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.

Platelet Glycoproteins in Platelet Aggregation

1979 ◽  
Author(s):  
David R. Phillips ◽  
Lisa K. Jennings ◽  
Harold H. Edwards
Keyword(s):  
Membrane Surface ◽  
Direct Interaction ◽  
Protein Composition ◽  
Actin Binding ◽  
Membrane Glycoproteins ◽  
Dense Cores ◽  
Insoluble Material ◽  
Filamentous Material ◽  
Membrane Components ◽  
Triton X

Thrombin stimulation alters the membrane surface of platelets so that specific components on the membrane surface interact. To identify such “aggregation factors”, tne exposed membrane proteins of washed platelets were labeled by lactoperoxidase-catalyzed iodination and tested for their association with cytoskeletal structures. Control, thrombin-stimulated (TS; nM thrombin in mM EDTA to prevent aggregation) and thrombin aggregated (TA; 2 mM Ca++) platelets were treated with 1% Triton X-100. The insoluble material (isolated by centrifugation) from TS platelets, but not unstimulated platelets, had clusters of filamentous material with dense cores about 1 μ in diameter. Each cluster appeared to arise from one platelet and contained proteins with the Mr of actin actin-binding protein and myosin plus a 56K and 90K protein. Triton extraction of TA platelets produced an insoluble material with a similar protein composition as that from TS platelets; however, the filamentous clusters remained- aggregration, indicating tnat membrane components which aggregate platelets were still present. Analysis of iodinated membrane components revealed that all were solubilized by Triton from control and TS platelets while two glycoproteins, termed IIb and III, remained with the filamentous material from TA platelets. This and the observation that platelets lacking IIb and III cannot aggregate [JCI. 60: 535 (1977)], indicate that one or both of these membrane glycoproteins are involved in the direct Interaction of platelets during aggregation.

Localization of VAMP Isoforms In Platelets Reveals Separate Granule Populations with Distinct Functions

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2015-2015
Author(s):  
Christian G Peters ◽  
Robert Flaumenhaft
Keyword(s):  
Plasma Membrane ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Leading Edge ◽  
Actin Binding ◽  
Membrane Repair ◽  
Platelet Activity ◽  
Membrane Flow ◽  
Microscopy Imaging ◽  
Platelet Spreading

Abstract Abstract 2015 Secretion of granules from platelets is essential for normal platelet activity in hemostasis and thrombosis. Platelet granules have different morphologies and different granule subpopulations contain distinct cargos. Platelet granules not only participate in the release of granule contents, but also appear to function to increase plasma membrane surface area during platelet spreading, morphogenesis, tether formation, and membrane repair. Whether different granule subpopulations mediate these distinct functions of the granule is currently not known. To judge whether specific subpopulations of granules are involved in different granule functions, we evaluated the subcellular localization of vesicle-associated membrane proteins (VAMPs) in resting and spread platelets using immunofluorescence confocal microscopy. Imaging showed that VAMP 3- and VAMP 8-containing granules segregate from one another into distinct granule populations (Pearson (R) correlation values (R = 0.143 ± .039, n=25). Fibrinogen and von Willebrand factor (vWF) also segregated from one another (R= 0.099 ± .027, n=15), but associated with granules containing either VAMP 3 or 8 (R = 0.447 ± 0.065 and R = 0.5485 ± 0.62, n=20, respectively). To visualize the granule contribution to membrane remodeling during spreading, granule membranes were selectively labeled using the membrane dye FM 1–43 followed by washout of external dye. Following platelet spreading, FM 1–43-labeled granules coalesced at the ends of pseudopodia and appeared as punctate areas of FM-1-43 staining on the plasma membrane. Quantitation of staining within spread platelets demonstrated that 92% of granules expressing VAMP 8 translocated with their associated cargo (vWF or fibrinogen) to the central granulomere. VAMP 3 also translocated with vWF and fibrinogen to the granulomere in spread platelets. In contrast, only 31% of granules expressing VAMP 5 localized to the granulomere of spread platelets, while 69% of VAMP 5 staining localized to pseudopodia and lamellipodia at the platelet periphery. Granules expressing VAMP 5 demonstrated little colocalization with vWF and fibrinogen in spread platelets. Similarly, VAMP 7 translocated to pseudopodia and lamellipodia during spreading, displaying punctuate staining at the leading edge of plasma membrane. Studies evaluating the expression of VAMP isoforms in nonpermeabilized platelets demonstrated that a portion of VAMP 5, but not VAMPs 3, 7, or 8, resides on the extracellular face of the plasma membrane. Flow cytometry confirmed the presence of VAMP 5 on the extracellular surface of platelets. These data demonstrate that different VAMP isoforms segregate to distinct platelet granule subpopulations and subcellular locals. The colocalization of VAMPs 3 and 8 with vWF and fibrinogen is consistent with previous data demonstrating a role for these VAMP isoforms in cargo release. In contrast, granules expressing VAMP 5 or VAMP 7, which contains an actin-binding login domain, translocate to areas of cytoskeletal remodeling and may contribute membrane to growing cytoskeletal structures as the platelet spreads. These studies indicate that separate granule populations defined by expression of distinct VAMP isoforms perform different functions in platelets. Disclosures: No relevant conflicts of interest to declare.

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 A possible mechanism for ezrin to establish epithelial cell polarity

2010 ◽  
Vol 299 (2) ◽  
pp. C431-C443 ◽  
Author(s):  
Lixin Zhu ◽  
James Crothers ◽  
Rihong Zhou ◽  
John G. Forte
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Membrane Surface ◽  
Terminal Region ◽  
Actin Binding ◽  
Apical Plasma Membrane ◽  
Specific Information ◽  
Basal Surface ◽  
Membrane Recruitment ◽  
Binding Forms

Ezrin is an important membrane/actin cytoskeleton linker protein, especially in epithelia. Ezrin has two important binding domains: an NH2-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin only after a conformational activation by phosphorylation at Thr567 of ezrin. The present experiments were undertaken to investigate the detailed cellular changes in the time course of expression of ezrin-T567 mutants (nonphosphorylatable T567A and permanent phospho-mimic T567D) in parietal cells and to assess ezrin distribution and its influence on the elaborate membrane recruitment processes of these cells. T567A mutant and wild-type (WT) ezrin were consistently localized to the apical plasma membrane, even with overexpression. On the other hand, T567D went first to apical membrane at early times and low expression levels, then accumulated mainly at the basal surface after 24 h. Overexpression of WT or T567A led to incorporation of internal membranes to apical vacuoles, while overexpression of T567D led to large incorporation of apical and intracellular membranes (including H-K-ATPase) to the basal surface. Differences in polar distribution of ezrin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections, forming the basis for a novel theory for ezrin as an organizer and regulator of membrane recruitment. A model simulating the cellular distribution of ezrin and its associated membrane- and F-actin-binding forms is given to predict redistributions observed with phosphorylation and mutant overexpression, and it can easily be modified as more specific information regarding binding constants and specific sites becomes available.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

scholarly journals Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

1994 ◽  
Vol 125 (2) ◽  
pp. 381-391 ◽  
Author(s):  
J Mulholland ◽  
D Preuss ◽  
A Moon ◽  
A Wong ◽  
D Drubin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Ultrastructural Level ◽  
Actin Binding ◽  
Cortical Actin ◽  
Actin Binding Proteins ◽  
Double Labeling ◽  
Wall Growth ◽  
Cortical Cytoskeleton

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

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.

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