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beyond the scope of this chapter. However, since all of the studies of phospholipases described in this chapter employed micelles or mixed micelles of substrates, the configurational effect on the physical properties should not be responsible for the observed stereospecificity. Acknowledgments The work in the laboratory of M.-D.T. was supported by a grant (GM30327) from the National Institutes of Health. The work in the laboratory of K.S.B. was supported by Grant CPBP.01.13.3.16 from the Polish Academy of Sciences.
[24] P h o s p h o l i p a s e A2: M i c r o i n j e c t i o n a n d Cell Localization T e c h n i q u e s B y D A F N A BAR-SAGI
Introduction P h o s p h o l i p a s e A 2 ( P L A 2 ) is a calcium-requiring esterase that catalyzes the hydrolysis of glycerophospholipids specifically at the sn-2 position to produce a fatty acid and a lysophospholipid.L2As mentioned elsewhere in this volume, the activity of PLA2 has been postulated to play an important regulatory role in several metabolic pathways. For example, PLA 2 catalyzes the release of arachidonic acid, the first and rate-limiting precursor in the biosynthesis of prostaglandins. In addition, the activity of the enzyme is part of the phosphoglyceride deacylation-reacylation cycle and as such mediates the rapid metabolic turnover of membrane phospholipids. Furthermore, there is increasing evidence in support of the participation of PLA2 in the generation of receptor-mediated transmembrane signals. This chapter is specifically concerned with two approaches, microinjection and cell localization, to analyze the biological properties of cellular PLA 2. As both approaches rely primarily on the availability of anti-PLA2 antibodies, methods for obtaining suitable anti-PLA2 antibodies are also included. t H. van den Bosch, Biochim. Biophys. Acta 604, 191 (1980). 2 H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Preparation and Characterization of Anfi-PLA2 Antibodies
Immunization As an immunogen, preparations of porcine pancreas PLA2 purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN) or Sigma Chemical Co. (St. Louis, MO) can be used. Although the pancreatic PLA2 represents the secreted form of the enzyme, the rationale for using it for raising antibodies against the intracellular form is based on the evidence that the two forms are immunochemically related. 3 The enzyme solution (10 mg/ml) is provided as an ammonium sulfate suspension (3.2 mol/liter, pH 15.5) and is dialyzed against phosphate-buffered saline (PBS). Fifty percent of the dialyzed solution is supplemented with sodium dodecyl sulfate (SDS) to a final concentration of 1% and boiled for 2-3 min. The "SDS-denatured" PLAz solution and the "native" PLA2 solution are each diluted to 1 mg/ml protein concentration with PBS, mixed at a ratio of ! : 1 (w/w), and injected subcutaneously into rabbits according to the following schedule: 50/zg of PLA2 in Freund's complete adjuvant on day 0, 100/xg of PLA2 in Freund's incomplete adjuvant at 10-day intervals over a 2-month period, and 100/xg booster injection in Freund's incomplete adjuvant every 4-5 weeks. Animals are bled at 1-month intervals after the sixth injection, and the titer of the serum is determined by the enzymelinked immunosorbent assay (using 50 ng of antigen per microtiter well). A high-titer serum is usually obtained within a 2 to 3-month period following the first injection.
Affinity Purification of Antibodies Homogeneous porcine pancreatic PLAz (10 mg) is coupled to 1 ml of Affi-Gel 10 (Bio-Rad, Richmond, CA) by the procedure recommended by the manufacturer. The coupling efficiency exceeds 90%. The resin is poured into a polypropylene Econo-Column (Bio-Rad), and the column is equilibrated with 75 mM HEPES (pH 7.5). Crude antiserum is clarified by low-speed centrifugation and passed through the column 3 times, and the column is then washed extensively with 75 mM HEPES (pH 7.5). Specifically bound antibodies are desorbed with 12 0.5-ml washes of 50 mM glycine-HC1 (pH 2.5), and the eluates are adjusted to neutral pH immediately by the addition of 0.5 M Tris (pH 7.5). Fractions are analyzed
3 M. Okamoto, T. Ono, H. Tojo, and T. Yamano, Biochem. Biophys. Res. Commun. 17,8, 788 (1985).
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PLA 2 MICROINJECTIONAND LOCALIZATION kDa
1
2 3 4 5
6
7 8
271
9 10 11 12
200 - 116 - 92-66--
43 m
31--
21 m glycine-HCI pH 2.5 ~
I
I anti-PLA 21gG
FIG. 1. Elution profile from a PLA2 affinity column of rabbit anti-PLA z IgG. Ten-microliter aliquots of each 0.5-ml fraction were analyzed on the gel.
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1). Combined eluates are dialyzed against PBS and concentrated 5- to 10-fold by centrifugal concentration (Centricon 10 microconcentrator, Amicon, Danvers, MA). After use, the column is rinsed extensively with 75 mM HEPES (pH 7.5) and stored in 75 mM HEPES containing 0.5% NAN3. The resin can be reused at least 5 times without noticeable effects on the quality of antibodies purified.
Characterization of Antibodies Two assays are used to establish the specificity of the antibody.
Immunoblotting. Cell extracts are fractionated by SDS-PAGE on a 12.5% gel. Proteins (100 ~g/lane) are transferred to nitrocellulose sheets
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by the blotting procedure described by Towbin et al. 4 The nitrocellulose sheets are incubated in a blocking solution [3% bovine serum albumin (BSA) in PBS] overnight and then incubated for 2 hr at 20° with antibody solution (20-50/zg/ml in 1% BSA in PBS). The sheets are rinsed 3 times for 5-10 min each in PBS, then incubated in peroxidase-conjugated goat anti-rabbit IgG (Cappel Laboratories, Downington, PA) diluted 1 : 1500 in 1% BSA in PBS, for 1 hr at 20°. The sheets are again washed 3 times for 5-10 rain each with PBS and then developed in 4-chloro-l-naphthol (3 mg/ ml of 4-chloro-l-naphthol in methanol diluted with 5 volumes of PBS to which 0.01 volume of 30% hydrogen peroxide is added). This method is very informative since it establishes both the presence and the molecular weight of the antigen and any cross-reacting material. In cases where the abundance of the antigen is very low, the sensitivity of the detection method can be amplified by using biotinylated goat anti-rabbit antibodies followed by streptavidin-alkaline phosphatase. Neutralizing Activity. The assay for neutralizing activity establishes the specificity of the antibody based on its ability to interfere with the enzymatic activity of the antigen. Cultured cells are labeled for 2 hr with 100/.~Ci/ml of carrier-free 3 2 p o 4 in phosphate-free DMEM (Dulbecco's modified eagle's medium). At the end of the labeling period, cells are washed 5 times with phosphate-free DMEM and harvested in ice-cold hypotonic lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaC1, 0.1% 2mercaptoethanol, 1% aprotinin). After incubation for 20 min on ice, cells are ruptured with 25-40 strokes (depending on cell type and density) of a Dounce homogenizer. Cell lysates are sedimented at low speed (500 g for 5 min at 4°) to remove nuclei, and the supernatant is centrifuged at I00,000 g for 30 min at 4°. The resulting membrane pellet is resuspended in incubation buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl) to a protein concentration of 0.2 mg/ml. Equal aliquots of the membrane suspension (100-200/zl) are preincubated for 2 hr at 4° with 1-15/xg of anti-PLA 2 IgG or control IgG. The reaction mixture is then supplemented with 5 mM C a C l 2 to trigger P L A 2 activity and incubated at 37° for various intervals. Reactions are terminated by the addition of an ice-cold mixture of methanol, chloroform, and HC1 (2 : 1 : 0.02, v/v), and phospholipids are extracted as described previously) Chromatographic separation of phospholipids is carried out on silica gel LK6D thin-layer chromatography (TLC) plates (Whatman, Inc, Clifton, N J). To resolve the products of PLA 2 activity, namely, lysophosphatidylcholine (lyso-PC) and lysophosphatidylethanolamine (lyso4 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 5 D. Bar-Sagi, J. P. Suhan, F. McCormick, and J. R. Feramisco, J. Cell Biol. 1116, 1649 (1988).
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PE), the solvent sysem of chloroform, methanol, ammonia, and water (45:30:3:5, v/v) is used. The 32p-labeled phospholipids are visualized by autoradiography and identified by cochromatography with standards detected with iodine vapor. The TLC plates are exposed to film for approximately 12 hr. Quantification of 32p incorporated into phospholipids is determined by scraping the labeled phospholipids off the plates and liquid scintillation counting. Total 32p incorporation into lipids is approximately 50,000 counts per minute (cpm) under these conditions. In this assay the activity of PLA 2 is time, temperature, and calcium dependent. Phosphatidylethanolamine is the preferred substrate for PLA 2 activity. 5 Inhibitory anti-PLA2 antibodies result in a dose-dependent inhibition of the accumulation of lysophosphoglycerides, whereas control antibodies have no effect on PLA 2 activity (Fig. 2). Similar results are obtained when the activity of PLA2 is monitored by the generation of free [3H]arachidonic acid following labeling of cells with [3H]arachidonic acid (1/zCi/ml) for 24 hr or by the hydrolysis of [14C]oleate labeled Escherichia coli substrate. Cell Injection The microinjection technique is a valuable tool for the assay of the functional significance of cellular proteins. In principle, this technique allows one to modulate the level and/or activity of a particular protein by introducing defined amounts of the protein or antibodies directed against the protein into the cells. In view of the paucity of specific PLA2 inhibitors, microinjection of inhibitory anti-PLA 2 antibodies makes this approach the method of choice to assay the role of phospholipase A2 in various cellular processes. The technique of microneedle injection has been described in detail elsewhere. 6In general, the procedure utilizes a glass capillary needle filled with the substance to be injected into the cell, a micromanipulator to place the needle into the cell, and a phase-contrast microscope to allow visualization of the injection process. Cells
Cells for injection can be grown on either glass coverslips or on petri dishes provided that they adhere tightly to the substrate. It is difficult to predict the suitability of a given cell type for microinjection. As a rule, large or tall cells are easier to inject, whereas small or flat cells are difficult and sometimes even impossible to inject. 6 A. Graessmann, M. Graessman, and C. Mueller, this series, Vol. 65, p. 816.
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I00
75
>
u 5O O
25
0
0
I
I
5
I0
15
~cj ontibody
FIG. 2. Inhibition of the activity of membrane-associated PLA2as a function of antibody concentration. After preincubation for 2 hr at 4° with the anti-PLA2antibodies (filled circles) or nonimmunerabbit anti-rat antibodies (open circles), phospholipaseA2activitywas assayed as described in the text. Results are expressed as percentages of the initial activity of the enzyme.
Sample Preparation The protein to be injected must be prepared in a buffer that will not have a deleterious effect on the cell. A variety of injection buffers have been used with success. 7 For example, a c o m m o n l y used buffer for antib o d y injection is c o m p o s e d of 10 m M NaH/PO4, 70 m M KCI, pH 7.2. Protein concentrations for the injections should be adjusted according to the purpose of the experiments. Concentration ranges of 1-15 mg/ml can be used. The lower end o f this range usually applies for the introduction 7 K. Wang, J. R. Feramisco, and J. F. Ash, this series, Vol. 85, p. 514.
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of proteins, whereas the higher concentrations generally apply to the injection of antibodies.
Microinjection Prior to injection, the protein solution is clarified by centrifugation for 5 min at 12,000 g at 4°. The solution is loaded into the needle by capillary action immediately prior to microinjection. Cells are removed from the incubator and are placed on the microscope stage. The area of injection can be marked by an ink circle using a marker objective. Cells are brought into focus, and the capillary is lowered until the tip is almost in focus. The cells are injected by further lowering the capillary tip and applying a gentle positive pressure. The injection process is marked by a slight swelling of the cell. The cytoplasm appears to lose contrast, whereas the nucleus gains contrast. These changes in appearance are transient, and within a few seconds injected cells should regain normal morphology. The volume injected per cell can be controlled by the amount of pressure applied on the syringe. It has been determined that the cytoplasmic injection of cells usually results in the introduction of 10-13 to 10-14 liter per cell. 6'8 Control injections should be done to determine the specificity of the effects monitored. In the case of microinjection of antibodies, control injections may include heat-denatured antibody, nonspecific antibody injected at the same concentration as the specific antibody, and buffer alone.
Analysis of Injected Cells The analytical potential of microinjection experiments depends on two parameters: (1) the ability to positively identify injected cells and (2) the availability of a single cell assay that can be monitored visually. As for the first parameter, the immunofluorescence technique is ideal for the identification of injected cells. The staining procedures for immunofluorescence are detailed below (Immunocytochemical Localization of PLA2). Following fixation and permeabilization of cells, injected rabbit anti-PLAz antibodies can be readily detected by staining with fluorescein-conjugated goat anti-rabbit IgG (Fig. 3). Antibody polypeptides injected at 5-10 mg/ml can be detected in cells up to 48 hr after injection. A summary of the results obtained in microinjection experiments using anti-PLAz antibodies (Table I) provides examples for cellular responses that can be measured at the single cell level. In each of the studies described, the observed effects were dose dependent and were not detected in control injections. 8 D. W. Stacey and V. G. Allfrey, Cell (Cambridge, Mass.) 9, 729 (1976).
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Immunocytochemical Localization of PLA 2 PLA 2 has been solubilized from several tissues by sonication,l indicating that the enzyme is not firmly bound to membrane structures. This makes it difficult to determine the cellular localization of the enzyme solely on the basis of biochemical fractionation. This section describes immunohistochemical techniques to determine the spatial aspects of PLA 2 distribution.
Preparation of Cellsfor Labeling It is most convenient to grow cells on coverslips which can then be processed and mounted on slides for observation. In the case of cells that adhere poorly to glass, adhesion and cell growth can be improved by cleaning the coverslips with chromic acid or coating the surface with a positively charged polymer. Cell density can influence cell morphology and may affect the quality of the signal, especially in high-resolution fluorescence microscopy. Cell debris adsorb antibodies nonspecifically. For these reasons it is suggested that standard conditions for culture labeling be established. Prior to staining, it is necessary to fix the cells in order to preserve structure and then permeabilize them to allow labeling reagents to reach intracellular targets. A large number of fixation procedures have been described in the literature, with varying degrees of preservation of antigenic site and cellular morphology. Details of those procedures that were optimal for anti-PLA 2 antibody generated in our laboratory are described here.
Indirect Immunofluorescence Cells plated on glass coverslips are washed twice with PBS and fixed at 20° for 30 min in 3.7% formaldehyde in PBS. Coverslips are rinsed twice with PBS, and cells are permeabilized by exposure to 0.2% Triton X-100 (Sigma) in PBS for 30 sec at 20°. The affinity-purified rabbit anti-PLAz antibody is used at a 1:50 dilution (20 /zg/ml) in PBS containing
Fro. 3. Detection of microinjected anti-PLA2 antibodies in rat embryo fibroblasts. Four cells in the field shown were injected with the antibody solution (5 mg/ml). At 5 hr postinjection, the cells were fixed, permeabilized, and stained with fluorescein-conjugated goat anti-rabbit IgG. (A) Fluorescence micrograph; (B) corresponding phase micrograph. Bar, 10/zm.
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TABLE I EFFECTS OF MICROINJECTIONOF ANTI-PEA2 ANTIBODIES
Cell type ras-Transformed
fibroblasts Normal fibroblasts (rat kidney or rat embryo cells) Rat peritoneal mast ceils Endothelial cells
Concentration of solution injected (mg/ml) 5 10
5 1
Assay
Results
Ref. °
Morphological reversion (cell flattening) Serum- or growth factorinduced stimulation of DNA synthesis b Ligand-induced exocytotic degranulation c Thrombin-induced sustained increase in cytosolic calcium d
+
1
No effect
2
-
3
-
4
Key to references: (1) D. Bar-Sagi, in "Cell Activation and Signal Initiation: Receptor and Phospholipase Control of Inositol Phosphate, PAF and Eicosanoid Production," p. 331. UCLA Symposium, Alan R. Liss, New York, 1989; (2) D. Bar-Sagi, unpublished observations (1988); (3) L. Graziadei and D. Bar-Sagi, in preparation (1990); (4) M. S. Goligorsky, D. N. Menton, A. Laszlo, and H. Lum, J. Biol. Chem. 264, 16771 (1989). b DNA synthesis is measured by [3H]thymidine incorporation and emulsion autoradiography. [3H]Thymidine (1/xCi/ml) is added to the medium within 1 hr after injection, and cells are further incubated for 12-24 hr. At the end of the incubation period the cells are fixed, and the dishes are coated with Nuclear Track Emulsion (NTB-2, Kodak) and processed for emulsion autoradiography. c Degranulation is monitored by light microscopic visualization of the extrusion of granules [D. Bar-Sagi and B. Gomperts, Oncogene 3, 463 (1988)]. a Concentration of cytosolic calcium in individual cells was monitored using the calcium indicator dye Fura-2.
0.5 mg/ml BSA. It should be noted, however, that the optimal concentration of the primary antibody varies widely, depending on titer and affinity, and therefore should be determined empirically. Incubation with the primary antibody is for 1 hr at 37 ° in a humidified atmosphere. Following two 10-min washes in PBS, cells are incubated with fluorescein-conjugated goat anti-rabbit antibody (Cappel) diluted 1 : 100 (10/xg/ml) in PBS containing 0.5 mg/ml BSA. Secondary antibody incubation is for I hr at 37 ° in a humidified atmosphere followed by two 10-min PBS washes and a distilled water rinse before mounting with Gelvatol (Monsanto Polymers and Petrochemicals Co., St. Louis, MO). 9 Reagents which slow down the photobleaching of fluorescein (N-propyl gallate or p-phenylenediamine) are generally added to the mounts. Before use, the secondary antibodies are 9 j. R. Feramisco and S. H. Blose, J. Cell Biol. 86, 608 (1980).
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preadsorbed with methanol-fixed NRK (normal rat kidney) monolayers and clarified using a Beckman Airfuge to reduce nonspecific stickiness. Cells are examined with a Zeiss photomicroscope III equipped for epifluorescence. Phase micrographs are recorded using Kodak (Rochester, NY) Technical Pan film, and fluorescence micrographs are recorded on Kodak Tri-X film. Controls are essential for interpreting immunofluorescence staining patterns. Some useful controls are the following: (1) omitting primary antibody from the staining procedure, (2) staining with nonimmune IgG, and (3) staining with specific IgG preadsorbed with antigen. All controls should give no specific staining pattern and be significantly dimmer than the experimental samples. It is particularly important to perform all controls in pilot experiments or whenever a new batch of antibodies or different cells are utilized.
Immunoelectron Microscopy Analysis of the cellular distribution ofPLA/at the electron microscopic level is extremely informative and should complement the analysis at the light microscopy level. A discussion of immunoelectron microscopy techniques, however, is beyond the scope of this chapter. In general, the preparation time for samples may take up to 1 week, and then the interpretation of results often calls for the assistance of an experienced electron microscopist. An important prerequisite for a meaningful ultrastructural analysis is a good preservation of cellular structures. However, good fixatives, while preserving fine structures, tend to alter or destroy antibody binding sites as well as to decrease accessibility of probes. Therefore, the effects of several different fixation procedures on the labeling pattern should be compared. Two staining methods can be employed: (I) indirect immunoperoxidase staining which utilizes peroxidase-conjugated secondary antibodies and (2) immunogold labeling which utilizes secondary antibodies attached to gold particles. The latter offers the possibility of using gold particles of different sizes (5-20 nm), thus allowing for the simultaneous labeling of two antigens in order to assess their spatial relationships. Acknowledgments We thank Madeline Wisnewski for secretarial assistance. This work was supported by National Institutes of Health Grant CA46370 and American Cancer Society Grant BC690.
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beyond the scope of this chapter. However, since all of the studies of phospholipases described in this chapter employed micelles or mixed micelles of substrates, the configurational effect on the physical properties should not be responsible for the observed stereospecificity. Acknowledgments The work in the laboratory of M.-D.T. was supported by a grant (GM30327) from the National Institutes of Health. The work in the laboratory of K.S.B. was supported by Grant CPBP.01.13.3.16 from the Polish Academy of Sciences.
[24] P h o s p h o l i p a s e A2: M i c r o i n j e c t i o n a n d Cell Localization T e c h n i q u e s B y D A F N A BAR-SAGI
Introduction P h o s p h o l i p a s e A 2 ( P L A 2 ) is a calcium-requiring esterase that catalyzes the hydrolysis of glycerophospholipids specifically at the sn-2 position to produce a fatty acid and a lysophospholipid.L2As mentioned elsewhere in this volume, the activity of PLA2 has been postulated to play an important regulatory role in several metabolic pathways. For example, PLA 2 catalyzes the release of arachidonic acid, the first and rate-limiting precursor in the biosynthesis of prostaglandins. In addition, the activity of the enzyme is part of the phosphoglyceride deacylation-reacylation cycle and as such mediates the rapid metabolic turnover of membrane phospholipids. Furthermore, there is increasing evidence in support of the participation of PLA2 in the generation of receptor-mediated transmembrane signals. This chapter is specifically concerned with two approaches, microinjection and cell localization, to analyze the biological properties of cellular PLA 2. As both approaches rely primarily on the availability of anti-PLA2 antibodies, methods for obtaining suitable anti-PLA2 antibodies are also included. t H. van den Bosch, Biochim. Biophys. Acta 604, 191 (1980). 2 H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Preparation and Characterization of Anfi-PLA2 Antibodies
Immunization As an immunogen, preparations of porcine pancreas PLA2 purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN) or Sigma Chemical Co. (St. Louis, MO) can be used. Although the pancreatic PLA2 represents the secreted form of the enzyme, the rationale for using it for raising antibodies against the intracellular form is based on the evidence that the two forms are immunochemically related. 3 The enzyme solution (10 mg/ml) is provided as an ammonium sulfate suspension (3.2 mol/liter, pH 15.5) and is dialyzed against phosphate-buffered saline (PBS). Fifty percent of the dialyzed solution is supplemented with sodium dodecyl sulfate (SDS) to a final concentration of 1% and boiled for 2-3 min. The "SDS-denatured" PLAz solution and the "native" PLA2 solution are each diluted to 1 mg/ml protein concentration with PBS, mixed at a ratio of ! : 1 (w/w), and injected subcutaneously into rabbits according to the following schedule: 50/zg of PLA2 in Freund's complete adjuvant on day 0, 100/xg of PLA2 in Freund's incomplete adjuvant at 10-day intervals over a 2-month period, and 100/xg booster injection in Freund's incomplete adjuvant every 4-5 weeks. Animals are bled at 1-month intervals after the sixth injection, and the titer of the serum is determined by the enzymelinked immunosorbent assay (using 50 ng of antigen per microtiter well). A high-titer serum is usually obtained within a 2 to 3-month period following the first injection.
Affinity Purification of Antibodies Homogeneous porcine pancreatic PLAz (10 mg) is coupled to 1 ml of Affi-Gel 10 (Bio-Rad, Richmond, CA) by the procedure recommended by the manufacturer. The coupling efficiency exceeds 90%. The resin is poured into a polypropylene Econo-Column (Bio-Rad), and the column is equilibrated with 75 mM HEPES (pH 7.5). Crude antiserum is clarified by low-speed centrifugation and passed through the column 3 times, and the column is then washed extensively with 75 mM HEPES (pH 7.5). Specifically bound antibodies are desorbed with 12 0.5-ml washes of 50 mM glycine-HC1 (pH 2.5), and the eluates are adjusted to neutral pH immediately by the addition of 0.5 M Tris (pH 7.5). Fractions are analyzed
3 M. Okamoto, T. Ono, H. Tojo, and T. Yamano, Biochem. Biophys. Res. Commun. 17,8, 788 (1985).
[24]
PLA 2 MICROINJECTIONAND LOCALIZATION kDa
1
2 3 4 5
6
7 8
271
9 10 11 12
200 - 116 - 92-66--
43 m
31--
21 m glycine-HCI pH 2.5 ~
I
I anti-PLA 21gG
FIG. 1. Elution profile from a PLA2 affinity column of rabbit anti-PLA z IgG. Ten-microliter aliquots of each 0.5-ml fraction were analyzed on the gel.
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1). Combined eluates are dialyzed against PBS and concentrated 5- to 10-fold by centrifugal concentration (Centricon 10 microconcentrator, Amicon, Danvers, MA). After use, the column is rinsed extensively with 75 mM HEPES (pH 7.5) and stored in 75 mM HEPES containing 0.5% NAN3. The resin can be reused at least 5 times without noticeable effects on the quality of antibodies purified.
Characterization of Antibodies Two assays are used to establish the specificity of the antibody.
Immunoblotting. Cell extracts are fractionated by SDS-PAGE on a 12.5% gel. Proteins (100 ~g/lane) are transferred to nitrocellulose sheets
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PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
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by the blotting procedure described by Towbin et al. 4 The nitrocellulose sheets are incubated in a blocking solution [3% bovine serum albumin (BSA) in PBS] overnight and then incubated for 2 hr at 20° with antibody solution (20-50/zg/ml in 1% BSA in PBS). The sheets are rinsed 3 times for 5-10 min each in PBS, then incubated in peroxidase-conjugated goat anti-rabbit IgG (Cappel Laboratories, Downington, PA) diluted 1 : 1500 in 1% BSA in PBS, for 1 hr at 20°. The sheets are again washed 3 times for 5-10 rain each with PBS and then developed in 4-chloro-l-naphthol (3 mg/ ml of 4-chloro-l-naphthol in methanol diluted with 5 volumes of PBS to which 0.01 volume of 30% hydrogen peroxide is added). This method is very informative since it establishes both the presence and the molecular weight of the antigen and any cross-reacting material. In cases where the abundance of the antigen is very low, the sensitivity of the detection method can be amplified by using biotinylated goat anti-rabbit antibodies followed by streptavidin-alkaline phosphatase. Neutralizing Activity. The assay for neutralizing activity establishes the specificity of the antibody based on its ability to interfere with the enzymatic activity of the antigen. Cultured cells are labeled for 2 hr with 100/.~Ci/ml of carrier-free 3 2 p o 4 in phosphate-free DMEM (Dulbecco's modified eagle's medium). At the end of the labeling period, cells are washed 5 times with phosphate-free DMEM and harvested in ice-cold hypotonic lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaC1, 0.1% 2mercaptoethanol, 1% aprotinin). After incubation for 20 min on ice, cells are ruptured with 25-40 strokes (depending on cell type and density) of a Dounce homogenizer. Cell lysates are sedimented at low speed (500 g for 5 min at 4°) to remove nuclei, and the supernatant is centrifuged at I00,000 g for 30 min at 4°. The resulting membrane pellet is resuspended in incubation buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl) to a protein concentration of 0.2 mg/ml. Equal aliquots of the membrane suspension (100-200/zl) are preincubated for 2 hr at 4° with 1-15/xg of anti-PLA 2 IgG or control IgG. The reaction mixture is then supplemented with 5 mM C a C l 2 to trigger P L A 2 activity and incubated at 37° for various intervals. Reactions are terminated by the addition of an ice-cold mixture of methanol, chloroform, and HC1 (2 : 1 : 0.02, v/v), and phospholipids are extracted as described previously) Chromatographic separation of phospholipids is carried out on silica gel LK6D thin-layer chromatography (TLC) plates (Whatman, Inc, Clifton, N J). To resolve the products of PLA 2 activity, namely, lysophosphatidylcholine (lyso-PC) and lysophosphatidylethanolamine (lyso4 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 5 D. Bar-Sagi, J. P. Suhan, F. McCormick, and J. R. Feramisco, J. Cell Biol. 1116, 1649 (1988).
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PE), the solvent sysem of chloroform, methanol, ammonia, and water (45:30:3:5, v/v) is used. The 32p-labeled phospholipids are visualized by autoradiography and identified by cochromatography with standards detected with iodine vapor. The TLC plates are exposed to film for approximately 12 hr. Quantification of 32p incorporated into phospholipids is determined by scraping the labeled phospholipids off the plates and liquid scintillation counting. Total 32p incorporation into lipids is approximately 50,000 counts per minute (cpm) under these conditions. In this assay the activity of PLA 2 is time, temperature, and calcium dependent. Phosphatidylethanolamine is the preferred substrate for PLA 2 activity. 5 Inhibitory anti-PLA2 antibodies result in a dose-dependent inhibition of the accumulation of lysophosphoglycerides, whereas control antibodies have no effect on PLA 2 activity (Fig. 2). Similar results are obtained when the activity of PLA2 is monitored by the generation of free [3H]arachidonic acid following labeling of cells with [3H]arachidonic acid (1/zCi/ml) for 24 hr or by the hydrolysis of [14C]oleate labeled Escherichia coli substrate. Cell Injection The microinjection technique is a valuable tool for the assay of the functional significance of cellular proteins. In principle, this technique allows one to modulate the level and/or activity of a particular protein by introducing defined amounts of the protein or antibodies directed against the protein into the cells. In view of the paucity of specific PLA2 inhibitors, microinjection of inhibitory anti-PLA 2 antibodies makes this approach the method of choice to assay the role of phospholipase A2 in various cellular processes. The technique of microneedle injection has been described in detail elsewhere. 6In general, the procedure utilizes a glass capillary needle filled with the substance to be injected into the cell, a micromanipulator to place the needle into the cell, and a phase-contrast microscope to allow visualization of the injection process. Cells
Cells for injection can be grown on either glass coverslips or on petri dishes provided that they adhere tightly to the substrate. It is difficult to predict the suitability of a given cell type for microinjection. As a rule, large or tall cells are easier to inject, whereas small or flat cells are difficult and sometimes even impossible to inject. 6 A. Graessmann, M. Graessman, and C. Mueller, this series, Vol. 65, p. 816.
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75
>
u 5O O
25
0
0
I
I
5
I0
15
~cj ontibody
FIG. 2. Inhibition of the activity of membrane-associated PLA2as a function of antibody concentration. After preincubation for 2 hr at 4° with the anti-PLA2antibodies (filled circles) or nonimmunerabbit anti-rat antibodies (open circles), phospholipaseA2activitywas assayed as described in the text. Results are expressed as percentages of the initial activity of the enzyme.
Sample Preparation The protein to be injected must be prepared in a buffer that will not have a deleterious effect on the cell. A variety of injection buffers have been used with success. 7 For example, a c o m m o n l y used buffer for antib o d y injection is c o m p o s e d of 10 m M NaH/PO4, 70 m M KCI, pH 7.2. Protein concentrations for the injections should be adjusted according to the purpose of the experiments. Concentration ranges of 1-15 mg/ml can be used. The lower end o f this range usually applies for the introduction 7 K. Wang, J. R. Feramisco, and J. F. Ash, this series, Vol. 85, p. 514.
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of proteins, whereas the higher concentrations generally apply to the injection of antibodies.
Microinjection Prior to injection, the protein solution is clarified by centrifugation for 5 min at 12,000 g at 4°. The solution is loaded into the needle by capillary action immediately prior to microinjection. Cells are removed from the incubator and are placed on the microscope stage. The area of injection can be marked by an ink circle using a marker objective. Cells are brought into focus, and the capillary is lowered until the tip is almost in focus. The cells are injected by further lowering the capillary tip and applying a gentle positive pressure. The injection process is marked by a slight swelling of the cell. The cytoplasm appears to lose contrast, whereas the nucleus gains contrast. These changes in appearance are transient, and within a few seconds injected cells should regain normal morphology. The volume injected per cell can be controlled by the amount of pressure applied on the syringe. It has been determined that the cytoplasmic injection of cells usually results in the introduction of 10-13 to 10-14 liter per cell. 6'8 Control injections should be done to determine the specificity of the effects monitored. In the case of microinjection of antibodies, control injections may include heat-denatured antibody, nonspecific antibody injected at the same concentration as the specific antibody, and buffer alone.
Analysis of Injected Cells The analytical potential of microinjection experiments depends on two parameters: (1) the ability to positively identify injected cells and (2) the availability of a single cell assay that can be monitored visually. As for the first parameter, the immunofluorescence technique is ideal for the identification of injected cells. The staining procedures for immunofluorescence are detailed below (Immunocytochemical Localization of PLA2). Following fixation and permeabilization of cells, injected rabbit anti-PLAz antibodies can be readily detected by staining with fluorescein-conjugated goat anti-rabbit IgG (Fig. 3). Antibody polypeptides injected at 5-10 mg/ml can be detected in cells up to 48 hr after injection. A summary of the results obtained in microinjection experiments using anti-PLAz antibodies (Table I) provides examples for cellular responses that can be measured at the single cell level. In each of the studies described, the observed effects were dose dependent and were not detected in control injections. 8 D. W. Stacey and V. G. Allfrey, Cell (Cambridge, Mass.) 9, 729 (1976).
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Immunocytochemical Localization of PLA 2 PLA 2 has been solubilized from several tissues by sonication,l indicating that the enzyme is not firmly bound to membrane structures. This makes it difficult to determine the cellular localization of the enzyme solely on the basis of biochemical fractionation. This section describes immunohistochemical techniques to determine the spatial aspects of PLA 2 distribution.
Preparation of Cellsfor Labeling It is most convenient to grow cells on coverslips which can then be processed and mounted on slides for observation. In the case of cells that adhere poorly to glass, adhesion and cell growth can be improved by cleaning the coverslips with chromic acid or coating the surface with a positively charged polymer. Cell density can influence cell morphology and may affect the quality of the signal, especially in high-resolution fluorescence microscopy. Cell debris adsorb antibodies nonspecifically. For these reasons it is suggested that standard conditions for culture labeling be established. Prior to staining, it is necessary to fix the cells in order to preserve structure and then permeabilize them to allow labeling reagents to reach intracellular targets. A large number of fixation procedures have been described in the literature, with varying degrees of preservation of antigenic site and cellular morphology. Details of those procedures that were optimal for anti-PLA 2 antibody generated in our laboratory are described here.
Indirect Immunofluorescence Cells plated on glass coverslips are washed twice with PBS and fixed at 20° for 30 min in 3.7% formaldehyde in PBS. Coverslips are rinsed twice with PBS, and cells are permeabilized by exposure to 0.2% Triton X-100 (Sigma) in PBS for 30 sec at 20°. The affinity-purified rabbit anti-PLAz antibody is used at a 1:50 dilution (20 /zg/ml) in PBS containing
Fro. 3. Detection of microinjected anti-PLA2 antibodies in rat embryo fibroblasts. Four cells in the field shown were injected with the antibody solution (5 mg/ml). At 5 hr postinjection, the cells were fixed, permeabilized, and stained with fluorescein-conjugated goat anti-rabbit IgG. (A) Fluorescence micrograph; (B) corresponding phase micrograph. Bar, 10/zm.
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TABLE I EFFECTS OF MICROINJECTIONOF ANTI-PEA2 ANTIBODIES
Cell type ras-Transformed
fibroblasts Normal fibroblasts (rat kidney or rat embryo cells) Rat peritoneal mast ceils Endothelial cells
Concentration of solution injected (mg/ml) 5 10
5 1
Assay
Results
Ref. °
Morphological reversion (cell flattening) Serum- or growth factorinduced stimulation of DNA synthesis b Ligand-induced exocytotic degranulation c Thrombin-induced sustained increase in cytosolic calcium d
+
1
No effect
2
-
3
-
4
Key to references: (1) D. Bar-Sagi, in "Cell Activation and Signal Initiation: Receptor and Phospholipase Control of Inositol Phosphate, PAF and Eicosanoid Production," p. 331. UCLA Symposium, Alan R. Liss, New York, 1989; (2) D. Bar-Sagi, unpublished observations (1988); (3) L. Graziadei and D. Bar-Sagi, in preparation (1990); (4) M. S. Goligorsky, D. N. Menton, A. Laszlo, and H. Lum, J. Biol. Chem. 264, 16771 (1989). b DNA synthesis is measured by [3H]thymidine incorporation and emulsion autoradiography. [3H]Thymidine (1/xCi/ml) is added to the medium within 1 hr after injection, and cells are further incubated for 12-24 hr. At the end of the incubation period the cells are fixed, and the dishes are coated with Nuclear Track Emulsion (NTB-2, Kodak) and processed for emulsion autoradiography. c Degranulation is monitored by light microscopic visualization of the extrusion of granules [D. Bar-Sagi and B. Gomperts, Oncogene 3, 463 (1988)]. a Concentration of cytosolic calcium in individual cells was monitored using the calcium indicator dye Fura-2.
0.5 mg/ml BSA. It should be noted, however, that the optimal concentration of the primary antibody varies widely, depending on titer and affinity, and therefore should be determined empirically. Incubation with the primary antibody is for 1 hr at 37 ° in a humidified atmosphere. Following two 10-min washes in PBS, cells are incubated with fluorescein-conjugated goat anti-rabbit antibody (Cappel) diluted 1 : 100 (10/xg/ml) in PBS containing 0.5 mg/ml BSA. Secondary antibody incubation is for I hr at 37 ° in a humidified atmosphere followed by two 10-min PBS washes and a distilled water rinse before mounting with Gelvatol (Monsanto Polymers and Petrochemicals Co., St. Louis, MO). 9 Reagents which slow down the photobleaching of fluorescein (N-propyl gallate or p-phenylenediamine) are generally added to the mounts. Before use, the secondary antibodies are 9 j. R. Feramisco and S. H. Blose, J. Cell Biol. 86, 608 (1980).
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preadsorbed with methanol-fixed NRK (normal rat kidney) monolayers and clarified using a Beckman Airfuge to reduce nonspecific stickiness. Cells are examined with a Zeiss photomicroscope III equipped for epifluorescence. Phase micrographs are recorded using Kodak (Rochester, NY) Technical Pan film, and fluorescence micrographs are recorded on Kodak Tri-X film. Controls are essential for interpreting immunofluorescence staining patterns. Some useful controls are the following: (1) omitting primary antibody from the staining procedure, (2) staining with nonimmune IgG, and (3) staining with specific IgG preadsorbed with antigen. All controls should give no specific staining pattern and be significantly dimmer than the experimental samples. It is particularly important to perform all controls in pilot experiments or whenever a new batch of antibodies or different cells are utilized.
Immunoelectron Microscopy Analysis of the cellular distribution ofPLA/at the electron microscopic level is extremely informative and should complement the analysis at the light microscopy level. A discussion of immunoelectron microscopy techniques, however, is beyond the scope of this chapter. In general, the preparation time for samples may take up to 1 week, and then the interpretation of results often calls for the assistance of an experienced electron microscopist. An important prerequisite for a meaningful ultrastructural analysis is a good preservation of cellular structures. However, good fixatives, while preserving fine structures, tend to alter or destroy antibody binding sites as well as to decrease accessibility of probes. Therefore, the effects of several different fixation procedures on the labeling pattern should be compared. Two staining methods can be employed: (I) indirect immunoperoxidase staining which utilizes peroxidase-conjugated secondary antibodies and (2) immunogold labeling which utilizes secondary antibodies attached to gold particles. The latter offers the possibility of using gold particles of different sizes (5-20 nm), thus allowing for the simultaneous labeling of two antigens in order to assess their spatial relationships. Acknowledgments We thank Madeline Wisnewski for secretarial assistance. This work was supported by National Institutes of Health Grant CA46370 and American Cancer Society Grant BC690.
