Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein.

By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D. Predescu, L. Saucan, L. Ghitescu, and G.E. Palade. 1997. Mol. Biol. Cell. 8:595-605) developed in our laboratory, we have isolated a caveolar subfraction from rat lung endothelium and we have partially characterized the proteins of this subfraction which include an apparently caveolae-specific glycoprotein we propose to call PV-1 (formerly known as gp68). The isolation and partial sequencing of PV-1, combined with the cloning of the full length PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single span type II integral membrane protein (438 amino acids long) which forms homodimers in situ; (b) the transmembrane domain of PV-1 is near the NH2 terminus defining a short cytoplasmic endodomain and a large COOH-terminal ectodomain exposed to the blood plasma; (c) PV-1 is N-glycosylated and its glycan antennae bear terminal nonreducing galactosyl residues in alpha1-3 linkage. PV-1 is expressed mostly in the lung but both the messenger RNA and the protein can be detected at lower levels also in kidney, spleen, liver, heart, muscle, and brain. No signal could be detected in testis and two lower molecular weight forms were detected in brain. Immunocytochemical studies carried out by immunodiffusion on rat lung with an anti-PV-1 polyclonal antibody directed against a COOH-terminal epitope reveal a specific localization of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae and confirm the extracellular orientation of the PV-1 COOH terminus.

Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

Plasma membrane: rapid isolation and exposure of the cytoplasmic surface by use of positively charged beads.

Erythrocytes were ionically attached to polylysine-coated beads 30 micrometers in diameter. The binding of the cells was so tenacious that lysis or disruption of the attached cells left the beads covered by plasma membranes whose cytoplasmic surface was exposed and accessible for further analysis.

Requirement for diacylglycerol and protein kinase C in HeLa cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading.

Release of arachidonic acid (AA) and subsequent formation of a lipoxygenase (LOX) metabolite(s) is an obligatory signal to induce spreading of HeLa cells on a gelatin substratum (Chun and Jacobson, 1992). This study characterizes signaling pathways that follow the LOX metabolite(s) formation. Levels of diacylglycerol (DG) increase upon attachment and before cell spreading on a gelatin substratum. DG production and cell spreading are insignificant when phospholipase A2 (PLA2) or LOX is blocked. In contrast, when cells in suspension where PLA2 activity is not stimulated are treated with exogenous AA, DG production is turned on, and inhibition of LOX turns it off. This indicates that the formation of a LOX metabolite(s) from AA released during cell attachment induces the production of DG. Consistent with the DG production is the activation of protein kinase C (PKC) which, as with AA and DG, occurs upon attachment and before cell spreading. Inhibition of AA release and subsequent DG production blocks both PKC activation and cell spreading. Cell spreading is also blocked by the inhibition of PKC with calphostin C or sphingosine. The inhibition of cell spreading induced by blocking AA release is reversed by the direct activation of PKC with phorbol ester. However, the inhibition of cell spreading induced by PKC inhibition is not reversed by exogenously applied AA. In addition, inhibition of PKC does not block AA release and DG production. The data indicate that there is a sequence of events triggered by HeLa cell attachment to a gelatin substratum that leads to the initiation of cell spreading: AA release, a LOX metabolite(s) formation, DG production, and PKC activation. The data also provide evidence indicating that HeLa cell spreading is a cyclic feedback amplification process centered on the production of AA, which is the first messenger produced in the sequence of messengers initiating cell spreading. Both DG and PKC activity that are increased during HeLa cell attachment to a gelatin substratum appear to be involved. DG not only activates PKC, which is essential for cell spreading, but is also hydrolyzed to AA. PKC, which is initially activated as consequence of AA production, also increases more AA production by activating PLA2.

Beta 1 integrins signal lipid second messengers required during cell adhesion.

Clustering of integrin receptors during cell adhesion stimulates signal transduction across the cell membrane. Second messengers are generated, activating cytosolic proteins and causing cytoskeletal assembly and rearrangement. HeLa cell adhesion to a collagen substrate has been shown to initiate an arachidonic acid-mediated signaling pathway, leading to the activation of protein kinase C (PKC) and cell spreading. To determine the role of integrin receptors in triggering this signaling pathway, monoclonal antibodies to beta 1 integrins were used to either cluster integrins on the cell surface or to provide an integrin-dependent substrate for cell adhesion. Using this approach, we have defined a pathway required for cell spreading that can be initiated by the ligation of integrins and leads to the activation of PKC. Specifically, our results indicate that clustering beta 1 integrins results in the activation of phospholipase A2 leading to the production of arachidonic acid and the activation of PKC.

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.

Extracellular calcium regulates HeLa cell morphology during adhesion to gelatin: role of translocation and phosphorylation of cytosolic phospholipase A2.

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55-65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.

Modulation of cell-substrate adhesion by arachidonic acid: lipoxygenase regulates cell spreading and ERK1/2-inducible cyclooxygenase regulates cell migration in NIH-3T3 fibroblasts.

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute approximately 25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.

Imporved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum.

The plasma membrane from Dictyostelium discoideum was routinely purified 35-fold by an improved technique using beads coated with positively charged polymers. Cells were attached to the beads and bare regions between the cells were neutralized with a polyanion. The neutralization decreased contamination of the bare regions by intracellular proteins released when cells were disrupted to leave behind beads coated by plasma membrane. The neutralization increased the purification as measured by membrane-bound 125I-labeled concanavalin A. Contamination by markers for various intracellular components was markedly decreased. Various bare-site neutralization reagents were evaluated and gave different results depending upon their charge density and molecular weight. The pH of the neutralization was critical. The optimum pH for cell attachment to beads, 5.0, had little effect as regard bare-site neutralization. A new procedure is given that optimizes the essential features for the plasma membrane isolation on beads.

Isolation of plasma membrane from eukaryotic cells on polylysine-coated polyacrylamide beads.

The conditions for covalently binding polylysine to polyacrylamide beads used for membrane isolation have been analyzed. Larger amounts of bead bound polylysine were required for maximimizing plasma membrane purification from HeLa cells than from Dictyostelium discoideum. The least was needed for erythrocytes. The amount of polylysine bound to the bead was dependent on the carboxyl content of the bead and on the concentration of the polylysine used during the bead-polylysine coupling reaction.

Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate.

The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains to carry out these functions: one attached to the substrate (ventral); another exposed to the media (dorsal); and an intracellular domain involved in endocytosis and secretion. A technique has been developed for the rapid isolation of these specific domains from HeLa cells immediately following adhesion to a gelatin substrate. The isolation procedure utilizes the tight binding of cationic colloidal silica to the dorsal plasma membrane domain of attached cells. Following silica binding and cell lysis, the silica-coated dorsal plasma membrane domain is readily separated from intracellular plasma membrane components by virtue of the high density of the silica pellicle, and the intact ventral plasma membrane domain remains attached to the gelatin substrate. Fluorescence and electron microscopy and biochemical studies using 125I-lactoperoxidase labeling, 125I-labeled wheat germ agglutinin binding, and [3H]-fucose incorporation into plasma membrane glycoproteins confirmed the separation of these three topologically distinct plasma membrane domains. The fractions isolated by the technique contained essentially all of the plasma membrane components present in intact cells. This unique membrane-isolation procedure is now being used to analyze membrane flow during plasma membrane domain formation accompanying cell adhesion to an extracellular matrix.

Coupling polylysine to glass beads for plasma membrane isolation.

Solid glass beads for use in isolating cell membranes were coated with a stable, covalently attached layer of polylysine. The optimal conditions for coating the bead surface were established and the beads were tested by measuring the attachment of human erythrocyte plasma membranes. When compared to other beads, such as those with absorbed polylysine or protamine, none retained red-cell membranes as well as glass beads with covalently linked polylysine.

Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation.

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.

Binding of plasma membrane glycoproteins to the cytoskeleton during patching and capping is consistent with an entropy-enhancement model.

Concentrations of concanavalin A that induced patching and capping of cell surface receptors on Dictyostelium discoideum also induce binding of the receptors to the cortical cytoskeleton, which was isolated by density-gradient centrifugation. The receptors were solubilized by deoxycholate, purified by affinity chromatography, and used to determine whether the receptors bound directly to the cytoskeletal protein, actin. As the concentration of actin was increased, many of the receptors became bound to purified filamentous rabbit muscle actin, even in the absence of concanavalin A. As in the ligation-induced binding of receptors to the cortical cytoskeleton in cells, concanavalin A induced much stronger binding of the purified receptors to filamentous actin. The results were consistent with a previously stated hypothesis that induction of receptor binding to the cytoskeleton during their patching and capping is driven by clustering the receptors, which reduces their translational entropy and by doing so enhances their avidity for the cytoskeleton.

Explanation of anomalous binding kinetics with a high yield immobilized enzyme system.

The activities of glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) from commercial preparations do not give typical adsorption curves upon immobilization on non-porous polyethylenimine-coated glass microbeads. The cause of this effect with glucose oxidase was investigated. Protein binding exhibited a rectangular hyperbolic adsorption isotherm, approaching saturation at high concentrations, however, enzyme activities did not. The isotherm for activities exhibited a maxima which corresponded to less than 50% saturation with regard to total protein adsorption. The enzyme preparation was found to contain small quantities of several low molecular weight impurities as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These impurities apparently compete with glucose oxidase for binding. When large excesses of protein are added to beads, the binding of impurities becomes significant and the amount of enzyme activity per unit of bead is reduced.

Membrane isolation on polylysine-coated glass beads. Asymmetry of bound membrane.

Erythrocyte membranes isolated on polylysine-coated glass beads exhibit many of the properties of the native membrane. Gel electrophoresis indicates that all major protein components of the membrane are retained during membrane isolation. The membrane integrity and accessibility of selected components was tested using non-penetrating probes. In general, membranes on beads displayed accessibility properties typical of inside-out vesicles. The accessibility of membrane acetylcholinesterase to assay reagents, as well as membrane accessibility to the actions of neuraminidase, trypsin and galactose oxidase-NaB3H4 demonstrated that the protoplasmic surface of membrane isolated on beads was exposed, while the extracellular surface was inaccessible. The differential accessibility of the membrane surfaces demonstrates the feasibility of investigating asymmetry of membranes isolated on cationic glass beads.

Evaluation of the silica microbead method for isolation of red beet protoplast plasma membrane sheets.

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet (Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K+-stimulated, Mg2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.

The prevalence of carbon-13 in respiratory carbon dioxide as an indicator of the types of endogenous substrate. The change from lipid to carbohydrate during the respiratory rise in potato slices.

Isotope discrimination is a common feature of biosynthesis in nature, with the result that different classes of carbon compounds frequently display different (13)C/(12)C ratios. The (13)C/(12)C ratio of lipid in potato tuber tissue is considerably lower than that for starch or protein. We have collected respiratory CO(2) from potato discs in successive periods through 24 hr from the time of cutting-an interval in which the respiration rate rises 3-5-fold. The (13)C/(12)C ratio of the evolved CO(2) was determined for each period, and compared with the (13)C/(12)C ratios of the major tissue metabolites. In the first hours the carbon isotope ratio of the CO(2) matches that of lipid. With time, the ratio approaches that typical of starch or protein. An estimation has been made of the contribution of lipid and carbohydrate to the total respiration at each juncture. In connection with additional observations, it was deduced that the basal, or initial, respiration represents lipid metabolism-possibly the alpha-oxidation of long chain fatty acids-while the developed repiration represents conventional tricarboxylic acid cycle oxidation of the products of carbohydrate glycolysis. The true isotopic composition of the respiratory CO(2) may be obscured by fractionation attending the refixation of CO(2) during respiration, and by CO(2) arising from dissolved CO(2) and bicarbonate preexisting in the tuber. Means are described for coping with both pitfalls.

Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs.

This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.