Growth cone repulsion to Netrin-1 depends on lipid raft microdomains enriched in UNC5 receptors.
Ontology highlight
ABSTRACT:
SUBMITTER: Hernaiz-Llorens M
PROVIDER: S-EPMC8004515 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Amyloid precursor protein (APP) at the plasma membrane is internalized via endocytosis and delivered to endo/lysosomes, where neurotoxic amyloid-β (Aβ) is produced via β-, γ-secretases. Hence, endocytosis plays a key role in the processing of APP and subsequent Aβ generation. β-, γ-secretases as well as APP are localized in cholesterol-enriched lipid raft microdomains. However, it is still unclear whether lipid rafts are the site where APP undergoes endocytosis and whether cholesterol levels affect this process. In this study, we found that localization of APP in lipid rafts was increased by elevated cholesterol level. We also showed that increasing or decreasing cholesterol levels increased or decreased APP endocytosis, respectively. When we labeled cell surface APP, APP localized in lipid rafts preferentially underwent endocytosis compared to nonraft-localized APP. In addition, APP endocytosis from lipid rafts was regulated by cholesterol levels. Our results demonstrate for the first time that cholesterol levels regulate the localization of APP in lipid rafts affecting raft-dependent APP endocytosis. Thus, regulating the microdomain localization of APP could offer a new therapeutic strategy for Alzheimer's disease.
| S-EPMC7727664 | biostudies-literature
Project description:In the brain, myelin is important in regulating nerve conduction and neurotransmitter release by providing insulation at axons. Myelin is a specialized yet continuous sheet structure of differentiated oligodendrocytes (OLs) that is enriched in lipids, specifically galactosylceramides (GalCer) originated at the endoplasmic reticulum (ER). GalCer are known to affect OL differentiation. However, the mechanism whereby GalCer affect OL differentiation is not well understood. Sigma-1 receptors (Sig-1Rs), shown by us to exist in detergent-insoluble lipid microdomains at lipid-enriched loci of ER in NG108 cells, are important in the compartmentalization/transport of ER-synthesized lipids and in cellular differentiation. In this study, we used rat primary hippocampal cultures and found that Sig-1Rs form GalCer-enriched lipid rafts at ER lipid droplet-like structures in the entire myelin sheet of mature OLs. In rat OL progenitors (CG-4 cells), levels of lipid raft-residing Sig-1Rs and GalCer increase as cells differentiate. Sig-1Rs also increase in OLs and myelin of developing rat brains. Sig-1R, GalCer, and cholesterol are colocalized and are resistant to the Triton X-100 solubilization. Treating cells with a Sig-1R agonist or targeting Sig-1Rs at lipid rafts by overexpression of Sig-1Rs in CG-4 cells enhances differentiation, whereas reducing Sig-1Rs at lipid rafts by transfection of functionally dominant-negative Sig-1Rs attenuates differentiation. Furthermore, Sig-1R siRNA inhibits differentiation. Our findings indicate that, in the brain, Sig-1Rs targeting GalCer-containing lipid microdomains are important for OL differentiation and that Sig-1Rs may play an important role in the pathogenesis of certain demyelinating diseases.
| S-EPMC522002 | biostudies-literature
Project description:Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-?-cyclodextrin (M?CD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.
| S-EPMC3284156 | biostudies-literature
Project description:NADPH oxidase (Nox)-dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D(1)-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58+/-3.3%) than in hypertensive rats (31+/-5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67(phox) in cells from normotensive rats. D(1)-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl-beta-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats.
| S-EPMC2879618 | biostudies-literature
Project description:The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b558, in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase.
| S-EPMC380990 | biostudies-literature
Project description:Lipid raft microdomains are enriched in sphingomyelin and cholesterol and function as platforms for signal transduction and as the site of budding of several enveloped viruses, including influenza virus. The influenza virus hemagglutinin (HA) glycoprotein, which mediates both viral-cell attachment and membrane fusion, associates intrinsically with lipid rafts. Residues in the HA transmembrane (TM) domain are important for raft association as sequence substitutions in the HA TM domain ablate HA association with rafts (nonraft HA). Cells expressing either WT or nonraft HA cause complete fusion (lipid mixing and content mixing) over widely varying HA expression levels. However, the number of fusion events measured for nonraft HA mutant protein at all HA surface densities was reduced to approximately 55% of the events for WT HA protein. Mutant influenza viruses were generated that contain the nonraft HA TM domain alterations. Electron microscopy experiments showed that WT HA was distributed at the cell surface in clusters of 200-280 nm in diameter, whereas nonraft HA was distributed mostly randomly at the plasma membrane. Nonraft HA virus showed reduced budding, contained reduced amounts of HA protein, was greatly reduced in infectivity, and exhibited decreased virus-membrane fusion activity. Cholesterol depletion of virus did not affect the ability of virions to cause either virus-cell lipid mixing or virus-mediated hemolysis, a surrogate for content mixing. Taken together, the data suggest that HA clusters in rafts to provide a sufficient concentration of HA in budding virus to mediate efficient virus-cell fusion.
| S-EPMC299746 | biostudies-literature
Project description:The fidelity of cAMP in controlling numerous cellular functions rests crucially on the precise organization of cAMP microdomains that are sustained by the scaffolding properties of adenylyl cyclase. Earlier studies suggested that AC8 enriches in lipid rafts where it interacts with cytoskeletal elements. However, these are not stable structures and little is known about the dynamics of AC8 secretion and its interactions. The present study addresses the role of the cytoskeleton in maintaining the AC8 microenvironment, particularly in the context of the trafficking route of AC8 and its interaction with caveolin1. Here, biochemical and live-cell imaging approaches expose a complex, dynamic interaction between AC8 and caveolin1 that affects AC8 processing, targeting and responsiveness in plasma membrane lipid rafts. Site-directed mutagenesis and pharmacological approaches reveal that AC8 is processed with complex N-glycans and associates with lipid rafts en route to the plasma membrane. A dynamic picture emerges of the trafficking and interactions of AC8 while travelling to the plasma membrane, which are key to the organization of the AC8 microdomain.
| S-EPMC6556161 | biostudies-literature
Project description:The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.
| S-EPMC3435554 | biostudies-literature
Project description:We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.
| S-EPMC4882513 | biostudies-literature
Project description:Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation.
| S-EPMC4399036 | biostudies-literature