scholarly journals The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation

2015 ◽  
Vol 83 (5) ◽  
pp. 2030-2042 ◽  
Author(s):  
Atasi De Chatterjee ◽  
Tavis L. Mendez ◽  
Sukla Roychowdhury ◽  
Siddhartha Das
Keyword(s):  
Lipid Rafts ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Cyst Formation ◽  
Neuraminidase Inhibitor ◽  
Major Constituent ◽  
Membrane Microdomains ◽  
Content Type ◽  
Cyst Production ◽  
Raft Domains

Although encystation (or cyst formation) is an important step of the life cycle ofGiardia, 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 fromGiardiaby 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 thatGiardiais able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation.

Nicotinic acetylcholine receptor α7 regulates cAMP signal within lipid rafts

2003 ◽  
Vol 285 (3) ◽  
pp. C567-C574 ◽  
Author(s):  
Jin Oshikawa ◽  
Yoshiyuki Toya ◽  
Takayuki Fujita ◽  
Masato Egawa ◽  
Junichi Kawabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Nicotinic Acetylcholine ◽  
Density Fractions

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR α7-subunit has a property of high Ca2+ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChRα7 existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR α5- and β2-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChRα7. Cholesterol depletion from plasma membranes with methyl-β-cyclodextrin redistributed nAChRα7 and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with α-bungarotoxin, a specific antagonist of nAChRα7, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChRα7 has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChRα7 with AC within plasma membranes. In addition, nAChRα7 may regulate the AC activity via Ca2+ within lipid rafts.

scholarly journals Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding

2005 ◽  
Vol 79 (11) ◽  
pp. 7077-7086 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Vesicular Stomatitis ◽  
Membrane Components

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line

2004 ◽  
Vol 32 (5) ◽  
pp. 777-779
Author(s):  
P. Pompach ◽  
P. Man ◽  
P. Novák ◽  
V. Havlíček ◽  
A. Fišerová ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Extraction Procedure ◽  
Sucrose Density Gradient ◽  
Cellular Adhesion ◽  
Jurkat Cells ◽  
Membrane Microdomains ◽  
Sucrose Density Gradient Centrifugation ◽  
Biologically Relevant ◽  
Jurkat T Cell

Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

scholarly journals Disruption of Lipid Rafts Interferes with the Interaction ofToxoplasma gondiiwith Macrophages and Epithelial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Karla Dias Cruz ◽  
Thayana Araújo Cruz ◽  
Gabriela Veras de Moraes ◽  
Tatiana Christina Paredes-Santos ◽  
Marcia Attias ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Host Cell ◽  
Lipid Rafts ◽  
Tyrosine Kinases ◽  
Plasma Membranes ◽  
Animal Cell ◽  
Cell Lineage ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Membrane Microdomains

The intracellular parasiteToxoplasma gondiican penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (MβCD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization ofT. gondiiin both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the LLC-MK2 cell lineage originated from kidney epithelial cells.

The Role of Lipid Rafts in the Assembly and Down-Regulation of the Factor X Activating Complex on the Activated Platelet Membrane.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1010-1010 ◽  
Author(s):  
Syed S. Ahmad ◽  
Ya-Chi Su ◽  
Peter N. Walsh
Keyword(s):  
Lipid Rafts ◽  
Gradient Centrifugation ◽  
Early Time ◽  
Sucrose Density Gradient ◽  
Platelet Membrane ◽  
Membrane Microdomains ◽  
Factor X ◽  
Sucrose Density Gradient Centrifugation ◽  
Time Points ◽  
Substrate Complex

Abstract In a recent study of the role of detergent-insoluble platelet membrane microdomains (lipid rafts) in the assembly of the factor X (FX) activating complex, we have shown, contrary to expectations, that the formation of lipid rafts after incubation of platelets with the thrombin receptor activation peptide (SFLLRN, 25 μM, for 30 min) results in the down-regulation, rather than assembly, of the enzyme-cofactor-substrate complex by sequestering FVIIIa and FX in rafts and separating them from FIXa, which is excluded from raft fractions isolated by sucrose density gradient centrifugation of triton X-100 (0.25%) solubilized platelets. Since the FX-activating complex is assembled rapidly on platelets after incubation with low concentrations of either thrombin (>1 nM) or SFLLRN (25 μM), we have now examined the kinetics of FX-activation complex assembly at early time points after exposure of platelets to agonists. Washed and gel-filtered human platelets, activated with SFLLRN (25 μM) in the presence of FVIIIa (5 U/ml, 1.5 nM) and FX (125 nM) rapidly (within 2 min) developed the capacity to support maximal rates of FIXa (1 nM) catalyzed FX activation that was transient and decayed to baseline within 5 min after exposure to agonist. At these early time points (0.5, 1 and 2 min), platelets activated with SFLLRN (25 μM) in the presence of 125I-labled FVIIIa (nM), FIXa (nM) or FX (nM) and analyzed by sucrose density gradient centrifugation after solubilization in triton X-100 (0.25%) were shown to sequester within the raft fractions ~15% of FIXa, ~15% of FVIIIa and ~15% of FX, whereas at later time points (5–30 min) only FVIIIa (~25%) and FX (~45%) were localized in rafts, from which FIXa was completely excluded. These results strongly suggest that platelet membrane microdomains (lipid rafts) form rapidly after exposure of platelets to PAR-1 agonists to colocalize the enzyme-cofactor-substrate complex, which is transient since at later time points FIXa dissociates from rafts that sequester the FVIIIa-FX complex to down-regulate FX activation.

scholarly journals Dynamics of putative raft-associated proteins at the cell surface

2004 ◽  
Vol 165 (5) ◽  
pp. 735-746 ◽  
Author(s):  
Anne K. Kenworthy ◽  
Benjamin J. Nichols ◽  
Catha L. Remmert ◽  
Glenn M. Hendrix ◽  
Mukesh Kumar ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Long Range ◽  
Dominant Factor ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Biochemical Fractionation ◽  
Associated Proteins ◽  
Raft Domains

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 μm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae

2005 ◽  
Vol 33 (5) ◽  
pp. 1131-1134 ◽  
Author(s):  
P.A. Insel ◽  
B.P. Head ◽  
H.H. Patel ◽  
D.M. Roth ◽  
R.A. Bundey ◽  
...  
Keyword(s):  
G Protein ◽  
Lipid Rafts ◽  
Plasma Membranes ◽  
Subcellular Fractionation ◽  
G Protein Coupled Receptors ◽  
Membrane Microdomains ◽  
Adult Cardiac Myocytes ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.

Membrane lipid rafts are required for D2 dopamine receptor signaling

2011 ◽  
Vol 26 (S2) ◽  
pp. 910-910 ◽  
Author(s):  
D. Sibley ◽  
L. Hazelwood ◽  
R. Roof ◽  
R.B. Free ◽  
Y. Han ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Gradient Centrifugation ◽  
Membrane Lipid ◽  
Receptor Protein ◽  
Membrane Microdomains ◽  
Membrane Cholesterol ◽  
Camp Accumulation ◽  
D2 Dopamine Receptor

IntroductionLipid rafts are specialized membrane microdomains enriched in cholesterol and sphingolipids and are important in the organization of receptor-protein complexes and the regulation of signaling.Objective/aimsGiven the emerging significance of lipids with respect to receptor structure and activation, we investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) signaling. As the D2 DAR is the molecular target for all antipsychotic drugs, more information about its signaling may help refine therapeutics for schizophrenia.MethodsD2 DAR constructs were expressed in HEK293T cells. Sucrose density fractionation resolved lipid rafts from other membrane components. Methyl-β-cyclodextrin (MCD) was used to deplete membrane cholesterol and to disrupt lipid rafts.ResultsDetergent solubilization followed by sucrose gradient centrifugation resolved lipid rafts from heavier membrane fractions. The D2 DAR was equally distributed amongst both the lipid raft and heavier membrane fractions. Pretreatment with MCD, however, eliminated both lipid raft markers and the D2 DAR from lipid raft fractions, although the receptor was still found in heavier membrane fractions. We also found that MCD treatment abolished D2 DAR-mediated inhibition of cAMP accumulation. In contrast D1 DAR-stimulated cAMP accumulation was unaffected by MCD treatment.ConclusionsOur current results show that the D2 DAR is distributed in multiple membrane microdomains, including cholesterol-rich lipid rafts. We found that extraction of cholesterol disrupted lipid rafts and also an eliminated D2 DAR-mediated signaling. Thus, we hypothesize that lipid rafts are critical for D2 DAR signaling to occur.

scholarly journals Monosialoganglioside GM1 Deficiency Inhibits the Neurotrophic Effects of GDNF by Disrupting Lipid Raft Assembly

2021 ◽  
Author(s):  
Shimin Jiang ◽  
Tai Zhou ◽  
Kejia Zhang ◽  
Yao Zhou ◽  
Zhongcheng Wang ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Gradient Centrifugation ◽  
Membrane Microdomains ◽  
Western Blot Assay ◽  
Caveolin 1 ◽  
Downstream Signaling ◽  
Proliferation And Differentiation ◽  
Lipid Raft Microdomains ◽  
Triton X

Abstract Recent studies have shown that monosialoganglioside GM1 deficiency can inhibit the signal transduction process of glial cell line-derived neurotrophic factor (GDNF), which plays an important role in the pathogenesis of Parkinson's disease (PD). However, its specific mechanism still needs to be explored. We inhibited the expression of GM1 by treating cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). CCK-8 assay, EdU cell proliferation assay and Western blot assay were used to evaluate the effect of GM1 deficiency on the proliferation and differentiation of SH-SY5Y cells induced by GDNF and on the GDNF-RET signaling pathway. Lipid rafts were isolated by Triton X-100 solubilization and OptiPrepTM density gradient centrifugation. The alterations of lipid raft assembly and the translocation of RET into lipid rafts were evaluated after PDMP treatment. We found that PDMP treatment inhibited the proliferation and differentiation of SH-SY5Y cells induced by GDNF and reduced the phosphorylation of RET and its downstream signaling molecules Erk and Akt. In addition, after PDMP treatment, caveolin-1 and flotillin-1, the prototypical markers of lipid rafts, diffused from lipid rafts to non-lipid raft microdomains, and GDNF-induced RET translocation into lipid rafts was also reduced. These alterations could be partially reversed by adding exogenous GM1. Our results suggest that ganglioside GM1 deficiency could compromise the neurotrophic effects and signals downstream of GDNF by altering the assembly of lipid raft membrane microdomains.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

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