A precursor of cytoplasmic actin in cultured Drosophila cells

In continuous lines of cultured Drosophila cells two forms of cytoplasmic actin, designated II and III, are detected after a 25- to 120-min pulse label with [35S]methionine. However, only one of these, actin II, accumulates in the cell. With the use of a pulse-chase protocol and two-dimensional gel electrophoresis, it has been found that actin III is synthesized as a precursor of the more stable cytoplasmic actin II. The half-life of actin III was estimated to be approximately 50 min.     

Changes in intramembranous particle topography and concanavalin A receptor mobility associated with myoblast differentiation.

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37 degrees C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.     

Isolation of a concanavalin A receptor from mouse L cells.

A cell surface glycoprotein receptor for concanavalin A (Con A) has been isolated from mouse L cells. The isolation procedure involved dissolving whole L cells in 0.3 M lithium diiodosalicylate and extracting with aqueous phenol. The Con A receptor, which was found in the aqueous phase of this extract, was further purified by affinity chromatography on a column of Con A-Sepharose; the receptor was adsorbed to Con A-Sepharose and eluted with 0.1 M methyl alpha-D-glucopyranoside or with 0.1 M methyl alpha-D-mannopyranoside, but not with other monosaccharides. The cell surface location of the Con A receptor purified in this way was confirmed by showing that it can be isolated from purified L cell plasma membranes and by demonstrating that it can be labeled from the exterior surface of intact L cells by the nonpenetrating galactose oxidase-KB3H4 system. Biochemical studies of the Con A receptor have shown that it migrates on sodium dodecyl sulfate-polyacrylamide gels as a single component having an apparent molecular weight of approximately 100,000. Its N-terminal amino acid is valine and it has carbohydrate attached at several (at least five) different sites along the polypeptide chain.     

The concanavalin a receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using ¹²⁵I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13·10⁶ M^?1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83·10⁶ M^?1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.     

Characterization of cytoplasmic glucocorticoid receptor in circulating human mononuclear cells

Human circulating mononuclear leukocytes were shown to have a specific 7.7 S glucocorticoid receptor in the cytoplasm utilizing a 5 to 20% sucrose gradient in low salt. The cytoplasmic receptor was shown to convert to a 4.1 S receptor upon exposure to high salt concentration (0.4 M KCl) or upon translocation to the nucleus. The dissociation constant (Kd) averaged 0.7 nM, the concentration of the cytoplasmic receptor averaged 179 fm/mg protein, and 2404 molecules bound per cell. Progesterone was found to displace 85% of labeled triamcinolone acetonide from the cytoplasmic receptor but no appreciable competition was found for 17β-estradiol or 5α-dihydrotestosterone (DHT). A significant correlation was found between the endogenous serum cortisol concentration and the cytoplasmic receptor capacity when expressed as molecules bound/cell (r = 0.64 p < 0.05).     

Discovering high mobility group A molecular partners in tumour cells

DNA-based activities rely on an extremely coordinated sequence of events performed by several chromatin-associated proteins which act in concert. High Mobility Group A (HMGA) proteins are non-histone architectural nuclear factors that participate in the regulation of specific genes but they are also believed to have a more general role in chromatin dynamics. The peculiarity of these proteins is their flexibility, both in terms of DNA-binding and in protein-protein interactions. Since these proteins act as core elements in the assembly of multiprotein complexes called enhanceosomes, and have already displayed the ability to interact with several different proteins, we started a proteomic approach for the systematic identification of their molecular partners. By a combination of affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry we have identified about twenty putative HMGA interactors which could be roughly assigned to three different classes: mRNA processing proteins, chromatin remodelling related factors and structural proteins. Direct HMGA interaction with some of these proteins was confirmed by glutathione-S-transferase pull-down assays and the HMGA domain involved was mapped. Blot-overlay experiments reveal that members of the HMGA family share most of their molecular partners but, interestingly, it seems that there are some cell-type specific partners. Taken together, these experimental data indicate that HMGA proteins are highly connected nodes in the chromatin protein network. Since these proteins are strongly implicated with cancer development, the identification of molecules able to perturb the HMGA molecular network could be a possible tool to interfere with their oncogenic activity.     

Receptor-stimulated actin polymerization requires cytoplasmic acidification in human PMNs

Cytoplasmic alkalinization has received considerable attention as a regulatory event In cell growth, transformation and signal transduction (Busa, W.B. (1986) Annu. Rev. Physiol. 48, 389-402 and Moolenar, W.H. (1986) Annu. Rev. Physiol. 48, 363-376). In contrast the current paper presents evidence for the role of an early cytoplasmic acidification, during signal transduction in the polymorphonuclear leukocyte (PMN). Following PMN stimulation with a chemotactic peptide, there is a significant decrease in cytoplasmic pH concomitant with a dramatic increase in cytoskeletal actin. The data indicate that this drop in pHi is necessary, but not sufficient, for signal transduction leading to cytoskeletal reorganization in these cells.     

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.     

A novel isoform of cytoplasmic actin that binds poly-L-proline.

An actin-like protein was purified to apparent homogeneity from chick-embryo homogenates and chick-embryo fibroblasts by the use of poly-L-proline-agarose affinity chromatography; we therefore refer to this protein as PBP (poly-L-proline-binding protein). PBP binds to deoxyribonuclease-agarose, co-migrates with known actin standards on SDS/polyacrylamide-gel electrophoresis, and has an amino acid composition similar to that of actin. Linear peptide maps after digestion with Staphylococcus aureus proteinase reveal its apparent homology with gamma-actin; however, isoelectric-focusing experiments show that PBP is clearly more acidic than any of the three major isoforms of actin. PBP polymerizes in the presence of ATP to form fibrillar structures resembling actin paracrystalline aggregates. In chick-embryo fibroblasts, immunofluorescence with antibodies to PBP shows that its distribution is cytoplasmic: perinuclear staining of the cytoplasm, generalized cytoplasmic staining and peripheral fibrillar structures are evident. In contrast, antibodies specific for the (alpha, gamma)-actins reveal the typical stress fibre structures characteristic of fibroblastic cells. PBP appears to constitute a novel isoform of cellular actin, distinct from the known actin isoforms in terms of its lower isoelectric point, its ability to bind poly-L-proline and its distinct subcellular localization.     

Inhibition of apoptosis in human tumour cells by okadaic acid.

Gamma-radiation, tetrandrine, bistratene A, and cisplatin were all found to induce pronounced morphological changes characteristic of apoptosis and extensive DNA fragmentation in the human BM13674 cell line 8 h after treatment. Apoptosis induced in BM13674 cells by these diverse agents was markedly inhibited by 1 microM okadaic acid, a tumour promoter that inhibits protein phosphatases 1 and 2A. This compound also inhibited the appearance of apoptosis in fresh human leukaemia cells that had been exposed to gamma-radiation. The inhibition of apoptosis was confirmed using fluorescence microscopy and DNA gel electrophoresis. Dephosphorylation of a limited number of proteins was shown to be associated with apoptosis and okadaic acid prevented these dephosphorylations. Previous studies on the BM13674 cell line showed that an inhibitor of protein synthesis failed to prevent apoptosis in these cells. The present data provides further support that posttranslational modification of proteins, in particular, phosphorylation/dephosphorylation status, plays an important role in inhibition/activation of programmed cell death in different human cells after exposure to several cytotoxic agents.     

Concanavalin A surface receptors and cytoplasmic actin in cell adhesion.

A double immunofluorescence staining technique to locate concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell was applied to monolayer cultures of rat foetal fibroblasts during cell detachment induced by trypsin and during cell attachment to glass substratum. Con A receptors were demonstrated by fluorescein-isothiocyanate-labelled Con A (FITC-Con A) and actin by specific anti-actin antibody (AAA) traced with rhodamine-labelled goat anti-human globulin (R-AHG). Untreated, control cells had an elongated shape, Con A receptors restricted to cell margins and prominent actin filaments. After 2 min treatment with 0.001% trypsin the cells became angular with Con A receptors in clusters and actin in a diffuse or aggreagate staining pattern. Progressive cell rounding followed and this was accompanied by the development of long, thin, arborized cell processes, studded with Con A receptors and containing fine actin filaments. Complete cell rounding preceded cell detachment. The sites of detached cells were marked by fine aggregates containing Con A receptors and actin. In cells attaching to a glass substratum, actin was present in a diffusely stained or aggregate pattern in round cells, in filaments restricted to cell margins in partially spread cells and in numerous filaments in fully spread cells. Con A receptors were present in clusters in round cells, in clusters or caps in partially spread cells and in cell margins in fully spread cells. Binding of FITC-Con A to partially spread cells resulted in dissolution of the few, newly formed, actin filaments. We believe our observations are consistent with the idea that actin filaments, formed during cell attachment, contribute towards the maintenance of cell adhesion by helping in the preservation of cell shape and by anchorage of Con A receptors at points of cell attachment to the substratum.     

Electrophoretic mobility of irradiated erythrocytes and Ehrlich ascites tumour cells.

The subunits of RNA polymerase I are partially resolved during density gradient centrifugation. An analysis of the relative subunit composition with respect to specific catalytic activity shows that the molar ratio of the 24,000 dalton subunit directly correlates with polymerase activity. Since this polypeptide is found also in polymerases II and III, it may be required for activity of all yeast nuclear RNA polymerases.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.