An analogue of the erythroid membrane skeletal protein 4.1 in nonerythroid cells.

Protein 4.1 is a crucial component of the erythrocyte membrane skeleton. Responsible for the amplification of the spectrin-actin interaction, its presence is required for the maintenance of erythrocyte integrity. We have demonstrated a 4.1-like protein in nonerythroid cells. An antibody was raised to erythrocyte protein 4.1 purified by KCl extraction (Tyler, J. M., W. R. Hargreaves, and D. Branton, 1979, Proc. Natl. Acad. Sci. USA, 76:5192-5196), and used to identify a serologically cross-reactive protein in polymorphonuclear leukocytes, platelets, and lymphoid cells. The cross-reactive protein(s) were localized to various regions of the cells by immunofluorescence microscopy. Quantitative adsorption studies indicated that at least 30-60% of the anti-4.1 antibodies reacted with this protein, demonstrating significant homology between the erythroid and nonerythroid species. A homologous peptide doublet was observed on immunopeptide maps, although there was not complete identity between the two proteins. When compared with erythrocyte protein 4.1, the nonerythroid protein(s) displayed a lower molecular weight--68,000 as compared with 78,000-and did not bind spectrin or the nonerythroid actin-binding protein filamin. There was no detectable cross-reactivity between human acumentin or human tropomyosin-binding protein, which are similarly sized actin-associated proteins, and erythrocyte protein 4.1. The possible origin and significance of 4.1-related protein(s) in nonerythroid cells are discussed.

Download Full-text

Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1

Blood ◽

10.1182/blood.v78.9.2438.2438 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2438-2443 ◽

Cited By ~ 23

Author(s):

JG Conboy ◽

R Shitamoto ◽

M Parra ◽

R Winardi ◽

A Kabra ◽

...

Keyword(s):

Amino Acids ◽

Mechanical Stability ◽

Membrane Skeleton ◽

Actin Binding ◽

Erythrocyte Morphology ◽

Protein 4.1 ◽

Hereditary Elliptocytosis ◽

Exon Expression ◽

Amino Acid Peptide ◽

Skeletal Protein

Abstract Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.

Download Full-text

Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1

Blood ◽

10.1182/blood.v78.9.2438.bloodjournal7892438 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2438-2443

Author(s):

JG Conboy ◽

R Shitamoto ◽

M Parra ◽

R Winardi ◽

A Kabra ◽

...

Keyword(s):

Amino Acids ◽

Mechanical Stability ◽

Membrane Skeleton ◽

Actin Binding ◽

Erythrocyte Morphology ◽

Protein 4.1 ◽

Hereditary Elliptocytosis ◽

Exon Expression ◽

Amino Acid Peptide ◽

Skeletal Protein

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.

Download Full-text

Distribution of actin, myosin, actin-binding protein and gelsolin in cultured lymphoid cells

Experimental Cell Research ◽

10.1016/0014-4827(82)90129-x ◽

1982 ◽

Vol 140 (2) ◽

pp. 395-400 ◽

Cited By ~ 7

Author(s):

R THORSTENSSON ◽

G UTTER ◽

R NORBERG ◽

A FAGRAEUS ◽

J HARTWIG ◽

...

Keyword(s):

Binding Protein ◽

Lymphoid Cells ◽

Actin Binding ◽

Actin Binding Protein

Download Full-text

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

Biochemical Journal ◽

10.1042/bj20041502 ◽

2005 ◽

Vol 388 (2) ◽

pp. 631-638 ◽

Cited By ~ 35

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.

Download Full-text

A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene

Development ◽

10.1242/dev.120.3.545 ◽

1994 ◽

Vol 120 (3) ◽

pp. 545-557 ◽

Cited By ~ 25

Author(s):

R.G. Fehon ◽

I.A. Dawson ◽

S. Artavanis-Tsakonas

Keyword(s):

Egf Receptor ◽

Normal Development ◽

Membrane Skeleton ◽

Septate Junction ◽

Cellular Functions ◽

Septate Junctions ◽

Protein 4.1 ◽

Skeletal Protein ◽

Developmental Role ◽

Primary Mutant

Protein 4.1 functions to link transmembrane proteins with the underlying spectrin/actin cytoskeleton. To permit a genetic analysis of the developmental role and cellular functions of this membrane-skeletal protein, we have identified and characterized its Drosophila homologue (termed D4.1). D4.1 is localized to the septate junctions of epithelial cells and is encoded by the coracle gene, a new locus whose primary mutant phenotype is a failure in dorsal closure. In addition, coracle mutations dominantly suppress Ellipse, a hypermorphic allele of the Drosophila EGF-receptor homologue. These data indicate that D4.1 is associated with the septate junction, and suggest that it may play a role in cell-cell interactions that are essential for normal development.

Download Full-text

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

10.1055/s-0038-1642926 ◽

1987 ◽

Cited By ~ 1

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.

Download Full-text

Anatomy of the red cell membrane skeleton: unanswered questions

Blood ◽

10.1182/blood-2014-12-512772 ◽

2016 ◽

Vol 127 (2) ◽

pp. 187-199 ◽

Cited By ~ 122

Author(s):

Samuel E. Lux

Keyword(s):

Cell Membrane ◽

Lipid Bilayer ◽

Membrane Surface ◽

Membrane Skeleton ◽

Actin Binding ◽

Red Cell ◽

Red Cell Membrane ◽

Associated Proteins ◽

Protein 4.1R ◽

Cell Skeleton

Abstract The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3–containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton’s physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.111.2.483 ◽

1990 ◽

Vol 111 (2) ◽

pp. 483-493 ◽

Cited By ~ 97

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.

Download Full-text

Identification of a membrane skeleton in platelets.

The Journal of Cell Biology ◽

10.1083/jcb.106.5.1525 ◽

1988 ◽

Vol 106 (5) ◽

pp. 1525-1538 ◽

Cited By ~ 102

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.

Download Full-text

Characterization of isoforms of protein 4.1 present in the nucleus

Biochemical Journal ◽

10.1042/bj2790581 ◽

1991 ◽

Vol 279 (2) ◽

pp. 581-585 ◽

Cited By ~ 44

Author(s):

I Correas

Keyword(s):

Molecular Mass ◽

Plasma Membranes ◽

Peptide Mapping ◽

Cell Types ◽

Immunoblot Analysis ◽

Membrane Skeleton ◽

High Molecular Mass ◽

Actin Binding ◽

Nuclear Staining ◽

Protein 4.1

Although protein 4.1 was originally identified as an element of the erythrocyte membrane skeleton, its presence in most mammalian cell types is now well described. Antibodies raised against erythrocyte protein 4.1 or synthetic peptides corresponding to the spectrin-actin-binding domain of protein 4.1 react with plasma membranes and, unexpectedly, nuclei of different cell types. Nuclear staining was further confirmed in isolated nuclei prepared from rat liver and human leukaemic cell lines. Immunoblot analysis of subcellular fractions derived from these cells revealed three prominent proteins, of 80, 135 and 145 kDa. The structural relationship of the high-molecular-mass proteins with erythrocyte protein 4.1 was demonstrated by peptide mapping. These results indicate that mammalian nucleated cells contain several isoforms of erythrocyte protein 4.1 and that some high-molecular-mass forms may primarily reside in the nucleus.

Download Full-text