Role Of Rafts Research Articles

A novel trigger for cholesterol-dependent smooth muscle contraction mediated by the sphingosylphosphorylcholine-Rho-kinase pathway in the rat basilar artery: a mechanistic role for lipid rafts.

Hyperlipidemia is a risk factor for abnormal cerebrovascular events. Rafts are cholesterol-enriched membrane microdomains that influence signal transduction. We previously showed that Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) induced by sphingosylphosphorylcholine (SPC) has a pivotal role in cerebral vasospasm. The goals of the study were to show SPC-Rho-kinase-mediated VSM contraction in vivo and to link this effect to cholesterol and rafts. The SPC-induced VSM contraction measured using a cranial window model was reversed by Y-27632, a Rho-kinase inhibitor, in rats fed a control diet. The extent of SPC-induced contraction correlated with serum total cholesterol. Total cholesterol levels in the internal carotid artery (ICA) were significantly higher in rats fed a cholesterol diet compared with a control diet or a β-cyclodextrin diet, which depletes VSM cholesterol. Western blotting and real-time PCR revealed increases in flotillin-1, a raft marker, and flotillin-1 mRNA in the ICA in rats fed a cholesterol diet, but not in rats fed the β-cyclodextrin diet. Depletion of cholesterol decreased rafts in VSM cells, and prevention of an increase in cholesterol by β-cyclodextrin inhibited SPC-induced contraction in a cranial window model. These results indicate that cholesterol potentiates SPC-Rho-kinase-mediated contractions of importance in cerebral vasospasm and are compatible with a role for rafts in this process.

Synaptic membrane rafts: traffic lights for local neurotrophin signaling?

Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signaling, plasticity, and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signaling. The tyrosine kinase neurotrophin receptors (Trk) and the low-affinity p75 neurotrophin receptor (p75NTR) are enriched in neuronal lipid rafts together with the intermediates of downstream signaling pathways, suggesting a possible role of rafts in neurotrophin signaling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

Heterodimerization With the Prostacyclin Receptor Triggers Thromboxane Receptor Relocation to Lipid Rafts

Prostacyclin and thromboxane mediate opposing cardiovascular actions through receptors termed IP and TP, respectively. When dimerized with IP, the TP shifts to IP-like function. IP localizes to cholesterol-enriched membrane rafts, but TP and IPTP heterodimer localization is not defined. We examined these receptors' membrane localization and the role of rafts in receptor function. Microdomain distribution of IP, TP, and IPTP heterodimers was examined in COS-7 cells by measuring energy transfer from renilla luciferase-fused receptors to fluorescently labeled rafts. IP raft association was confirmed. TP was raft excluded, but redistributed to rafts upon dimerization with IP. Signaling of the IP and IPTP heterodimer, but not TP alone, was suppressed after raft disruption by cholesterol depletion. Cholesterol enrichment also selectively suppressed IP and IPTP function. Native IP and IPTP signaling in smooth muscle cells and macrophages were similarly sensitive to cholesterol manipulation, whereas macrophages from hypercholesterolemic mice displayed suppressed IP and IPTP function. IP and TP function within distinct microdomains. Raft incorporation of TP in the IPTP heterodimer likely facilitates its signaling shift. We speculate that changes in IP and IPTP signaling after perturbation of membrane cholesterol may contribute to cardiovascular disease associated with hypercholesterolemia.

T Cell Signal Regulation by the Actin Cytoskeleton

T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase Lck. Conversely, the membrane phosphatase CD45, which activates Src family kinases, is excluded, and this is necessary to avoid quenching of T cell receptor phosphosignals. Data suggest that this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B, which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following the addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.

Disruption of Lipid Rafts Enhances the Effect of Lactobacilli on the Production of Tumor Necrosis Factor-Alpha in Mononuclear Blood Cells

We investigated, to determine whether disruption of lipid rafts would influence the effect of three selected strains of Lactobacillus on TNF-alpha production by peripheral blood mononuclear cells. Two strains increased TNF-alpha production; and the third one reduced cytokine levels. Disruption of rafts changed the immunomodulatory effect of the three strains. This is the first report on a potential role of rafts in lactobacilli-cell interaction.

Domain-specific lipid distribution in macrophage plasma membranes

Lipid rafts, defined as cholesterol- and sphingolipid-rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo, and regulation is based largely on the study of isolated lipid rafts; however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma membrane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based and detergent-free methods can generate raft fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin-1, such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X-100-resistant rafts are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonication-derived nonraft domains to generate new detergent-resistant rafts. The role of rafts in regulating the biological activities of macrophage plasma membrane proteins may require careful reevaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques.

Lipid rafts in cytokine signaling

Lipid rafts are established as critical structures for a variety of cellular processes, including immune cell activation. Beyond their importance for initial immune cell activation at the immunological synapse, lipid rafts are now also being recognized as important sites for cytokine and growth factor signal transduction, both in immune cells as part of secondary regulatory processes, and in non-immune cells. This review summarizes current knowledge regarding the roles of rafts in cytokine signaling and emphasizes the need for measures to better standardize the study of rafts.

The order of rafts

The EuroConference on Microdomains, Lipid Rafts and Caveolae was held in Tomar, Portugal, from 17 to 22 May 2003. This joint EURESCO Conference/EMBO workshop was organized by G. van Meer and K. Simons and was the second meeting in a series that was initiated by K. Fiedler in 2001. ![][1] The plasma membrane was described as a fluid mosaic in the early 1970s by Singer & Nicolson (1972). Since then, several studies that were designed to elucidate the temporal and spatial architecture of the plasma membrane have provided a more complicated picture (reviewed in Edidin, 2003a). Many of these studies indicate that the plasma membrane is at the very least a mosaic of compartments that is maintained by an active cytoskeleton mesh. The membrane raft hypothesis proposes another type of compartmentalization (Simons & Ikonen, 1997; Simons & van Meer, 1988), in which specific lipids may dynamically associate with each other to form platforms that are important for membrane protein sorting and the formation of signalling complexes. This conference focused on the lateral domains that occur in biomembranes. In this report, we discuss new developments in the understanding of the lateral segregation of lipids that have been obtained from studies in artificial membranes and the parallel efforts to visualize lipidic assemblies in living cell membranes. We also report on new information about the roles of rafts in several cellular processes, such as in the sorting of membrane constituents during vesicular trafficking and in signal transduction, especially in immunological processes and caveolae formation and function. ### Raft structure In artificial membranes. To address the role of lipids in the formation of membrane rafts, studies have been performed with artificial membranes, and these suggest that homogeneous membrane bilayers might be an exception rather than a rule (Edidin, 2003b; McConnell & Vrljic, 2003). In general, … [1]: /embed/graphic-1.gif

Raf kinases in lung tumor development.

The specific response of a cell to an extracellular signal is mediated by signaling cascades that relay the excitation of one or more receptor(s) to the co-ordinated activity of a set of effector molecules which regulate cell division and growth and prevent an intrinsic cell death program. The small G protein Ras is an essential component in the transduction of extracellular signals. Raf is a serine/threonine protein kinase and a direct downstream effector of Ras, which is activated to its GTP-bound state in response to various ligands binding to their cognate receptors. Raf kinases are located at the head of one of the best characterized signaling cascades, the Raf-MEK-ERK kinase cascade which is involved in many signaling processes (Fig. 1). In multicellular organisms, the family of Raf kinases consists of three members, ARaf, BRaf and CRaf. Although all of these Raf kinases phosphorylate MEK, they differ in the way they are activated (Hagemann and Rapp, 1999). For example, cAMP potentiates BRaf kinase activity but inhibits CRaf (Erhardt et al., 1995; Cook and McCormick, 1993; Dhillon et al., 2002). Genetic and biochemical data also indicate that isoform specific functions exist for Raf kinases which correlate with specific protein interactions (Hagemann and Rapp, 1999). Recent findings regarding interaction of Raf with lipid membrane microdomains (Hekman et al., 2002; Raabe and Rapp, 2002) and the discovery of Rab binding scaffold proteins that may be regulated by MAP kinases in their ability to activate actin comet formation for vesicle transport (Kerkhoff et al., 2001; Eden et al., 2002) have suggested to us a revised model for membrane translocation of Raf (Fig. 2). The model incorporates suggestions on the role of rafts in protein sorting (Simons and Ikonen, 1997) and anticipates a self organizing element in the maturation of signaling complexes. As pictured in Fig. 2, joint sorting of Raf, Ras and upstream signal regulators, e.g., growth factor receptors together with elements of the Ras cycle as well as effectors of Ras/Raf signaling such as MEK and ERK (perhaps

Lipid rafts, major histocompatibility complex molecules, and immune regulation

Glycolipid-enriched membrane microdomains (“rafts”) are critical sites for signal transduction and other processes such as intracellular transport. While the participation of T-cell rafts in the formation of the immunological synapse is well established, the role of rafts on antigen-presenting cells (APCs) as well as the relationship between these domains and major histocompatibility complex (MHC) molecules is less clearly defined. We therefore investigated whether MHC class I or II molecules are found in rafts of the human macrophage-monocytic cell line U937. We detected the preferential localization of MHC class II, but not class I, molecules in rafts. Furthermore, raft disruption resulted in a decrease in constitutive protein tyrosine phosphorylation events in U937 cells. Our findings are reviewed in the context of results from other groups who also found important associations of MHC class II molecules with APC rafts. Additional, and at times contradictory, findings by others regarding the relationship between rafts and MHC molecules are also discussed. It is concluded that class II MHC molecules can localize in rafts of APCs and that this localization may be relevant for APC function and thus immune regulation.

Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3.

1. Rabbit ileal Na+-absorbing cell Na+-H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-soluble fractions and the other approximately 50% with Triton X-100-insoluble fractions; approximately 33% of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2. The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+-H+ exchange activity. 3. Disrupting rafts by removal of cholesterol with methyl-beta-cyclodextrin (MbetaCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbetaCD treatment. 4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.

Microdomains, lipid rafts and caveolae (San Feliu de Guixols, Spain, 19-24 May 2001).

The first full-length meeting on membrane rafts and caveo-lae, organized by Klaus Fiedler and Gerrit van Meer and spon-sored by EURESCO, washeld in the spectacularly situatedEden Roc hotel in San Feliu de Guixols, Spain, overlookingthe rugged Costa Brava coastline. Rafts and caveolae remainuncertain bedfellows. Although rafts (lipid microdomains de-fined by tight acyl chain packing) have a high affinity for cav-eolae (plasma membrane pits formed by caveolin proteins),the two are distinguishable; rafts can exist and function out-side of caveolae. Similarly, it is not yet clear how rafts areinvolved in caveolar function. At least some functions of cav-eolae appear to be mediated by binding of caveolins to otherproteins, with no obvious requirement for the unique raft lipidenvironment. We will focus here on the following themes,which were especially prominent at the meeting: raft struc-ture, the function of rafts and caveolae in transport along theexocytic and endocytic pathways, the role of rafts in sig-naling, and the function and dynamics of caveolin proteins.

NA +/H +-exchanger 3 (NHE3) is present in lipid rafts in the ileal absorptive cell brush border: A role for rafts and the actin cytoskeleton in endocytosis of NHE3

NA +/H +-exchanger 3 (NHE3) is present in lipid rafts in the ileal absorptive cell brush border: A role for rafts and the actin cytoskeleton in endocytosis of NHE3