scholarly journals Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

1997 ◽  
Vol 8 (5) ◽  
pp. 855-869 ◽  
Author(s):  
Z Xiao ◽  
P N Devreotes
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
G Protein ◽  
Biochemical Characterization ◽  
Surface Membrane ◽  
Actin Binding ◽  
Membrane Microdomains ◽  
Immunogold Electron Microscopy ◽  
Membrane Structures

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.

scholarly journals Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes

2003 ◽  
Vol 373 (1) ◽  
pp. 125-132 ◽  
Author(s):  
Gert H. HANSEN ◽  
Jens PEDERSEN ◽  
Lise-Lotte NIELS-CHRISTIANSEN ◽  
Lissi IMMERDAL ◽  
E. Michael DANIELSEN
Keyword(s):  
Lipid Raft ◽  
Membrane Trafficking ◽  
Brush Border ◽  
Surface Membrane ◽  
Ruthenium Red ◽  
Border Region ◽  
Membrane Microdomains ◽  
Immunogold Electron Microscopy ◽  
Cholesterol Depletion ◽  
Membrane Structures

The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid ‘rafts’. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo GM1, and they were sensitive to cholesterol depletion by methyl-β-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50–100 nm, and penetrated up to 1 μm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border.

scholarly journals Vesicular Transport Is Not Required for the Cytoplasmic Pool of Cholera Toxin To Interact with the Stimulatory Alpha Subunit of the Heterotrimeric G Protein

2004 ◽  
Vol 72 (12) ◽  
pp. 6826-6835 ◽  
Author(s):  
Ken Teter ◽  
Michael G. Jobling ◽  
Randall K. Holmes
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cholera Toxin ◽  
G Protein ◽  
Vesicular Transport ◽  
Cytotoxic Action ◽  
Anterograde Transport ◽  
Heterotrimeric G Protein ◽  
Α Subunit ◽  
Vesicular Traffic

ABSTRACT Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic A1 polypeptide of CT (CTA1) then crosses the ER membrane, enters the cytosol, ADP-ribosylates the stimulatory α subunit of the heterotrimeric G protein (Gsα) at the cytoplasmic face of the plasma membrane, and activates adenylate cyclase. The cytosolic pool of CTA1 may reach the plasma membrane and its Gsα target by traveling on anterograde-directed transport vesicles. We examined this possibility with the use of a plasmid-based transfection system that directed newly synthesized CTA1 to either the ER lumen or the cytosol of CHO cells. Such a system allowed us to bypass the CT retrograde trafficking itinerary from the cell surface to the ER. Previous work has shown that the ER-localized pool of CTA1 is rapidly exported from the ER to the cytosol. Expression of CTA1 in either the ER or the cytosol led to the activation of Gsα, and Gsα activation was not inhibited in transfected cells exposed to drugs that inhibit vesicular traffic. Thus, anterograde transport from the ER to the plasma membrane is not required for the cytotoxic action of CTA1.

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.

scholarly journals Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells.

1996 ◽  
Vol 133 (3) ◽  
pp. 543-558 ◽  
Author(s):  
A Müsch ◽  
H Xu ◽  
D Shields ◽  
E Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mdck Cells ◽  
Current Model ◽  
Gh3 Cells ◽  
Plasma Membrane Proteins ◽  
Vesicular Release ◽  
Vesicular Stomatitis

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.

scholarly journals Identification of novel therapeutic targets for histone 3 mutated children’s brain tumour, using unique tumour cell surface proteomic signatures

2021 ◽  
Vol 23 (Supplement_4) ◽  
pp. iv9-iv9
Author(s):  
Anya Snary ◽  
Richard Grundy ◽  
Rob Layfield ◽  
Ruman Rahman ◽  
Farhana Haque
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Cell Surface ◽  
Brain Tumours ◽  
Expression Patterns ◽  
Surface Membrane ◽  
Therapeutic Targets ◽  
Molecular Signature ◽  
Histone 3 ◽  
Cell Membrane Proteins

Abstract Aims Improvements in the treatments for childhood and adolescent brain tumours, High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), have not advanced much and they continue to carry a very poor prognosis. These brain tumours are now defined by mutations affecting histone 3 proteins, indeed 80% of DIPGs harbour histone H3.1 and H3.3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. We hypothesized that the histone 3 mutant tumours will have distinct mutation specific surfactome (cell membrane proteins) signature. Method We therefore analysed the cell surface proteomics of pHGG and DIPG, in order to identify novel targets for therapy. We have at first isolated the cell membrane fractions from a range of patient cells carrying different histone 3 mutations (G34R, G34V), relative to wild type histone 3. A comparative quantitative mass-spectrometry analyses of these cell surface membrane fractions is then performed. Results The results obtained to date demonstrated unique differential cell membrane expression patterns which correlated to specific mutation type. For example, increased expression of Ras-related proteins Rab-3, Rab-3D is detected only in histone H3.3-G34R mutated cell line in comparison. Conclusion Identification and analyses of these unique cell membrane proteins’ association with specific in H3.3 mutation in pHGG, will help to identify precise mutation specific therapeutic targets, benefiting the patients to receive therapy based on tumour’s molecular signature.

Differential partitioning of plasma membrane proteins into the triton X-100-insoluble cytoskeleton fraction during concanavalin A-induced receptor redistribution

1989 ◽  
Vol 92 (1) ◽  
pp. 85-91
Author(s):  
W.F. Patton ◽  
M.R. Dhanak ◽  
B.S. Jacobson
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Concanavalin A ◽  
Temporal Order ◽  
Surface Glycoprotein ◽  
Plasma Membrane Proteins ◽  
Cell Surface Glycoprotein ◽  
Extensive Cell ◽  
Triton X

The plasma membrane proteins of Dictyostelium discoideum were characterized with respect to their partitioning into the Triton-insoluble cytoskeleton fraction of the cell during concanavalin A-induced capping. Two fractions of plasma membrane-associated concanavalin A were identified; one that immediately associated with the cytoskeleton fraction via cell surface glycoproteins, and one that partitioned with the cytoskeleton only after extensive cell surface glycoprotein cross-linking. Three major classes of polypeptides were found in the plasma membrane that differed with respect to their partitioning properties into the cytoskeleton fraction. The temporal order of association of the polypeptides with the cytoskeleton during concanavalin A-induced capping corresponded to the strength of their association with the cytoskeleton fraction as determined by pH and ionic strength elution from unligated cytoskeletons.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

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