Membrane-cytoskeleton Interactions Research Articles

Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organising cytoskeleton upon platelet activation

Vinculin, a 130-kDa protein discovered in chicken gizzard smooth-muscle cells and subsequently also described in platelets, is believed to be involved in membrane-cytoskeleton interactions. In this study we investigated vinculin distribution in human blood platelets. Two skeletal fractions and a remaining cytosolic fraction were prepared with a recently described Triton X-100 lysis buffer causing minimal post-lysis breakdown by proteolysis. The presence of vinculin was demonstrated in the membrane skeleton and cytosol of resting and thrombin-activated human platelets. Upon thrombin stimulation vinculin also appeared in the cytoskeleton. this cytoskeletal incorporation was completed during the early stages of platelet aggregation and secretion, when the uptake of myosin, actin-binding protein and talin was still not maximal. We conclude therefore, that vinculin may play an important role in the structural (re)organisation of the human platelet cytoskeleton upon platelet activation.

A 32 kDa lipocortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation

A 32 kDa protein isolated from human mononuclear cells is a member of the lipocortin family, a new group of Ca2+-dependent lipid-binding proteins thought to be involved in the regulation of phospholipase A2, in exocytosis and in membrane-cytoskeleton interactions. Purification of this protein was based on its ability to associate with membrane phospholipids in a Ca2+-dependent manner and its capacity to inhibit purified phospholipase A2 from pig pancreas. Using immunological detection, we show that it is present in various cells involved in the inflammatory and coagulation processes. We present extensive amino acid data that strongly suggest that this protein is identical with a recently described inhibitor of blood coagulation, with endonexin II and with lipocortin V. Sequence alignment with other known proteins show a significant degree of homology with lipocortins I and II, the substrates of the epidermal-growth-factor receptor tyrosine kinase and the oncogene pp60src tyrosine kinase respectively, and with protein II. The possible physiological role of this 32 kDa lipocortin is discussed.

Actin polymerization induces a shape change in actin-containing vesicles.

We have encapsulated actin filaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least approximately 0.5 micron long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane-cytoskeleton interactions, and cytomechanics.

Carbachol-induced protein phosphorylation in parietal cells: regulation by [Ca2+

1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), an intracellular calcium [( Ca2+]i) chelator, was used to investigate the role of [Ca2+]i in acid secretory activity and protein phosphorylation in parietal cells from rabbit. Chelation of extracellular calcium [( Ca2+]o) with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not prevent the initial carbachol-induced elevation of [Ca2+]i as measured with the fluorescent Ca2+ probe, fura-2, and only partially inhibited [14C]-aminopyrine (AP) accumulation, an indirect indicator of acid secretory activity. [Ca2+]i chelation with BAPTA/AM eliminated carbachol-stimulated increases in [Ca2+]i and AP accumulation but only transiently reduced histamine stimulation of AP accumulation. Carbachol increased phosphorylation of a 36-kDa, pI approximately 7 protein (pp36) and transient phosphorylation of a 28-kDa, pI approximately 5 protein (pp28), whereas histamine increased phosphorylation of 40-kDa, pI approximately 6.5 (pp40) and 27-kDa, pI approximately 6.2 (pp27) proteins. Phosphorylation of pp36 and pp28 were mimicked by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, ionomycin, respectively. Two other phosphoproteins with molecular weights of 66,000 and pIs of 5.7 and 5.9 were also phosphorylated in response to TPA and carbachol. Chelation of [Ca2+]i and [Ca2+]o blocked carbachol-induced phosphorylation of pp28 and pp36 and ionomycin phosphorylation of pp28 but not TPA-stimulated phosphorylation of pp36 or the two pp66s or histamine-stimulated phosphorylation of pp27 or pp40. Chelation of [Ca2+]i alone did not block increases in [Ca2+]i or phosphorylation of pp28 in response to ionomycin. Both pp28 and pp36 were localized in both microsomal and cytosolic fractions of cells, which suggests involvement in cytoskeleton-membrane interactions. These phosphoproteins could be common elements of Ca2+-dependent stimulus-secretion coupling as similar proteins were phosphorylated by carbachol and cholecystokinin (CCK) in chief cells. Based on data with TPA and ionomycin, both protein kinase C and an as yet unidentified Ca2+-dependent protein kinase(s) appear to be activated upon stimulation with cholinergic agonists and CCK.

Three actin-binding proteins are developmentally regulated in rat liver plasma membranes

Actin-binding membrane proteins (linking microfilaments to the cell membrane) are involved in cytoskeleton-membrane interactions which are supposed to undergo profound changes during cell proliferation and development. In this study 8 polypeptides were shown to bind F-actin directly in the liver cell membranes of mature rats. From these, the abundance of three polypeptides, of 130, 50 and 36 kDa, was observed to increase considerably during postnatal development, which indicates a developmental change in the cytoskeleton-membrane interactions.

Lymphoma protein kinase C is associated with the transmembrane glycoprotein, GP85, and may function in GP85-ankyrin binding

In this study, several complementary techniques have been used to investigate the involvement of a protein kinase C (PKC) molecule in the plasma membrane-cytoskeleton interactions that occur in mouse T-lymphoma cells. Our data indicate that the lymphoma plasma membrane contains a 78-kDa polypeptide that exists in a complex with one of the major transmembrane glycoproteins, GP85 (a wheat germ agglutinin-binding protein). This membrane-associated 78-kDa protein appears to have PKC-like properties based on the following criteria: 1) it cross-reacts with a specific antibody raised against brain PKC; 2) it has a pI of 5.6-5.8, which is similar to that of the PKC described previously in other cell types; and 3) it displays characteristic PKC enzymatic activity by phosphorylating histone H1 in a Ca2+- and phospholipid-dependent manner. Double immunocytochemical staining experiments reveal that the lymphoma PKC-like molecules translocate from the cytoplasm to the cell membrane and accumulate directly underneath receptor capped structures following addition of various ligands. Studies we have done to identify the cellular substrate(s) of the lymphoma plasma membrane-associated PKC have shown that GP85 is preferentially phosphorylated in isolated membrane preparations following addition of the PKC activator, TPA (phorbol-12-O-tetradecanoyl-phorbol 13-acetate), but not the biologically inactive TPA analogue, 4 alpha-PDD (4 alpha-phorbol 12,13-didecanoate). In addition, we have found that GP85 can be phosphorylated by purified brain protein kinase C. Analysis of the resulting phosphoamino acids indicates that phosphorylation of GP85 occurs primarily at serine residues, occurs in minor amounts (approximately 5%) at threonine residues, and does not occur at tyrosine residues. These data indicate that the lymphoma GP85 is a substrate for PKC. Furthermore, we have established that phosphorylation of GP85 by PKC enhances its binding affinity with the membrane linker molecule, ankyrin. These findings suggest that PKC-mediated phosphorylation of GP85 may be an important part of the lymphoma plasma membrane-cytoskeleton interaction.

Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis.

Using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and its cationic derivative, 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene, we evaluated membrane fluidity in living polymorphonuclear leukocytes and in erythrocytes of psoriatic patients. Our results have shown that erythrocyte membranes of psoriatic patients exhibit a decrease of fluidity. These changes were not associated with any relevant modifications of the cholesterol to phospholipid molar ratio. Moreover, we observed a decrease in polymorphonuclear leukocytes membrane fluidity associated with changes in chemotactic migration. Our results indicate changes of membrane fluidity involving membranes different from the epidermal cells and suggest the hypothesis of a defective membrane-cytoskeleton interaction in psoriasis.

Lateral diffusion of proteins and lipids in the plasma membrane of rose protoplast

Lateral diffusion measurements have been made on lipids and proteins in the plasma membrane of live protoplasts derived from rose (Rosa sp. “Paul's Scarlet”) suspension-cultured cells. Two different fluorescent lipid probes exhibited markedly different diffusion rates, indicating possible heterogeneity in the lipid domain of the membrane. Membrane proteins were labeled directly with covalently-reactive fluorophores, and factors that might perturb the lateral diffusion of these labeled proteins were investigated. Treatment of the protoplasts with various cytoskeleton-disrupting drugs generally had little effect on protein diffusion, although treatment with oryzalin, a microtubule-disrupting drug, did slightly reduce the mobile fraction of membrane proteins. Elevation of the CaCl2 concentration in the medium from 1 mM to 10 mM significantly reduced the mobile fraction of membrane proteins and also increased the fraction of protoplasts that were able to regenerate cell walls and divide in culture. These results are discussed in relation to reported evidence of lipid domains in the plasma membranes of other cells and protoplasts. The relative importance of lipid domains and membrane-cytoskeleton interaction in governing protein diffusion is considered.

The function of the major cytoskeletal components of the brush border

In order to carry out a detailed analysis of the structure and function of the cytoskeleton in eucaryotic cells as well as of cytoskeleton membrane interactions, cell and molecular biologists need an appropriate biological model systems. The complex cellular functions achieved by these microfilaments and the lack of numerous suitable models to approach these questions have certainly severely hampered rapid progress in this field.

The detection of a spectrin-like protein in [formula omitted] with a polyclonal antibody

A rabbit serum raised against sheep erythrocyte spectrin stained mainly the flagella of epimastigote forms of the parasitic protozoan Trypanosoma cruzi . In Western blots of T. cruzi proteins solubilized with Nonidet P-40 it recognized mainly a polypeptide doublet with an apparent molecular weight of about 240 kDa, while a pre-immune rabbit serum and a tubulin monoclonal antibody did not. The results are consistent with the idea that a spectrin-like protein may be involved in cytoskeleton-membrane interactions in flagella of T.cruzi .

Membrane-cytoskeleton interactions in the flagellum: a 240,000 Mr surface-exposed glycoprotein is tightly associated with the axoneme in Chlamydomonas moewusii.

The flagellar surface of Chlamydomonas moewusii is a dynamic structure involved in several adhesive and motile events. In this report, we describe for the first time the flagellar membrane components of vegetative C. moewusii. A glycoprotein (or pair of glycoproteins) with an apparent molecular weight of 240 x 10(3) is the dominant flagellar protein (other than the tubulins) in this species of Chlamydomonas. Both a rabbit polyclonal antibody (designated P-19) and the lectin concanavalin A recognize this 240K (K = 10(3) Mr) glycoprotein on nitrocellulose transblots of flagellar proteins. Fluorescence microscopic studies using these same two probes suggest that the 240K glycoprotein is exposed at the flagellar surface. Direct evidence that the 240K glycoprotein is exposed at the flagellar surface is provided by vectorial labelling with a N-hydroxysuccinamide derivitized biotin reagent (NHS-LC-biotin). Nonionic detergent extraction of isolated flagella fails to solubilize most of the 240K glycoprotein, although it completely removes the flagellar membranes as demonstrated by transmission electron microscopy. Furthermore, immunofluorescence microscopy of isolated axonemes demonstrates that both P-19-defined epitopes and surface-biotinylated proteins continue to be associated with the axoneme structure after detergent treatment. These observations demonstrate that the 240K flagellar protein is a glycoprotein that is both exposed at the flagellar surface and tightly coupled to the underlying cytoskeleton (axoneme). Because of its cell surface orientation and axonemal linkage, it is likely that the 240K glycoprotein plays an important role in the adhesive and/or motile phenomena exhibited by the C. moewusii flagellar surface.

Talking point Amphitropic proteins: a new class of membrane proteins

Specific lipids play crucial roles in signal transmission across membranes and in the modulation, regulation and membrane-association of proteins. Several proteins, previously described as soluble cytoplasmic proteins, appear to interact both specifically with lipids and directly with the hydrophobic part of membranes, indicating a reversible association of these proteins with membranes under appropriate conditions. Here it is proposed that such proteins belong to a new class of membrane proteins defined as the ‘amphitropic’ proteins. From this new concept emerge important implications for studies of cytoskeleton-membrane interactions, transmembrane signaling, and the organization of the cytoskeleton.

Membrane-cytoskeleton interactions: Inhibition of odontoblast differentiation by a monoclonal antibody directed against a membrane protein

Abstract It is known that high-molecular-weight (HMW) membrane proteins mediate interactions with constituents of the extracellular matrix and/or with cytoskeletal elements. To study participation of HMW membrane proteins in odontoblast or ameloblast differentiation, an immunological approach has been adopted. Antibodies directed against membrane proteins (M r, 110–190) from mouse embryos have been produced by the hybridoma technique. Supernatants of hybridoma cultures were screened for their ability to stain dental tissues and also tested for their biological activities on dental cells in primary culture or on developing tooth germs in organ culture. An IgM monoclonal antibody, MC16A16, directed against a 165-kDa antigen present in plasma membrane preparations, reacted strongly with the dental epithelial cells cultured on glass, but not mesenchymal cells which maintained vinculin-containing focal contacts. This antibody, which affected the organization of dental-cell micro-filaments in primary culture, also inhibited the polarization of odontoblasts, but not that of ameloblasts.

Phosphatidylinositol cycle and its possible involvement in the regulation of cytoskeleton-membrane interactions.

Receptor-mediated activation of many cells, including blood platelets, leads to changes at the cytoplasmic side of the membrane. In platelets, phospholipases, such as phospholipase C and phospholipase A2, have been shown to become activated. From phospholipids they generate the second messengers diacylglycerol and inositol phosphate(s) and fatty acids, respectively. At the same time, actin polymerization and reorganization of actin filaments into bundles and networks occurs. Here, the association of lipids, radiolabeled either with saturated (palmitic acid) or unsaturated (arachidonic acid) fatty acids, with the cytoskeletons of resting and activated human blood platelets was studied. The relative binding of lipid components to the cytoskeleton of activated platelets labeled with palmitic acid is six times higher than that of platelets labeled with arachidonic acid. Analysis of lipids associated with isolated cytoskeletons of resting and activated platelets (labeled with palmitic acid) showed a 30-fold increase in the binding of labeled lipids to the cytoskeletal structures during activation. Both diacylglycerol and fatty acids were found to be associated with the cytoskeleton of activated platelets. Gel filtration, chromatofocusing, and immunoprecipitation studies demonstrated tight binding of these lipids to alpha-actinin. alpha-Actinin is one of the proteins that rapidly becomes associated with the cytoskeleton during platelet aggregation; it is also one of the molecules proposed to act as an actin-membrane linker. The results reported indicate a possible participation of alpha-actinin, fatty acids, and the phosphoinositide-derived second messenger diacylglycerol in the regulation of cytoskeleton-membrane interactions. Together with the results of others they suggest a possible involvement of the phosphatidylinositol cycle in the assembly of actin filaments and their anchoring to membranes.

Plasmalemmal undercoat: The cytoskeleton supporting the plasmalemma.

The plasmalemmal undercoat can be defined as the electron-dense material of layered organization closely applied to the cytoplasmic surface of the plasma membrane (plasmalemma) as revealed by thin-section electron microscopy. Though the structures which fulfill this criterion occur widely, most of them have not received the attention they may deserve. The undercoat is a special form of the cytoskeleton-membrane interaction, though it constitutes a part of the cytoskeleton. The significance of the plasmalemmal undercoat may be primarily to provide a structural support for the membrane. With this support, cells can perform many important functions on their limited or whole surfaces. The undercoat may provide a mechanical support to the plasmalemma so that the membrane may acquire rigidity, strength and elasticity. Through association with the membrane, the undercoat may regulate the distribution of integral membrane proteins to form and maintain various functional domains on the plasmalemma. The undercoat may further provide attachment sites for cytoskeletal fibrous components such as actin filaments, intermediate filaments and microtubules.

The 180-kD component of the neural cell adhesion molecule N-CAM is involved in cell-cell contacts and cytoskeleton-membrane interactions.

N-CAM180, the molecular form of the three neural cell adhesion molecules (N-CAM) with the largest cytoplasmic domain, is accumulated at sites of cell-cell contact (cell bodies, neurites, growth cones) in cultures of neuroblastoma and cerebellum. At these sites the cytoskeleton-membrane linker protein brain spectrin and actin are also accumulated. Brain spectrin copurifies with N-CAM180 by immunoaffinity chromatography and binds specifically to N-CAM180 but not to N-CAM140 or N-CAM120 in a solid-phase binding test. These observations indicate an association of N-CAM180 with the cytoskeleton in vivo. This association may underlie the reduced lateral mobility of N-CAM180 in the surface membrane compared to N-CAM140 (Pollerberg et al. 1986). Together with the fact that N-CAM180 is only expressed after termination of neuron migration in vivo (Persohn and Schachner, unpublished) these results suggest a role for N-CAM180 in stabilization of cell contacts.

Association of vinculin to the platelet cytoskeleton during thrombin-induced aggregation

Vinculin is a protein generally believed to be involved in membrane-cytoskeleton interaction, and its presence in platelets has been verified earlier. Here we show that in resting bovine platelets, vinculin is not associated with the Triton-insoluble cytoskeletal fraction but becomes incorporated into it during the thrombin-induced activation process. The incorporation starts around the same time as the release reaction and only after the shape change and the first phase of aggregation have taken place. Its time course parallels the cytoskeletal association of actin and certain other contractile proteins. Vinculin is a minor component of platelet cytoskeleton and only about 10 % of the total platelet vinculin becomes incorporated into the Triton X-100 residue.

The Torpedo electrocyte: a model system for the study of receptor-cytoskeleton interactions.

We have used the electrocyte of Torpedo electric organ as a model system for the study of AchR stabilization in the postsynaptic membrane. Attention was focused on membrane cytoskeleton interactions in particular on a peripheral protein of 43 KD that is believed to participate in AchR immobilization. Using immunocytochemical methods, we have shown that the cortical skeleton in Torpedo electrocyte displays a local differentiation proper for each specialized domain of the plasma membrane. In the postsynaptic membrane, characterized by an accumulation and a geometrical organization of the receptors in the plane of the membrane, the 43 KD protein participates in a submembraneous coating or "postsynaptic densities" that strictly codistribute with the AchR. The 43 KD protein might also account for the anchoring of intermediate-sized filaments. The organization of the postsynaptic domain appears readily different from that of the non-innervated one where the membrane folds are maintained by a cortical meshwork of cytoskeletal proteins such as ankyrin, spectrin and oligomeric actin. In conclusion, the asymmetrical organization of the cortical skeleton in the electrocyte offers a unique opportunity for the study of the specific aspects of membrane-skeleton interactions that take place in the postsynaptic domain.

Immunological detection of an analogue of the erythroid protein 4.1 in endothelial cells

Endothelial cells (EC) of arterial and venous origin were investigated by indirect immunofluorescence and immunoautoradiography for the presence of red cell membrane 4.1-like protein. By immunofluorescence, EC exhibited a relatively uniform fluorescent staining sometimes of a reticular pattern, distributed over the entire cell. All controls were negative. Immunoblot analysis of EC revealed a cross reactive band of a molecular weight comparable to that of the erythrocyte band 4.1. These findings indicate that endothelial cells of arterial and venous origin express a polypeptide immunologically related to the erythrocyte protein 4.1, which may play an important role in membrane-cytoskeleton interactions.

Aberrant activation and regulation of the oxidative burst in neutrophils with Mol glycoprotein deficiency.

Patients whose cells are deficient in the glycoproteins LFA-1, Mol, and p150,95 have recurrent infections and pronounced abnormalities in neutrophil adherence, aggregation, chemotaxis, and phagocytosis. We characterized activation and regulation of oxidative metabolism of Mol-deficient neutrophils. These cells failed to depolarize or to produce O2- or H2O2 normally when stimulated by opsonized zymosan. The chemotactic peptide formyl methionyl-leucyl-phenylalanine depolarized Mol-deficient neutrophils normally but caused supernormal production of O2- and H2O2, a result of a prolonged burst in oxidative metabolism. Phorbol myristate acetate depolarized Mol-deficient neutrophils at a nearly normal rate but evoked release of significantly less O2- and H2O2 than from normal PMN. The aberrant activation and regulation of the oxidative burst in Mol-deficient neutrophils are considered in light of recently emerging concepts in the cell biology of this process, and the possibility that these abnormalities reflect a defect in the cytoskeleton-membrane interaction is discussed.