scholarly journals Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells

2004 ◽  
Vol 380 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Galina RADEVA ◽  
Frances J. SHAROM
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Lipid Rafts ◽  
Receptor Protein ◽  
Membrane Microdomains ◽  
Plasma Membrane Vesicles ◽  
Leukaemia Cell ◽  
Leukaemia Cell Line ◽  
Isolation And Characterization ◽  
Triton X

Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135–170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.

scholarly journals Identification of a novel member of the Rab8 family from the rat basophilic leukaemia cell line, RBL.2H3

1996 ◽  
Vol 109 (6) ◽  
pp. 1265-1274 ◽  
Author(s):  
J. Armstrong ◽  
N. Thompson ◽  
J.H. Squire ◽  
J. Smith ◽  
B. Hayes ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Northern Blot Analysis ◽  
Intracellular Localization ◽  
Transient Transfection ◽  
The Novel ◽  
Leukaemia Cell ◽  
Marked Contrast ◽  
Epitope Tag ◽  
Leukaemia Cell Line

We describe the cloning of a cDNA from the rat basophilic leukaemia cell line (RBL.2H3) encoding a novel member of the Rab family of small GTP binding proteins. The novel clone, which we call Rab8b, is most highly related to the Rab8 family with substantial divergence in the variable C-terminal domain. Northern blot analysis reveals highest levels of expression of Rab8b in the spleen, testis and brain, which is in marked contrast to the tissue distribution of Rab8. The Rab8b cDNA was modified to introduce a c-myc epitope tag at the extreme N terminus of the protein, and transient transfection studies were performed to analyse the intracellular localization of Rab8b by confocal microscopy. Transient expression of the c-myc/Rab8b fusion protein in both PC12 and RBL.2H3 cells shows staining of both the plasma membrane and ill-defined vesicular structures, and in the case of RBL.2H3 cells appears to induce striking outgrowths of the plasma membrane.

Purification and sequencing of glycosylation variants of BSF-1, as a MAF, from the EL-4 leukaemia cell line

1989 ◽  
Vol 17 (1) ◽  
pp. 65-74 ◽  
Author(s):  
C. Sutton ◽  
P. Depledge ◽  
L. Bawden ◽  
A. Carne ◽  
M. Meltzer ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukaemia Cell ◽  
Leukaemia Cell Line

scholarly journals Vindesine receptors in cells of a human leukaemia cell line

1982 ◽  
Vol 46 (3) ◽  
pp. 392-396 ◽  
Author(s):  
K Totsuka ◽  
K Oshimi ◽  
H Mizoguchi
Keyword(s):  
Cell Line ◽  
Leukaemia Cell ◽  
Leukaemia Cell Line ◽  
Human Leukaemia Cell ◽  
Human Leukaemia ◽  
In Cells

Induction of erythroid differentiation in the human leukaemia cell line K562

Nature ◽  
1979 ◽  
Vol 278 (5702) ◽  
pp. 364-365 ◽  
Author(s):  
LEIF C. ANDERSSON ◽  
MIKKO JOKINEN ◽  
CARL G. GAHMBERG
Keyword(s):  
Cell Line ◽  
Erythroid Differentiation ◽  
Leukaemia Cell ◽  
Leukaemia Cell Line ◽  
Human Leukaemia Cell ◽  
Human Leukaemia

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.

Association of repeat sequences with integrated retroviruses in a murine leukaemia cell line

1996 ◽  
Vol 20 (5) ◽  
pp. 421-427
Author(s):  
Ellen S.-P. Ho ◽  
Barbara van Leeuwen ◽  
Helen C. O'Neill
Keyword(s):  
Cell Line ◽  
Leukaemia Cell ◽  
Leukaemia Cell Line ◽  
Repeat Sequences

scholarly journals Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds

2013 ◽  
Vol 16 (6) ◽  
pp. 1361-1371 ◽  
Author(s):  
Caroline Nothdurfter ◽  
Sascha Tanasic ◽  
Barbara Di Benedetto ◽  
Manfred Uhr ◽  
Eva-Maria Wagner ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Lipid Rafts ◽  
Gabaa Receptor ◽  
Lipid Raft ◽  
Tricyclic Antidepressant ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Receptor Modulation ◽  
Gabaa Receptor Subunits ◽  
Triton X

Abstract Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

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