scholarly journals Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane.

1990 ◽  
Vol 111 (2) ◽  
pp. 483-493 ◽  
Author(s):  
J E Fox ◽  
C D Austin ◽  
J K Boyles ◽  
P K Steffen
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Membrane Skeleton ◽  
Membrane Properties ◽  
Actin Binding ◽  
Ionophore A23187 ◽  
Membrane Interactions ◽  
Membrane Glycoproteins ◽  
Actin Binding Protein ◽  
Hydrolysis Of

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this change on plasma membrane properties were examined. The agents that were used were ionophore A23187 and dibucaine. Both agents activated calpain (the Ca2(+)-dependent protease), resulting in the hydrolysis of actin-binding protein and decreased association of actin with membrane glycoproteins. Disruption of actin-membrane interactions was accompanied by the shedding of procoagulant-rich microvesicles from the plasma membrane. The shedding of microvesicles correlated with the hydrolysis of actin-binding protein and the disruption of actin-membrane interactions. When the calpain-induced disruption of actin-membrane interactions was inhibited, the shedding of microvesicles was inhibited. These data are consistent with the hypothesis that association of the membrane skeleton with the plasma membrane maintains the integrity of the plasma membrane, preventing the shedding of procoagulant-rich microvesicles from the membrane of unstimulated platelets. They raise the possibility that the procoagulant-rich microvesicles that are released under a variety of physiological and pathological conditions may result from the dissociation of the platelet membrane skeleton from its membrane attachment sites.

scholarly journals Identification of a membrane skeleton in platelets.

1988 ◽  
Vol 106 (5) ◽  
pp. 1525-1538 ◽  
Author(s):  
J E Fox ◽  
J K Boyles ◽  
M C Berndt ◽  
P K Steffen ◽  
L K Anderson
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Protein ◽  
Amorphous Material ◽  
Three Dimensional ◽  
Amorphous Layer ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Filamentous Form ◽  
Actin Binding Protein

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

THE DISTRIBUTION OF GP lb AND THE STABILITY OF THE PLASMA MEMBRANE ARE DEPENDENT UPON AN INTACT MEMBRANE SKELETON

1987 ◽  
Author(s):  
J K Boyles ◽  
JE B Fox ◽  
M C Berndt
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Protein ◽  
Protein A ◽  
Cell Aggregation ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Gold Particles ◽  
Actin Binding Protein ◽  
The Stability

Platelets are know to have a cytoskeleton of actin filaments. We have presented evidence that they also have a membrane skeleton linked to the cytoskeletal filaments and that the membrane skeleton is linked to GP Ib-IX on the plasma membrane via actin-binding protein. In the current study, electron microscopy of thick (0.2 ym) epoxy sections was used to identify the distribution of GP lb. After various treatments, platelets were fixed and incubated with affinity-purified GP lb antibody and colloidal gold-labeled Protein-A. The entire cell surface was covered with a network of short intersecting chains of relatively evenly spaced gold particles. This was true of platelets in blood dripped directly from a vein into fixative, of washed discoid platelets, and of platelets activated by thrombin, ionophore, or cold under conditions in which aggregation did not occur. This pattern was not affected by the size of the gold label, the immunocytochemical protocol, or the fixative. The number of gold particles per cell was between 10,000 and 20,000, indicating a 1:1 ratio of label to GP lb. The distribution of GP lb was not affected by a level of cyto-chalasin B sufficient to disrupt the actin filaments of the platelet cytoskeleton. Proteolysis of actin-binding protein is known to be induced by treatment of platelets with dibucaine and by platelet activation (with either ionophore or thrombin) under conditions in which cell aggregation occurs. These same treatments caused GP lb to cluster. They also produced platelets with unstable membranes that vesiculated when the cells were subjected to shear force during centrifugation or osmotic-ally stressed during fixation. These studies show that both the distribution of GP lb and membrane stability are dependent upon the integrity of actin-binding protein and the membrane skeleton. In the high-shear environment of the blood vessel, the membrane skeleton and its linkage to GP Ib-IX and the cytoskeleton may be essential for proper platelet function.

scholarly journals Identification of membrane proteins mediating the interaction of human platelets

1980 ◽  
Vol 86 (1) ◽  
pp. 77-86 ◽  
Author(s):  
D Phillips ◽  
L Jennings ◽  
H Edwards
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Membrane Surface ◽  
Direct Interaction ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Insoluble Residue ◽  
Membrane Glycoproteins ◽  
Actin Binding Protein ◽  
Activated Platelets

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

scholarly journals αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

2005 ◽  
Vol 388 (2) ◽  
pp. 631-638 ◽  
Author(s):  
Björn ROTTER ◽  
Odile BOURNIER ◽  
Gael NICOLAS ◽  
Didier DHERMY ◽  
Marie-Christine LECOMTE
Keyword(s):  
Binding Protein ◽  
Focal Adhesions ◽  
Membrane Skeleton ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Src Homology 3 ◽  
Src Homology 3 Domain ◽  
Src Homology

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

Isoform specific expression of the neuronal F-actin binding protein, drebrin, in specialized cells of stomach and kidney epithelia

2000 ◽  
Vol 113 (2) ◽  
pp. 325-336 ◽  
Author(s):  
B.H. Keon ◽  
P.T. Jedrzejewski ◽  
D.L. Paul ◽  
D.A. Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Cultured Cells ◽  
Binding Protein ◽  
Specific Pattern ◽  
Actin Binding ◽  
Apical Plasma Membrane ◽  
Specific Expression ◽  
Actin Binding Protein ◽  
Acid Secreting

To further understand the functional role that the F-actin binding protein, drebrin (developmentally regulated brain protein), plays in the regulation of F-actin, we characterized its expression in non-neuronal cells. Using nanoelectrospray mass spectrometry methods, we initially identified drebrin in non-neuronal cultured cells. Using a drebrin-specific monoclonal antibody, we were able to detect drebrin protein in several different cell lines derived from fibroblasts, astrocytomas, and simple epithelia, but not in cell lines derived from stratified epithelia. Double-label immunofluorescence experiments of cultured cell monolayers revealed the localization of drebrin at the apical plasma membrane together with a pool of submembranous F-actin. Immunoblot analysis of mouse organs revealed that, in addition to its high levels of expression in brain, drebrin was present in stomach and to a lesser degree in kidney, colon, and urinary bladder. Drebrin protein detected in the non-brain organs migrated faster through SDS-PAGE gels, indicating that the lower molecular weight embryonic brain isoform (E2) may be the prominent isoform in these organs. RT-PCR experiments confirmed the specific expression of the E2 isoform in adult stomach, kidney, and cultured cells. In situ immunofluorescence experiments revealed a cell-type specific pattern in both stomach and kidney. In stomach, drebrin was specifically expressed in the acid-secreting parietal cells of the fundic glands, where it accumulated at the extended apical membrane of the canaliculi. In kidney, drebrin was expressed in acid-secreting type A intercalated cells, where it localized specifically to the apical plasma membrane. Drebrin was expressed as well in the distal tubule epithelial cells where the protein was concentrated at the luminal surface and present at the interdigitations of the basolateral membranes.

IDENTIFICATION OF GLYCOPROTEIN Ib8 AS THE Mr = 24,000 PLATELET POLYPEPTIDE PHOSPHORYLATED BY AGENTS THAT ELEVATE CYCLIC AMP

1987 ◽  
Author(s):  
J E B Fox ◽  
C C Reynolds ◽  
J K Boyles ◽  
R A Abel ◽  
M M Johnson
Keyword(s):  
Cyclic Amp ◽  
Platelet Function ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Inhibitory Effects ◽  
Actin Binding Protein ◽  
Triton X ◽  
Β Chain

Platelet function is inhibited by agents that elevate intracellular cyclic AMP concentrations, presumably as a result of the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are of Mr = 250,000, Mr = 51.000 (P51), Mr = 36,000 (P36), Mr = 24,000 (P24), and Mr = 22.000 (P22). The Mr = 250,000 polypeptide is actin-binding protein, but the identity of the other polypeptides 1s unknown. In the present study, we identified the P24 polypeptide. Platelets were radiolabeled with [32P]P1 and then Incubated for 2-5 min in the presence or absence of 5 μM prostaglandin E1 (PGE1). The PGE1-induced phosphorylation of P24 was detected on autoradiograms of SDS-gels. Since P24 has been shown to be membrane-associated, its molecular weight was compared with those of known membrane proteins. P24 comigrated with the β-chain of purified GP Ib on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ibβ is disulfide-linked to GP Ibα and migrates with Mr = 170,000). Like GP Ibβ, P24 was associated with actin filaments in Triton X-100 lysates. Both GP Ibβ and P24 were selectively associated with filaments of the membrane skeleton and were released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (that lacked GP Ib) to PGE1 resulted in phosphorylation of actin-binding protein, P51, P36, and P22, but not P24. We conclude that P24 is GP Ibβ. To determine whether phosphorylation of GP Ibβ is responsible for the inhibitory effects of PGE1 on platelets, we compared the action of PGE1 on control platelets with that on Bernard-Soulier platelets. One of the ways in which PGE1 inhibits platelet activation is by inhibiting the polymerization of actin. While PGE1 inhibited actin polymerization in control platelets, it did not in Bernard-Soulier platelets. We conclude that GP Ibβ is phosphorylated by agents that elevate cyclic AMP and that phosphorylation of this glycoprotein results in inhibition of platelet function.

Presence of Clathrin at Adhesion Sites in Phagocytosing Macrophages

1982 ◽  
Vol 40 ◽  
pp. 114-117
Author(s):  
J. Aggeler ◽  
J.E. Heuser ◽  
Z. Werb
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Binding Protein ◽  
Cell Spreading ◽  
Actin Binding ◽  
Dense Network ◽  
Actin Binding Protein ◽  
Adhesion Sites ◽  
Cytoplasmic Surface

Phagocytosis of particles by macrophages may be similar to cell spreading on a substratum, in that a dense network of actin filaments appears beneath the plasma membrane in both cases. When viewed in broken-open or detergent- extracted cells, cytoskeletal filaments are observed to form focal attachments to the plasma membrane and to the cytoplasmic surface of phagosomes. Hartwig et al. have presented a model of phagocytosis in which an actin-binding protein alters the organization of subplasmalemma1 actin filaments in such a way that the plasma membrane is forced up over the particle to form the phagosome. Their evidence indicates that similar actin-binding proteins may function during cell spreading.

scholarly journals The pH-sensitive Actin-binding Protein Hisactophilin ofDictyosteliumExists in Two Isoforms Which Both Are Myristoylated and Distributed between Plasma Membrane and Cytoplasm

1995 ◽  
Vol 270 (2) ◽  
pp. 596-602 ◽  
Author(s):  
Frank Hanakam ◽  
Christoph Eckerskorn ◽  
Friedrich Lottspeich ◽  
Annette Müller-Taubenberger ◽  
Wolfram Schäfer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Ph Sensitive ◽  
Actin Binding ◽  
Actin Binding Protein
Sign in / Sign up

Export Citation Format

Share Document