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

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.

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.

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 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.

MONOCLONAL ANTIBODIES RECOGNISING PROTEINS OF THE OUTER AND INNER SURFACE OF THE PLATELET PLASMA MEMBRANE

1987 ◽  
Author(s):  
J M Wilkinson ◽  
N Hack ◽  
L I Thorsen ◽  
J A Thomas
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Binding Protein ◽  
Cytoskeletal Proteins ◽  
Membrane Preparation ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Major Surface ◽  
Surface Glycoproteins

Platelet membrane preparations can be fractionated into two major subpopulations by free flow electrophoresis and these have been shown to correspond to the plasma membrane and the endoplasmic reticulum of the platelet. The plasma membrane fraction can be shown, by two-dimensional electrophoresis, to contain the major surface glycoproteins together with considerable amounts of actin and actin-associated proteins such as the 250 kDa actin-binding protein (filamin), P235 (talin), myosin, α-actinin and tropomyosin (Hack, N. … Crawford, N., Biochem. J. 222, 235 (1984). These cytoskeletal proteins are associated with the cytoplasmic face of the plasma membrane and probably interact with transmembrane glycoproteins. We have raised monoclonal antibodies to the purified plasma membrane preparation in order to investigate the nature of these glycoprotein-cytoskeletal interactions. In two fusion experiments, out of 804 tested, 104 hybrids secreted antibody to the membrane preparation and of these 24 were selected for further study. Initial assays were by ELISA using either the membrane preparation or whole fixed platelets as the target antigen. The specificity of the antibodies was investigated further by immunoblotting of SDS gels of total platelet proteins prepared under reducing and nonreducing conditions, by immunofluorescence, by immunohisto-chemistry and by crossed immunoelectrophoresis. The majority of the antibodies recognise major surface glycoproteins; of these, four bind to glycoprotein Ib under all conditions examined while another seven recognise the glycoprotein IIb/IIIa complex as detected by crossed immunoelectrophoresis. Three antibodies recognise the actin binding protein and these cross-react with the smooth muscle protein filamin in a number of different species. Further characterisation of these antibodies in both structural and functional terms will be presented.We are grateful to the Smith and Nephew Foundation for financial support for these studies

scholarly journals EPI64 regulates microvillar subdomains and structure

2006 ◽  
Vol 175 (5) ◽  
pp. 803-813 ◽  
Author(s):  
Abraham Hanono ◽  
Damien Garbett ◽  
David Reczek ◽  
David N. Chambers ◽  
Anthony Bretscher
Keyword(s):  
Plasma Membrane ◽  
High Resolution ◽  
Light Microscopy ◽  
Binding Protein ◽  
Actin Binding ◽  
Pdz Domains ◽  
Variable Regions ◽  
Actin Binding Protein ◽  
Knock Down ◽  
Microvillar Structure

EPI64 is a TBC domain–containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants

1996 ◽  
Vol 109 (7) ◽  
pp. 1825-1835 ◽  
Author(s):  
M. Stoeckelhuber ◽  
A.A. Noegel ◽  
C. Eckerskorn ◽  
J. Kohler ◽  
D. Rieger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Biochemical Properties ◽  
Actin Binding ◽  
Dependent Manner ◽  
Wild Type ◽  
Coding Region ◽  
Actin Binding Protein ◽  
Significant Difference ◽  
Ph Dependent

Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner. To further characterize this interaction we isolated hisactophilin overexpression (hisII+) and hisactophilin minus (his-) mutants. D. discoideum contains two hisactophilin isoforms; both genes are independently transcribed and carry a short intron at the same position of the coding region. The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of a total 118 amino acids. After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression. The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette. The properties of the hisII+ and his- cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions. However, the hisII+ transformants revealed a significant difference to wild-type cells and his- cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor. HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his- cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin. Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane. The fact that cells deficient in hisactophilin again lose resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration.

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 Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin

1994 ◽  
Vol 6 (3) ◽  
pp. 389-400 ◽  
Author(s):  
Bjorn K. Drobak ◽  
Peter A.C. Watkins ◽  
Rudolf Valenta ◽  
Stephen K. Dove ◽  
Clive W. Lloyd ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Plant Plasma Membrane
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