Isolation of chromaffin cell plasma membranes on polycationic beads

We have tried to define which proteins of chromaffin cell plasma membranes are facing the cytoplasm by surface labelling a selectively oriented membrane preparation.Viable chromaffin cells were isolated by collagenase treatment of bovine adrenals. Plasma membranes from these cells were isolated on polycationic beads by the method of Jacobson and Branton (Jacobson, B.S. and Branton, D. (1977) Science 195, 302–304). The purity and orientation of the membranes were defined by biochemical and morphological criteria. The membranes, with their external side apposed to the bead surface, were enriched about 10-fold with respect to a whole cell homogenate, and contained only small amounts of contaminating organelles. Surface specific iodination of membranes on beads with 1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluril (Iodogen), followed by polyacrylamide gel electrophoresis, allowed the identification of cytoplasmically exposed proteins. A different pattern was observed when intact cells were labelled prior to membrane isolation. The advantages and possible uses of this immobilized membrane preparation are discussed.     

Evaluation of methods for the isolation of plasma membranes displaying guanosine 5'-triphosphate-dependence for the regulation of adenylate cyclase activity: potential application to the study of other guanosine 5'-triphosphate-dependent transduction systems

The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH3 cells, NBP2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.     

Genetic studies of the lac repressor. VI. The B116 repressor: an altered lac repressor containing amino acid specified by both the trp and lacI leader regions.

Human erythrocytes were treated with the diazanium salt of oligodeoxythymidylic acid 5′-p-aminophenylphosphate, a reagent unable to penetrate the plasma membrane. The oligomers, covalently linked to the cell surface, were extended by treatment with terminal deoxynucleotidyl transferase in the presence of deoxythymidine triphosphate. The cells then hybridized readily to columns of polyriboadenylic acid-agarose. We expect this technology to be of value for cell sorting, for the isolation of proteins accessible at the surfaces of cells, and for the synthesis of a variety of DNA-protein polymers.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Isolated adipocytes: an assessment of cell surface changes during their preparation

A method is described, based on the detection of adipocyte-specific cell surface antigens, which allows assessment of the relative surface damage incurred by the cells when they are prepared under a variety of conditions. Using the method it is possible to develop, for any set of reagents, a set of cell isolation conditions (collagenase concentration, time of incubation) which will produce minimally damaged cells which exhibit high levels of specific cell surface immunoreactivity. Under certain conditions a recovery from limited surface damage can be achieved, although, when cells are prepared under more extreme conditions irreversible surface damage occurs. The surface morphology of the cells as revealed by scanning electron microscopy, is also clearly affected by the conditions of cell isolation. The method has been used to define the conditions necessary for the isolation of cells to be used in the study of subtle biochemical responses.     

Plasma membrane folds on the mast cell surface and their relationship to secretory activity

Changes in the surface morphology of secreting mast cells have been followed by scanning electron microscopy. Mast cells isolated from the rat peritoneal cavity have folds of plasma membrane that form snake-like ridges on their surfaces. Fold length varies considerably from cell to cell, whereas fold width and depth appear to remain relatively constant. To assess the possible relationship between secretory activity and surface folding, a seimquantitative method was used for measuring fold length in control and secreting populations. A positive correlation is found between secretion of histamine and the extent of membrane folds on the mast cell surface. The source of the membrane required for fold formation is probably secretory granule membrane incorporated into the plasma membranene as a result of exocytosis. Furthermore, a distinct cell type devoid of surface folds, designated as a raspberry-type cell, is found to occur as an integral part of a normal population of mast cells. This cell type is resistant to stimulation by polymyxin.     

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.     

Specificity of an Antiserum Produced Against Pseudomicrothorax dubius Trichocysts Prepared by a Rapid Isolation Method

ABSTRACT. A rapid method was developed for the isolation of Pseudomicrothorax dubius ciliary and trichocyst fractions which were characterized by SDS-PAGE followed by combined silver and Coomassie blue staining. Antibodies were prepared against the trichocyst fraction and employed to label Lowicryl thin sections of cells. Trichocysts were strongly labeled, as were the surfaces of the plasma and ciliary membranes. Immunoblots of the trichocyst fraction revealed labeling of major bands at 16–29 kD, characteristic of the trichocyst proteins. On immunoblots of the ciliary fraction, approximately eight bands were labeled, including the major cell surface glycoprotein, the immobilization antigen. Ciliary proteins not located on the membrane surface, such as the tubulins, were not labeled. Absorption of the antiserum against fixed P. dubius cells eliminated the cell surface labeling on Lowicryl sections and on immunoblots of the ciliary fraction. The major trichocyst protein bands were as strongly labeled as with the nonabsorbed antiserum. Labeling of several of the minor, higher molecular weight bands of the trichocyst fraction was eliminated, indicating that they are cell surface contaminants. Of the two major structural components of the trichocyst, the shaft and the arms, the antiserum is shown to react nearly exclusively with the shaft proteins on both Lowicryl sections and immunoblots.     

Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies

We examined epithelial cell surface polarity in subconfluent and confluent Madin-Darby canine kidney (MDCK) cells with monoclonal antibodies directed against plasma membrane glycoproteins of 35,000, 50,000, and 60,000 mol wt. The cell surface distribution of these glycoproteins was studied by immunofluorescence and immunoelectron microscopy. At the ultrastructural level, the electron-dense reaction product localizing all three glycoproteins was determined to be uniformly distributed over the apical and basal cell surfaces of subconfluent MDCK cells as well as on the lateral surfaces between contacted cells; however, after formation of a confluent monolayer, these glycoproteins could only be localized on the basal-lateral plasma membrane. The development of cell surface polarity was followed by assessing glycoprotein distribution with immunofluorescence microscopy at selected time intervals during growth of MDCK cells to form a confluent monolayer. These results were correlated with transepithelial electrical resistance measurements of tight junction permeability and it was determined by immunofluorescence that polarized distributions of cell surface glycoproteins were established just after electrical resistance could be detected, but before the development of maximal resistance. Our observations provide evidence that intact tight junctions are required for the establishment of the apical and basal- lateral plasma membrane domains and that development of epithelial cell surface polarity is a continuous process.     

Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin

An indirect affinity chromatography procedure utilizing biotinylated lectins and designed for the specific isolation of surface glycoproteins is described. The method is illustrated with intact acute leukemic lymphoblastic cells (ALL cells) with biotin-epsilon-aminocaproyl-concanavalin A (biocap-Con A) and streptavidin-Sepharose 4B. Biocap-Con A, containing on average 27 biotin residues per tetrameric lectin molecule, is used to isolate Con A-binding glycoproteins from the surface of [35S]methionine-radiolabeled intact cells. The biocap-Con A/glycoprotein complexes, after solubilization in detergent, are retrieved on immobilized streptavidin. The surface glycoproteins isolated from intact ALL cells by this method are subjected to two-dimensional gel electrophoresis and detected by autoradiography. More than fifty Con A-binding glycoproteins can be separated from the ALL cells. These glycoproteins retrievable from the cell surface were compared to those retrieved by the indirect affinity chromatography procedure from isolated plasma membrane fractions. Certain groups of glycoproteins present in the fraction isolated from intact cells were not detected in that from the plasma membrane preparations. The advantage of using the biocap-con A/streptavidin system with intact cells rather than isolated plasma membranes for the detection of surface glycoproteins is discussed.     

Cell surface expression and orientation in membranes of the 44-amino-acid SH protein of simian virus 5.

Antiserum was raised against a synthetic peptide containing the N-terminal hydrophilic domain of the small hydrophobic protein (SH) of simian virus 5 (SV5) and used to characterize properties of the SH protein. SH demonstrated properties of an integral membrane protein. Indirect immunofluorescence experiments showed that the protein is involved in the exocytotic pathway, and isolation of plasma membranes from SV5-infected cells showed an enrichment of SH, indicating that SH is transported to the infected-cell surface. Biochemical analysis of the orientation of SH in membranes by proteolysis of intact SV5-infected cell surfaces and intracellular microsomal vesicles indicated that SH is oriented in membranes with its N-terminal hydrophilic domain exposed on the cytoplasmic face of the plasma membrane and the C terminus of approximately five amino acid residues exposed at the cell surface. These data are discussed with respect to positive-acting signals being necessary in the ectodomain of SH for cell surface expression.     

A cell-binding, immunoglobulin-like protein from human plasma. II. Cell-binding activity

Cell adhesion to plastic surfaces coated with a new high-molecular-mass immunoglobulin-like protein from normal human plasma was studied. Mouse subdermal fibroblasts, hamster kidney cells, human umbilical vein endothelial cells, and human skin fibroblasts were found to become attached to the surface, but cancer cells derived from human stomach cancer and human breast cancer did not. The appearance of the attached cells differed from that of cells attached to surfaces coated with fibronectin or concanavalin A. The cell adhesion to the surfaces coated with the protein was inhibited by goat anti-human IgM. Furthermore, the binding of the protein to the cell surfaces was demonstrated by the indirect immunofluorescence method. It is concluded that this protein is a new cell-binding protein.     

Involvement of vesicle coat material in casein secretion and surface regeneration

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.     

Surfaces designed to control the projected area and shape of individual cells

Materials with spatially resolved surface chemistry were designed to isolate individual mammalian cells to determine the influence of projected area on specific cell functions (e.g., proliferation, cytoskeletal organization). Surfaces were fabricated using a photolithographic process resulting in islands of cell binding N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) separated by a nonadhesive interpenetrating polymer network [poly (acrylamide-co-ethylene glycol); P (AAm-co-EG)]. The surfaces contained over 3800 adhesive islands/cm2, allowing for isolation of single cells with projected areas ranging from 100 microns 2 to 10,000 microns 2. These surfaces provide a useful tool for researching how cell morphology and mechanical forces affect cell function.     

WGA-QD probe-based AFM detects WGA-binding sites on cell surface and WGA-induced rigidity alternation

A strategy involving the conjugation of fluorescent quantum dot (QD) with wheat germ agglutinin (WGA) acting as fluorescent and topographic probes prior to cell surface staining is developed for fluorescence microscopy and atomic force microscopy (AFM). This strategy provided at least two advantages: (a) an amplified fluorescence of WGA-QD aggregates, strongly resistant to photobleaching, ensures repeated/real-time observations of the probe-labeled cells by fluorescence microscopy; (b) the enlarged size of WGA-QD probe makes it possible for labeled WGA to be distinguished from other membrane proteins by AFM. Here, the random distribution of WGA-binding sites on non-crosslinked cells and the uneven or polarized reorganization due to WGA-induced crosslinking on cell surfaces were studied using AFM-detectable WGA-QD probe. Moreover, we developed a method to rapidly detect the WGA-induced rigidity alternation of the whole cells, which is efficient and has the potentiality of being developed to a useful tool in clinical diagnosis.     

Identification of a heparin-releasable lipoprotein lipase binding protein from endothelial cells.

Triglycerides in circulating plasma lipoproteins are hydrolyzed by lipoprotein lipase (LPL) which is thought to bind to proteoglycans on the luminal endothelial cell surface. Previous studies from this laboratory using LPL-Sepharose affinity chromatography identified a 220-kDa LPL binding proteoglycan. Using ligand blotting with 125I-LPL, we now report a 116-kDa LPL binding protein in plasma membrane preparations of endothelial cells. 125I-LPL binding to this protein was abolished by addition of unlabeled LPL. When the cell surface of endothelial cells was labeled with biotin, a 116-kDa protein was biotinylated. Furthermore, the biotinylated 116-kDa protein bound to LPL-Sepharose and eluted with 0.4 M NaCl suggesting that the 116-kDa LPL binding protein is present on the cell surface. When detergent extracts of endothelial cells were applied to LPL-Sepharose in the presence of 0.15 M NaCl, the 116-kDa, but not the 220-kDa, protein still bound to LPL-Sepharose. The 116-kDa protein was not labeled with 35SO4 and eluted from DEAE-cellulose prior to proteoglycans, suggesting that it is not a proteoglycan. However, a 116-kDa endothelial cell surface protein was metabolically labeled with [35S]methionine. This protein was dissociated from the cell surface by incubating cells with heparin (50 units/ml)-containing buffer. After heparin treatment of endothelial cells, LPL binding to and internalization by the cells decreased greater than 70% compared to control cells. These results suggest that endothelial cells synthesize a heparin-releasable, high affinity 116-kDa LPL binding protein. We postulate that this protein is associated with proteoglycans on luminal endothelial surfaces and mediates LPL binding, internalization, and recycling. We name this protein hrp (heparin-releasable protein)-116.     

Surface-Enhanced Raman Spectroscopy of the Endothelial Cell Membrane

We applied surface-enhanced Raman spectroscopy (SERS) to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.     

Competitive protein adsorption on micro patterned polymeric biomaterials, and viscoelastic properties of tailor made extracellular matrices

Cell adhesion on biomaterial surfaces and the vitality of anchorage dependent cells is affected by several parameters of an adsorbate layer which is intentionally or spontaneously formed. Surface pre-treatments and several conditioning steps prior and during to the cell/biomaterial contact affect the composition, orientation, quantity and viscoelasticity of the interfacing layer between cells and biomaterial. This work was performed to elucidate the response of cells on two modified biomaterial surfaces based on protein or carbohydrate adsorbates: (a) Masked UV irradiations opened a simple route to obtain chemically patterned substrates controlling serum protein adsorption and cell adhesion. It is possible to achieve structures of subcellular size and to produce immobilized gradients. In order to examine the protein matrix deposited on these substrates we applied a quartz microbalance technique (QCM-D) capable to extract viscoelastic data in addition to the mass uptake during plasma protein deposition. It was found that the quantity and viscosity of surface bound albumin is lowered when the surface is modified (patterned) by UV exposure. Hence, the UV modification promotes the competitive adsorption of cell adhesion proteins from the media or upon secretion by the cells and yields to the observed cell patterns. (b) Another tissue engineering technique, using immobilized, modified and/or cross linked hyaluronic acid (HA), an important extra cellular matrix component in vivo, is also examined by QCM-D. Our data demonstrate that HA can be modified by an activation with a carbodiimide, followed by the application of an alpha,omega-bisamino polyethyleneglycol. The QCM-D data can be interpreted as a stiffening of the HA layer combined with the release of hydration water. Further, the hydration state and the viscoelastic behaviour of surface bound ultrathin HA hydrogels was examined. Quantification of viscoelastic parameters of thin films of ECM by QCM-D is valuable for the interpretation of durotaxis, describing effects of mechanical substrate parameters on the adhesion and motility of cells.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

Ammonia plasma treatment of polystyrene surfaces enhances proliferation of primary human mesenchymal stem cells and human endothelial cells

The control of surface properties is a substantial step in the development and improvement of biomaterials for clinical applications as well as for their use in tissue engineering. Interaction of the substrate surface with the biochemical or biological environment is crucial for the outcome of the applied biomaterial and therefore should meet specific requirements regarding the chemical composition, wettability, elasticity, and charge. In this study, we examined the effect of chemical groups introduced by low pressure plasma treatments of polystyrene surfaces on the cell behavior of primary human mesenchymal stem cells (hMSCs) and dermal microvascular endothelial cells (hDMECs). X-ray photoelectron spectroscopy analysis and contact angle measurements were employed to evaluate ammonia-, carbon dioxide-, and acrylic acid-plasma modifications to substrate surfaces. HMSCs and hDMECs were analyzed simultaneously to identify the most suitable surface functionalization for each cell type. Significantly higher cell proliferation was detected on ammonia plasma-treated surfaces. Cell-material interaction could be shown on all created interfaces as well as the expression of typical cell markers. Hence, the applied plasma treatment presents a suitable tool to improve culture condition on polystyrene for two important cell types (hMSCs and hDMECs) in the field of tissue engineering.