Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts

Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit anti-mouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the sections are covered by methyl cellulose and exposed to paraformaldehyde vapour at 80 degrees C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) alpha- and beta-tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.     

Relationship of heparan sulfate proteoglycans to the cytoskeleton and extracellular matrix of cultured fibroblasts

The distribution of heparan sulfate proteoglycans (HSPG) on cultured fibroblasts was monitored using an antiserum raised against cell surface HSPG from rat liver. After seeding, HSPG was detected by immunofluorescence first on cell surfaces and later in fibrillar deposits of an extracellular matrix. Cell surface HSPG aligned with microfilament bundles of rat embryo fibroblasts seen by phase-contrast microscopy but was diffuse on transformed rat dermal fibroblasts (16C cells) which lack obvious stress fibers. Focal adhesions isolated from either cell type and monitored by interference reflection microscopy showed a concentration of HSPG labeling with respect to the rest of the membrane. Increased labeling in these areas was also seen for fibronectin (FN) by using an antiserum that detects both plasma and cell-derived FN. Double immunofluorescent staining of fully adherent rat embryo fibroblast cells showed some co-distribution of HSPG and FN, and this was confirmed by immunoelectron microscopy, which detected HSPG at localized areas of dorsal and ventral cell membranes, overlapping cell margins, and in the extracellular matrix. During cell shape changes on rounding and spreading, HSPG and FN may not co- distribute. Double labeling for actin and either HSPG or FN showed a closer correlation of actin with HSPG than with FN. The studies are consistent with HSPG being closely involved in a transmembrane cytoskeletal-matrix interaction; the possibility that HSPG coordinates the deposition of FN and other matrix components with cytoskeletal organization is discussed.     

Early expression of bone matrix proteins in osteogenic cell cultures.

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.     

Interferon enhances fibronectin expression in various cell types.

Fibronectin (FN), a normal plasma and extracellular matrix glycoprotein, plays a significant role in various phases of wound healing. At wound site FN is synthesized locally by various cell types involved in the healing process (viz. epithelial, endothelial, fibroblast and macrophage cells) or deposited from the plasma. The present study was undertaken to investigate the in vitro effect of IFN on FN synthesis as well as release in the culture medium by various cell types. Indirect immunofluorescence and immunoelectron microscopy studies, using specific antibodies, revealed that IFN treatment resulted in significantly more staining for FN as compared to untreated control cells. Metabolic labeling with 35S-methionine, immunoprecipitation and SDS-page studies showed an increase in FN synthesis and release by IFN treated cells. In addition, to determine whether this increased synthesis was reflected at mRNA levels, poly (A)+ RNA was isolated from human lung epithelial cells (A549) and probed with FN specific cDNA. We found that IFN treatment increased the level of FN mRNA.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Simultaneous cell surface phenotype and cell cycle analysis of lymphocytes by flow cytometry

A method for the simultaneous measurement of cell surface components and nucleic acids (DNA and RNA) of human lymphocytes by flow cytometry has been developed, thereby providing a means of analyzing cell surface changes during the various phases of the cell cycle. Unfixed cells were coated with fluorescein-conjugated concanavalin A (F Con A) or surface antigen-specific antibody, fixed sequentially with paraformaldehyde and methanol, treated with specific nucleases, and then stained with propidium iodide. Neither portion of the procedure (cell surface staining, nucleic acid staining) interfered significantly with the other. Cell cycle phases of phytohemagglutinin-stimulated human lymphocytes as determined by this method were comparable with those identified by acridine orange staining. Cell cycle-specific blocking agents were used to additionally demonstrate the specificity of the staining procedure. Simultaneous measurement of cell cycle phase and detection of surface receptors for Con A and T lymphocyte surface determinants was performed with this method.     

Detection of insulin-regulated GLUT4-translocation by the insertion of a protein C epitope in L6 myoblasts

Insulin stimulates glucose transport by translocation of the membrane glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. GLUT4 is highly expressed in adipose tissue and skeletal muscle. We have constructed a cDNA containing the human GLUT4 inserted by a 12 amino acid protein C epitope in the first extracellular (exofacial) domain of the human GLUT4 (GLUT4-PC). Stable expression of GLUT4-PC in L6 myoblasts (L6-GLUT4-PC) was confirmed in immunofluorescence using monoclonal antibodies against protein C. The protein C staining yielded labeling in perinuclear vesicles strongly co-localizing with GLUT4 detected with antibodies directed against the endofacial part of GLUT4. The L6-GLUT4-PC cells were further characterized in a direct cell-based enzyme-linked immunosorbent assay by the use of beta-galactosidase. Cell surface binding of monoclonal protein C antibodies was detected with beta-galactosidase-conjugated secondary antibodies and chlorophenolred-beta-D-galactopyranoside (CPRG) as substrate in 2% paraformaldehyde fixed cells. In this assay, stimulation with insulin created a rapidly detectable recruitment of GLUT4-PC to the cell surface. This cell-based enzyme-linked immunosorbent GLUT4 assay was shown to be comparable with that of previously reported radioactive assays.     

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

Summary The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit antimouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the paraformaldehyde vapour at 80° C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) - and -tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.Presented in part at the 9th European Congress on Electron Microscopy, York, England, September 4–9, 1988     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Leishmania donovani: immunochemical localization and secretory mechanism of soluble acid phosphatase

Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.     

免疫酶双重染色中抗体洗脱的进一步研究

本实验进一步检查了三种常用的洗脱方法从切片上除去免疫酶组织化学染色后的抗体的效果。结果表明,PAP法染色后,氧化法的抗体洗脱完全,效果可靠;酸洗法和酸洗/二甲基甲酰胺法的效果视不同抗体而不同,二甲基甲酰胺单用或用于酸洗后似无效。所用方法均不能从ABC法染色的垂体组织切片上完全除去抗体复合物。因之,将ABC法用于第一抗体来源于同一动物种的双重染色中,来显示第一种抗原时,要特别注意排除假性双标记。     

Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells

Various modifications to the immunofluorescent labeling procedures for microtubules in plant cells have been compared using cell cultures of Vicia hajastana Grossh. Using serial section electron microscopic reconstructions as a reference, we have chosen as our standard procedure a method that maximizes both the preservation of the cytoskeleton and the proportion of cells staining, while minimizing the degree of nonspecific staining. The critical steps of the procedure include stabilization of the cytoskeleton, cell wall permeabilization, and cell extraction. To maintain structural integrity during the procedure, it is necessary to stabilize the cytoskeleton with paraformaldehyde. To facilitate antibody penetration into the cell, it was necessary that the walls be made permeable via partial enzymatic digestion. Detergent extraction of cells increased the proportion of cells staining and decreased the level of nonspecific binding of the antibodies. The procedures detailed in this article provide a good starting point for the application of immunofluorescent labeling techniques to other plant systems.     

Constitutive and UV-induced fibronectin degradation is a ubiquitination-dependent process controlled by beta-TrCP

Loss of fibronectin (FN) assembly in the extracellular matrix has long been recognized as a feature of cellular transformation. However, such assembly is regulated not only by FN synthesis but also by its post-translational modifications. The mechanism controlling FN protein stability has remained unclear so far. Recently it was demonstrated that FN matrix turnover occurs intracellularly at the lysosome following caveolin-1-dependent endocytosis. Although FN was reported to undergo ubiquitindependent degradation, the ubiquitin ligase responsible for FN ubiquitination is unknown. In this study, we have identified beta-TrCP as the ubiquitin ligase for lysosomal degradation of FN. We found two conserved beta-TrCP recognition motif (DSGVVYS and DSGSIVVS) in the primary amino acid sequence of human, mouse, and rat FN. Down-regulation of either beta-TrCP1 or beta-TrCP2 by small interference (siRNA) caused significant accumulation of FN. Immunolocalization studies showed intracellular accumulation of FN in beta-TrCP siRNA-treated cells without showing much alteration in its matrix association. We also observed that exposure of cells to UV irradiation effectively down-regulated FN following increased ubiquitination, which was significantly inhibited either by lysosomal inhibitor or by siRNA-mediated down-regulation of beta-TrCP. Taken together, constitutive FN degradation, as well as UV-induced degradation, is ubiquitination dependent and controlled by beta-TrCP.     

Mode of orthophosphate uptake and ATP labeling by mammalian cells

Incubation of HeLa cells with [³²P]orthophosphate results in more rapid labeling of the γ-phosphorus of ATP than of the intracellular pool of orthophosphate. The specific radioactivity of ATP equals that of extracellular orthophosphate after 2h of incubation. A similar pattern of labeling is seen with human erythrocytes when incubated at physiological concentrations of orthophosphate (2 mM) and pH 7.4–7.8. At lower pH, 6.8–7.2, the rate of orthophosphate uptake increases and exceeds the rate of labeling of ATP. These data are explained by the existence of a primary system for ATP uptake which involves the mediation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. Phosphate first enters the cell as 1,3-diphosphoglyceric acid, is then transferred to ATP, and then enters the intracellular orthophosphate pool. At lower pH monovalent orthophosphate also enters the erythrocyte by a process not involving glyceraldehyde-3-phosphate dehydrogenase.     

Improved procedures for immunoferritin labeling of ultrathin frozen sections

In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross- linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.     

Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection

Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.