Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells

The effects of plasma fibronectin (pFn) and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on adhesion and cytoskeletal organization of human erythroleukemia (HEL) cells were studied. HEL cells, that normally grow in suspension, attached rapidly on pFn-coated growth substratum and some cells showed spreading. Upon exposure to TPA most of the cells adhered and showed some degree of spreading also when plated on plastic. The spread cells showed mostly peripheral accumulations of F-actin in addition to actin fibers seen in some of the cells. When the cells were plated in the presence of TPA on pFn or on pFn-fragments, containing the cell binding site, all the cells adhered rapidly, spread extensively, organized prominent F-actin stress fibers and typical ventral plaques of vinculin and alpha-actinin. Both proteins were revealed also in the suspended cells by Western blot analysis. When plated on substratum coated with other pFn-fragments or laminin, the HEL cells did not adhere or spread. Both adhesion on pFn as well as formation of stress fibers in the presence of TPA could be prevented by the synthetic peptide Arg-Gly-Asp-Ser (RGDS). HEL cells were also able to organize typical ventral fibrillar arrays of Fn. Immunostaining and metabolic labeling experiments showed that the cells did not contain or synthesize Fn, indicating that the plaques were formed from exogenous pFn by the cells. The results suggest that Fn and TPA synergistically induce the organization of the actomyosin system in HEL cells by promoting the formation of prominent actin stress fibers and focal adhesion sites.     

Characterization of the nucleotide tight-binding sites of the isolated mitochondrial F1-ATPase

The properties of the nucleotides tightly bound with mitochondrial F1-ATPase were examined. One of three bound nucleotide molecules is localized at the site with Kd approximately 10(-7) M and released with koff approximately 0.1 s-1. The second nucleotide molecule is bound with the enzyme with Kd approximately 10(-8) M and koff for its dissociation is 3 X 10(-4) s-1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1-ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1-ATPase catalytic site is the high rate (koff greater than or equal to 0.1 s-1) of its spontaneous release.     

Role of the polar head group stereoconfiguration in the cation-induced aggregation of dimyristoylphosphatidylglycerol vesicles

Cation-induced aggregation of small unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG), the corresponding 3' stereoisomer (3'-DMPG), and their 1:1 mixture was studied as a function of the concentration of different mono- and divalent cations. The order of efficiency, Na+ greater than Li+ greater than K+ greater than Cs+, of the monovalent cations to induce the aggregation of DMPG vesicles is the same for both stereoisomers and their mixture. However, significant differences in the Na+-induced aggregation of 1'-DMPG and 3'-DMPG were evident. The threshold concentration of aggregation by Na+ was 0.35 M for 3'-DMPG, 0.55 M for 1'-DMPG, and 0.50 M for the mixed liposomes. Such difference in the aggregation of DMPG stereoisomers was not observed for the other mono- and divalent cations. The higher affinity of 3'-DMPG for Na+ is suggested to be due to a slightly different favored conformation of the head group glycerol moiety. Aggregation of the stereoisomers by 1 M NaCl was identical, indicating that the differences in the affinity of 1'-DMPG and 3'-DMPG for sodium can be overcome by very high ionic strength. Inclusion of 20 mol % cholesterol in vesicles enhanced the aggregation of 1'-DMPG and decreased the aggregation of 3'-DMPG by Na+ and thus abolished the difference between the two stereoisomers.     

Expression of intermediate filaments (IF) in tissues and cultured cells

Intermediate filaments are found in most nucleated cells as part of their cytoskeleton. Intermediate filaments are formed by different proteins in cells of major tissues types. Therefore, antibodies against intermediate filaments can be used in tissue typing, in the analysis of cell lineages during development and in the elucidation of the origin of unknown tumors.     

Dolichos biflorus agglutinin (DBA) reacts selectively with mast cells in human connective tissues

Dolichos biflorus agglutinin (DBA) binds to N-acetyl-D-galactosamine (GalNAc) residues in glycoconjugates and agglutinates erythrocytes carrying blood group antigen A. In cryostat sections of various tissues from blood group-specified humans, fluorochrome-coupled DBA bound preferentially to fusiform connective tissue cells and to certain epithelial cells. The connective tissue cells were identified as mast cells by their typical metachromasia in consecutive staining with toluidine blue. Double labeling with DBA and conjugated avidin revealed two distinct populations of mast cells. In several tissues the DBA-reactive cells likewise displayed uniform avidin reactivity. In intestinal mucosa, however, morphologically distinct DBA-binding mast cells were found, which were labeled with the avidin conjugates only in specially fixed paraffin sections. DBA did not bind to vascular endothelial cells, which could be identified by double staining with antibodies to factor VIII-related antigen. Labeling with Helix pomatia agglutinin (HPA), another blood group A-reactive lectin, resulted in distinct blood group-dependent fluorescence of the endothelia. Sophora japonica agglutinin (SJA), a blood group B-reactive lectin, labeled vascular endothelial cells in tissues from blood group A, AB, and B donors. HPA and SJA reacted with small mast cells in the gastrointestinal mucosa but failed to label large mast cells in any of the tissues. These results indicate that the blood group reactivity of lectins, as determined by erythroagglutination, is not necessarily consistent with their reactivity with blood group determinants in tissue sections. Moreover, DBA conjugates appear to be a reliable probe for detection of mast cells in various human connective tissues.     

Phospholipase A2 assay using an intramolecularly quenched pyrene-labeled phospholipid analog as a substrate

A phospholipid analog 1-palmitoyl-2-6(pyren-1-yl)hexanoyl-sn-glycero-3-phospho-N- (trinitrophenyl)aminoethanol (PPHTE) in which pyrene fluorescence is intramolecularly quenched by the trinitrophenyl group was used as a substrate for pancreatic phospholipase A2. Upon phospholipase A2 catalyzed hydrolysis of this molecule pyrene monomer fluorescence emission intensity increased as a result of the transfer of the pyrene fatty acid to the aqueous phase. Optimal conditions for phospholipase A2 hydrolysis of PPHTE were similar to those observed earlier for other pyrenephospholipids (T. Thuren, J. A. Virtanen, R. Verger, and P. K. J. Kinnunen (1987) Biochim. Biophys. Acta 917, 411-417). Although differential scanning calorimetry revealed no thermal phase transitions for PPHTE between +5 and +60 degrees C the Arrhenius plot of the enzymatic hydrolysis of the lipid showed a discontinuity at 30 degrees C. The molecular origin of this discontinuity remains at present unknown. To study the effects of dimyristoylphosphatidylcholine (DMPC) phase transition at 23.9 degrees C on phospholipase A2 reaction PPHTE was mixed with DMPC in a molar ratio of 1:200 in small unilamellar vesicles. The hydrolysis of DMPC-PPHTE vesicles was measured by following the increase in pyrene monomer fluorescence emission due to phospholipase A2 action on PPHTE. Below the phase transition of DMPC the enzymatic reaction exhibited a hyperbolic behavior. At the transition as well as at slightly higher temperatures a lag period was observed. The longest lag period was approximately 20 min. Above 26 degrees C no lag time could be observed. However, the reaction rates were slower than below the phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reorganization of intermediate filament cytoskeleton in induced metanephric mesenchyme cells is independent of tubule morphogenesis

The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells.     

Interferon affects the formation of adhesion plaques in human monocyte cultures

When monocytes isolated from human blood adhere to glass substratum, actin- and vinculin-containing punctate plaques rapidly appear at the ventral surface of the cells. We show here that highly purified human leukocyte interferon (IFN) can inhibit formation of these adhesion plaques in a dose-dependent manner. Complete inhibition was obtained when 300 IU/ml IFN were added into the cell-seeding medium. Plaques already formed in the absence of IFN were only partially affected by subsequent addition of IFN into the culture medium. Prevention by IFN of the formation of the adhesion plaques was associated with loosened attachment of the cells to the substratum. Effect of IFN on cellular morphology was complex. At higher doses, IFN added to the cultures within 24 h of seeding almost completely inhibited the differentiation of monocytes to macrophages and most of the cells remained rounded. At lower doses, however, an enhancement of the bipolar spreading was seen and the end result was a culture with predominantly elongated fibroblastoid cells. The latter cells, unlike the fibroblastoid cells in untreated monocyte-macrophage cultures, were completely devoid of the actin plaques, while the reorganization of vimentin-type intermediate filaments took place in a normal manner. These results further support the view that the actin- and vinculin-containing plaques have a role in mediating firm adherence of human monocytes to growth substratum.     

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.