-cell receptor signalling by membrane microdomainsIMMUNOLOGY, Issue 4 2004Tahir M. Razzaq Summary There is now considerable evidence suggesting that the plasma membrane of mammalian cells is compartmentalized by functional lipid raft microdomains. These structures are assemblies of specialized lipids and proteins and have been implicated in diverse biological functions. Analysis of their protein content using proteomics and other methods revealed enrichment of signalling proteins, suggesting a role for these domains in intracellular signalling. In T lymphocytes, structure/function experiments and complementary pharmacological studies have shown that raft microdomains control the localization and function of proteins which are components of signalling pathways regulated by the T-cell antigen receptor (TCR). Based on these studies, a model for TCR phosphorylation in lipid rafts is presented. However, despite substantial progress in the field, critical questions remain. For example, it is unclear if membrane rafts represent a homogeneous population and if their structure is modified upon TCR stimulation. In the future, proteomics and the parallel development of complementary analytical methods will undoubtedly contribute in further delineating the role of lipid rafts in signal transduction mechanisms. [source] CD20-mediated apoptosis: signalling through lipid raftsIMMUNOLOGY, Issue 2 2002Julie P. Deans Summary CD20 is an effective target for therapeutic B-cell depletion with monoclonal antibodies. One proposed mechanism of action is direct cytotoxicity mediated via tyrosine kinase-dependent signalling pathways activated upon CD20 cross-linking. The association of CD20 with membrane microdomains known as lipid rafts, enriched in src-family tyrosine kinases and other signalling effectors, suggests an indirect mechanism of anti-CD20-induced apoptosis in which activation of src-family kinases occurs as a consequence of lipid raft clustering. [source] Membrane Photolithography: Direct Micropatterning and Manipulation of Fluid Phospholipid Membranes in the Aqueous Phase Using Deep-UV Light,ADVANCED MATERIALS, Issue 14 2004K. Yee A wet photolithography approach using light-activated, localized, oxidative chemistry can directly pattern fluid phospholipid bilayers submerged in aqueous phases. Targeted incorporation of secondary components within pattern voids (see Figure) allows many membrane dynamical processes to be probed and optically defined arrays of holes, functional membrane microdomains, and proteins embedded in a lipidic background can be designed. [source] Mechanisms for recycling and biosynthesis of endogenous cannabinoids anandamide and 2-arachidonylglycerolJOURNAL OF NEUROCHEMISTRY, Issue 4 2008Ekaterina A. Placzek Abstract The mechanisms of endogenous cannabinoid biosynthesis are not completely understood. We hypothesized that anandamide could be recycled by the cell to form new endocannabinoid molecules and released into the extracellular space. We determined that new endocannabinoids derived from exogenous anandamide or arachidonic acid were synthesized and released from RBL-2H3 cells in response to ionomycin. Treatment of RBL-2H3 cells with nystatin and progesterone, agents that disrupt organization of lipid raft/caveolae, resulted in the attenuation of anandamide and 2-arachidonyl glycerol synthesis and/or release in response to stimulation with ionomycin suggesting a role for these membrane microdomains in endocannabinoid biosynthesis. Furthermore, anandamide synthesis may be independent of N -acyl phosphatidylethanolamine phospholipase D as expression of the enzyme was not detected in RBL-2H3 cells. We also established that extracellular calcium is necessary for endocannabinoid biosynthesis because release of intracellular calcium stores alone does not promote endocannabinoid biosynthesis. Next, we examined the role of calcium as a ,switch' to activate the synthesis of anandamide and simultaneously reduce uptake. Indeed, [3H] anandamide uptake was reduced in the presence of calcium. Our findings suggest a mechanism indicative of calcium-modulated activation of anandamide synthesis and simultaneous termination of uptake. [source] The different forms of PNS myelin P0 protein within and outside lipid raftsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Anna Fasano Abstract It is now well established that plasma membranes, such as the myelin sheath, are made of different microdomains with different lipid and protein composition. Lipid rafts are made mainly of sphingolipids and cholesterol, whereas the non-raft regions are made mainly of phosphoglycerides. Most myelin proteins may distribute themselves in raft and non-raft microdomains but the driving force that gives rise to their different distribution is not known yet. In this paper, we have studied the distribution of protein zero (P0), the most representative protein of PNS myelin, in the membrane microdomains. To this end, we have purified P0 from both non-raft (soluble P0, P0-S) and raft (P0-R) regions of PNS. Purified proteins were analyzed by two-dimensional gel electrophoresis and identified and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A detailed structural description of the two P0 forms is given in terms of amino acid sequence, post-translational modifications, and composition of associated lipids. Our findings suggest that structural differences between the two proteins, mainly related to the glycogroups, might be responsible for their different localization. [source] Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complexJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Grazia Paola Nicchia Abstract Altered aquaporin-4 (AQP4) expression has been reported in brain edema, tumors, muscular dystrophy, and neuromyelitis optica. However, the plasma membrane organization of AQP4 and its interaction with proteins such as the dystrophin-associated protein complex are not well understood. In this study, we used sucrose density gradient ultracentrifugation and 2D blue native/sodium dodecyl sulfate,polyacrylamide gel electrophoresis and showed the expression of several AQP4 multi-subunit complexes (pools) of different sizes, ranging from , 1 MDa to ,500 kDa and containing different ratios of the 30/32 kDa AQP4 isoforms, indicative of orthogonal arrays of particles of various sizes. A high molecular weight pool co-purified with dystrophin and ,-dystroglycan and was drastically reduced in the skeletal muscle of mdx3cv mice, which have no dystrophin. The number and size of the AQP4 pools were the same in the kidney where dystrophin is not expressed, suggesting the presence of dystrophin-like proteins for their expression. We found that AQP2 is expressed only in one major pool of ,500 kDa, indicating that the presence of different pools is a peculiarity of AQP4 rather than a widespread feature in the AQP family. Finally, in skeletal muscle caveolin-3 did not co-purify with any AQP4 pool, indicating the absence of interaction of the two proteins and confirming that caveolae and orthogonal arrays of particles are two independent plasma membrane microdomains. These results contribute to a better understanding of AQP4 membrane organization and raise the possibility that abnormal expression of specific AQP4 pools may be found in pathological states. [source] Partitioning of the plasma membrane Ca2+ -ATPase into lipid rafts in primary neurons: effects of cholesterol depletionJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Lei Jiang Abstract Spatial and temporal alterations in intracellular calcium [Ca2+]i play a pivotal role in a wide array of neuronal functions. Disruption in Ca2+ homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca2+ -ATPase (PMCA) is a high affinity Ca2+ transporter that plays a crucial role in the termination of [Ca2+]i signals and in the maintenance of low [Ca2+]i essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or ,lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca2+ signaling in the central nervous system. [source] Localization of synaptic proteins involved in neurosecretion in different membrane microdomainsJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Elena Taverna Abstract A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Cav2.1 (P/Q-type) channels and soluble N -ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Cav2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-,-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery. [source] Protein,membrane interactions: blood clotting on nanoscale bilayersJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009J. H. MORRISSEY Summary., The clotting cascade requires the assembly of protease,cofactor complexes on membranes with exposed anionic phospholipids. Despite their importance, protein,membrane interactions in clotting remain relatively poorly understood. Calcium ions are known to induce anionic phospholipids to cluster, and we propose that clotting proteins assemble preferentially on such anionic lipid-rich microdomains. Until recently, there was no way to control the partitioning of clotting proteins into or out of specific membrane microdomains, so experimenters only knew the average contributions of phospholipids to blood clotting. The development of nanoscale membrane bilayers (Nanodiscs) has now allowed us to probe, with nanometer resolution, how local variations in phospholipid composition regulate the activity of key protease,cofactor complexes in blood clotting. Furthermore, exciting new progress in solid-state NMR and large-scale molecular dynamics simulations allow structural insights into interactions between proteins and membrane surfaces with atomic resolution. [source] Proteomic analysis of detergent-resistant membranes from Candida albicansPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue S1 2006María Insenser Abstract Lipid rafts are membrane microdomains with a higher amount of saturated fatty acids and sterols than the rest of the membrane. They are more resistant to the action of non-anionic detergents, and are called, for this reason, detergent-resistant membranes (DRMs). Lipid rafts are involved in many cellular processes, like signaling, cytokinesis, response to environment, etc., and therefore must contain important proteins. We have obtained a fraction enriched in proteins from Candida albicans DRMs. The sample has been analyzed by SDS-PAGE and 29 proteins have been identified including markers for lipid rafts in Saccharomyces cerevisiae, like Pma1p and a glycosylphosphatidylinositol (GPI)-anchored protein belonging to the Phr family. Ecm33p, a GPI-anchored protein involved in cell wall biogenesis, has been found for the first time in lipid rafts. We have also identified proteins implicated in protein glycosylation, like the mannosyltransferases Mnn7p, Pmt2p and Mnt1p; proteins involved in lipid metabolism, like Erg11p and Scs7p; and heat shock proteins, like Ssa1p and Hsp90p. Most of the proteins identified are located in plasma, mitochondrial, Golgi or ER membranes, supporting the postulated existence of lipid-raft domains in all the membranes. [source] A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thalianaTHE PLANT JOURNAL, Issue 1 2007Bationa Shahollari Summary Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica - insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica -induced growth promotion and enhanced seed production in Arabidopsis thaliana. [source] Cholesterol-rich membrane microdomains mediate cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal-distending toxinCELLULAR MICROBIOLOGY, Issue 5 2006Kathleen Boesze-Battaglia Summary We have previously shown that Actinobacillus actinomycetemcomitans cytolethal-distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt-mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1-enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl ,-cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed. [source] | |||||||||||||