scholarly journals Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

1998 ◽  
Vol 141 (4) ◽  
pp. 929-942 ◽  
Author(s):  
Thomas Harder ◽  
Peter Scheiffele ◽  
Paul Verkade ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Protein Tyrosine Kinase ◽  
Cholesterol Depletion ◽  
Placental Alkaline Phosphatase ◽  
Glycosyl Phosphatidylinositol ◽  
Lipid Domain ◽  
Polarized Cells ◽  
T Lymphoma ◽  
Raft Domains ◽  
Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.

scholarly journals Cell-surface attachment of pedestal-forming enteropathogenicE. coliinduces a clustering of raft components and a recruitment of annexin 2

2002 ◽  
Vol 115 (1) ◽  
pp. 91-98
Author(s):  
Nicole Zobiack ◽  
Ursula Rescher ◽  
Sven Laarmann ◽  
Silke Michgehl ◽  
M. Alexander Schmidt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Surface ◽  
Actin Binding ◽  
Surface Attachment ◽  
Glycosyl Phosphatidylinositol ◽  
Annexin 2 ◽  
Endosomal Membranes ◽  
Membrane Components ◽  
Functional Receptor ◽  
Pedestal Formation

Annexin 2 is a Ca2+-regulated membrane- and F-actin-binding protein implicated in the stabilization or regulation of membrane/cytoskeleton contacts, or both, at the plasma membrane and at early endosomal membranes. To analyze the dynamic nature of such action we investigated whether annexin 2 could be found at sites of localized actin rearrangements occurring at the plasma membrane of HeLa cells infected with noninvading enteropathogenic Escherichia coli (EPEC). We show that adherent EPEC microcolonies, which are known to induce the formation of actin-rich pedestals beneath them, specifically recruit annexin 2 to the sites of their attachment. Mutant EPEC (EPECtir), which lack a functional receptor for intimate attachment (Tir, translocated intimin receptor) and which fail to produce full pedestal formation, are still capable of recruiting annexin 2 to the bacterial contact sites. Accumulation of annexin 2 at sites of EPEC or EPECtir attachment is accompanied by a recruitment of the annexin 2 protein ligand S100A10. EPEC and EPECtir attachment also induces a concentration of cholesterol and glycosyl phosphatidylinositol-anchored proteins at sites of bacterial contact. This indicates that membrane components present in rafts or raft-like microdomains are clustered upon EPEC adherence and that annexin 2 is recruited to the cytoplasmic membrane surface of such clusters, possibly stabilizing raft patches and their linkage to the actin cytoskeleton beneath adhering EPEC.

scholarly journals Transient anchorage of cross-linked glycosyl-phosphatidylinositol–anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides

2006 ◽  
Vol 175 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Yun Chen ◽  
William R. Thelin ◽  
Bing Yang ◽  
Sharon L. Milgram ◽  
Ken Jacobson
Keyword(s):  
Transmembrane Protein ◽  
Cross Linking ◽  
Cholesterol Depletion ◽  
Transmembrane Conductance Regulator ◽  
Membrane Biology ◽  
Glycosyl Phosphatidylinositol ◽  
Caveolin 1 ◽  
Outer Leaflet ◽  
Membrane Components ◽  
Open Question

How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol–anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle–induced clusters of both Thy-1 and CD73, a 5′ exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.

scholarly journals Induction of Caveolae in the Apical Plasma Membrane of Madin-Darby Canine Kidney Cells

1999 ◽  
Vol 148 (4) ◽  
pp. 727-740 ◽  
Author(s):  
Paul Verkade ◽  
Thomas Harder ◽  
Frank Lafont ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Cross Linking ◽  
Cholesterol Depletion ◽  
Placental Alkaline Phosphatase ◽  
Coated Pits ◽  
Apical Side ◽  
Associated Proteins

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929–942), only small (∼100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly (∼10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

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 Influence of the glycocalyx and plasma membrane composition on amphiphilic gold nanoparticle association with erythrocytes

Nanoscale ◽  
2015 ◽  
Vol 7 (26) ◽  
pp. 11420-11432 ◽  
Author(s):  
Prabhani U. Atukorale ◽  
Yu-Sang Yang ◽  
Ahmet Bekdemir ◽  
Randy P. Carney ◽  
Paulo J. Silva ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Plasma Membrane ◽  
Gold Nanoparticle ◽  
Erythrocyte Membranes ◽  
Membrane Composition ◽  
Membrane Components

Amphiphilic gold nanoparticles spontaneously insert into erythrocyte membranes; we characterize this association as a function of key plasma membrane components.

Association of the hepatic IP3 receptor with the plasma membrane: relevance to mode of action

1993 ◽  
Vol 265 (6) ◽  
pp. C1588-C1596 ◽  
Author(s):  
L. Feng ◽  
N. Kraus-Friedmann
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Plasma Membrane Fraction ◽  
T Lymphoma Cells ◽  
T Lymphoma ◽  
Triton X ◽  
Brain Receptor ◽  
The Brain

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

scholarly journals RGS4 and RGS2 Bind Coatomer and Inhibit COPI Association with Golgi Membranes and Intracellular Transport

2000 ◽  
Vol 11 (9) ◽  
pp. 3155-3168 ◽  
Author(s):  
Brandon M. Sullivan ◽  
Kimberly J. Harrison-Lavoie ◽  
Vladimir Marshansky ◽  
Herbert Y. Lin ◽  
John H. Kehrl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Rgs Proteins ◽  
Placental Alkaline Phosphatase ◽  
Protein Signaling ◽  
Golgi Membranes

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit β′-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on Giα. In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi–plasma membrane or intra-Golgi transport.

scholarly journals Phospholipid flippases enable precursor B cells to flee engulfment by macrophages

2018 ◽  
Vol 115 (48) ◽  
pp. 12212-12217 ◽  
Author(s):  
Katsumori Segawa ◽  
Yuichi Yanagihashi ◽  
Kyoko Yamada ◽  
Chigure Suzuki ◽  
Yasuo Uchiyama ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
B Cell ◽  
Developmental Stages ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Wild Type ◽  
Immature B Cells ◽  
T Lymphoma ◽  
Precursor B Cells

ATP11A and ATP11C, members of the P4-ATPases, are flippases that translocate phosphatidylserine (PtdSer) from the outer to inner leaflet of the plasma membrane. Using the W3 T lymphoma cell line, we found that Ca2+ ionophore-induced phospholipid scrambling caused prolonged PtdSer exposure in cells lacking both the ATP11A and ATP11C genes. ATP11C-null (ATP11C−/y) mutant mice exhibit severe B-cell deficiency. In wild-type mice, ATP11C was expressed at all B-cell developmental stages, while ATP11A was not expressed after pro−B-cell stages, indicating that ATP11C−/y early B-cell progenitors lacked plasma membrane flippases. The receptor kinases MerTK and Axl are known to be essential for the PtdSer-mediated engulfment of apoptotic cells by macrophages. MerTK−/− and Axl−/− double deficiency fully rescued the lymphopenia in the ATP11C−/y bone marrow. Many of the rescued ATP11C−/y pre-B and immature B cells exposed PtdSer, and these cells were engulfed alive by wild-type peritoneal macrophages, in a PtdSer-dependent manner. These results indicate that ATP11A and ATP11C in precursor B cells are essential for rapidly internalizing PtdSer from the cell surface to prevent the cells’ engulfment by macrophages.

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