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.

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.

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.

The role of lipid rafts in signalling and membrane trafficking in T lymphocytes

2001 ◽  
Vol 114 (22) ◽  
pp. 3957-3965
Author(s):  
Miguel A. Alonso ◽  
Jaime Millán
Keyword(s):  
T Lymphocytes ◽  
Epithelial Cells ◽  
Lipid Rafts ◽  
T Cell Activation ◽  
Membrane Trafficking ◽  
Intracellular Transport ◽  
Cell Activation ◽  
Membrane Microdomains ◽  
Caveolin 1 ◽  
Lipid Species

Combinatorial association of different lipid species generates microheterogeneity in biological membranes. The association of glycosphingolipids with cholesterol forms membrane microdomains – lipid rafts – that are involved in specialised pathways of protein/lipid transport and signalling. Lipid rafts are normally dispersed in cellular membranes and appear to require specialised machinery to reorganise them to operate. Caveolin-1 and MAL are members of two different protein families involved in reorganisation of lipid rafts for signalling and/or intracellular transport in epithelial cells. T cell activation induces a rapid compartmentalisation of signalling machinery into reorganised rafts that are used as platforms for the assembly of the signalling complex. Costimulatory molecules participate in this process by providing signals that mobilise raft lipids and proteins, and remodel the cytoskeleton to the contact site. As in epithelial cells, rafts are used also as vesicular carriers for membrane trafficking in T lymphocytes. Furthermore, there are potential similarities between the specialised protein machinery underlying raft-mediated processes in T lymphocytes and polarised epithelial cells.

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.

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.

scholarly journals Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells

2019 ◽  
Vol 20 (16) ◽  
pp. 3904 ◽  
Author(s):  
Edismauro Garcia Freitas Filho ◽  
Luiz Augusto Marin Jaca ◽  
Lilian Cristiane Baeza ◽  
Célia Maria de Almeida Soares ◽  
Clayton Luiz Borges ◽  
...  
Keyword(s):  
Mast Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Protein Composition ◽  
Enrichment Analysis ◽  
Functional Enrichment Analysis ◽  
Cell Types ◽  
Cellular Membrane ◽  
Functional Enrichment ◽  
Membrane Microdomains

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

scholarly journals Structure-Function Analysis of Lyn Kinase Association with Lipid Rafts and Initiation of Early Signaling Events after Fcɛ Receptor I Aggregation

2001 ◽  
Vol 21 (24) ◽  
pp. 8318-8328 ◽  
Author(s):  
Martina Kovářová ◽  
Pavel Tolar ◽  
Ramachandran Arudchandran ◽  
Lubica Dráberová ◽  
Juan Rivera ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Immunoglobulin E ◽  
Function Analysis ◽  
Membrane Microdomains ◽  
Lyn Kinase ◽  
Signaling Events ◽  
Syk Protein Tyrosine Kinase

ABSTRACT The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcɛRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, incis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcɛRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca2+. However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcɛRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcɛRI does not exhibit an increased association with lipid rafts, suggest that FcɛRI phosphorylation and early activation events can be initiated outside of lipid rafts.

scholarly journals Stem Cells and Rat Liver Carcinogenesis: Contributions of Confocal and Electron Microscopy

1998 ◽  
Vol 46 (5) ◽  
pp. 613-626 ◽  
Author(s):  
Phyllis M. Novikoff ◽  
Ana Yam
Keyword(s):  
Epithelial Cells ◽  
Connective Tissue ◽  
Cell Lineage ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Nodule Formation ◽  
Surrounding Connective Tissue ◽  
Multiple Cell ◽  
Polarized Epithelial Cells ◽  
Parenchymal Hepatocytes

Microscopic analysis in combination with cytochemistry and immunocytochemistry has revealed the presence of four cell types not previously described in the portal area and parenchyma of the liver from an experimental rodent hepatocarcinogenic rat model. Within the intrahepatic bile ductules, which proliferate after administration of chemical carcinogens and partial hepatectomy, small, undifferentiated nonpolarized, nonepithelial cells with a blast-like phenotype and polarized epithelial cells different from the polarized epithelial cells that typically line the walls of the bile ductules were found. In the connective tissue stroma surrounding the bile ductules, nonpolarized epithelial cells with hepatocyte phenotype were found. In the parenchyma, subpopulations of bile ductule epithelial cells that established ATPase-positive bile canalicular structures, including the formation of desmosomes and tight junctions, with parenchymal hepatocytes within the hepatic lobule were found. These observations raise the following questions in this model. Are there undifferentiated progenitor cells with stem cell-like properties within bile ductules? What are the interrelations of the newly described cell types with each other, with parenchymal hepatocytes, with preneoplastic nodules, and with hepatomas? Do the heterogeneous cell types within the bile ductules, in the surrounding connective tissue, and within the hepatic cords represent intermediate stages of single or multiple cell lineage pathways leading to hepatocyte differentiation, liver regeneration, and/or preneoplastic nodule formation?

scholarly journals KCa3.1-dependent uptake of the cytotoxic DNA-binding dye Hoechst 33258 into cancerous but not healthy cervical cells

2020 ◽  
pp. jbc.RA120.013997
Author(s):  
Maurish Bukhari ◽  
Han Deng ◽  
Darren Sipes ◽  
Marisa Ruane-Foster ◽  
Kayla Purdy ◽  
...  
Keyword(s):  
Dna Binding ◽  
Epithelial Cells ◽  
Cancer Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Nuclear Accumulation ◽  
Hoechst 33258 ◽  
Direct Stimulation ◽  
Significant Difference ◽  
Cervical Cells

The poor and nonselective penetration of current chemotherapeutics across the plasma membranes of cancer cells, which is necessary for the targeted disruption of the intracellular machinery, remains a major pharmaceutical challenge. In several cell types, including mast cells and macrophages, exposure to extracellular ATP is known to stimulate passive entry of large and otherwise membrane impermeable cationic dyes, which is usually attributed to conduction through ionotropic P2X receptors. Here, we report that elevations in cytosolic Ca2+ stimulate the rapid uptake and nuclear accumulation of a DNA-binding fluorescent cation, Hoechst 33258 (H33258), in cervical cancer cells. The H33258 uptake was dependent on activation of intermediate conductance Ca2+-activated K+ channels (KCa3.1), and direct stimulation of the channel with the activators SKA 31 and DCEBIO was sufficient to induce cellular uptake of H33258 directly. In contrast to the results from cancerous cervical cells, KCa3.1-dependent H33258 uptake was rarely observed in epithelial cells derived from the ectocervix and transformation zone of healthy cervical tissue. Furthermore, whole-cell patch clamp experiments and assessment of membrane potential using the slow voltage-sensitive dye DiSBAC2(3) revealed a significant difference in functional KCa3.1 activity between cancerous and healthy cervical epithelial cells, which correlated strongly with the incidence of KCa3.1-dependent H33258 uptake. Finally, we show that activation of KCa3.1 channels caused a modest but significant sensitization of cancer cells to the growth suppressant effects of H33258, lending plausibility to the idea of using KCa3.1 channel activators to enhance cell penetration of small cationic toxins into cancer cells expressing these channels.

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