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[45] L o c a l i z a t i o n o f P e p t i d e G r o w t h F a c t o r s in t h e N u c l e u s By
BRUNO GABRIEL, V~RONIQUE BALDIN, ANNA MARIA ROMAN, ISABELLE BOSC-BIERNE, JACQUELINE NOAILLAC-DEPEYRE, HERV~ PRATS, JUSTIN TEISSIi~, GI~RARD BOUCHE, a n d FRAN¢~OIS AMALRIC
Introduction Work in several laboratories has shown that various peptide hormones and growth factors are associated with nuclei of target cells. This has usually been demonstrated by biochemical or immunocytologicai detection of the hormone or growth factor in the nucleus. For platelet-derived growth factor (PDGF) and related molecules, ~'2 the signal for nuclear targeting has also been identified. The accumulated data have been largely ignored because they do not fit with the current concept that all endocytosed polypeptides are destroyed by lysosomes, while the signal transduction is carried out by second messengers. Nuclear localization of peptide hormones and growth factors requires the existence of an alternative transport pathway, resulting in delivery of the polypeptide to the nucleus rather than to lysosomes. In addition, nuclear localization suggests that these polypeptides may exert some of their biological effects directly at the nuclear level. 3'4 In this chapter, we describe the procedures used to demonstrate the localization of basic fibroblast growth factor (bFGF) in the nucleus and the nucleolus of proliferative primary cultures of adult bovine aortic arch endothelial (ABAE) cells; b F G F is mitogenic for a wide variety of mesoderm- and neuroectoderm-derived cells) The methodologies consist of the use of radioiodinated growth factors followed by cellular fractionation and utilization of immunocytochemistry and indirect immunofluorescence techniques. Because of the low level of fluorescence, a video-enhanced fluorescence microscope coupled to a digitized image processor 6 is used to quantify fluorescein-conjugated anti-FGF antibodies at the single cell level and to compare fluorescence intensities in the various domains of the cell I B. A. Lee, D. W. Maher, M. Hannink, and D. Donoghue, Mol. Cell. Biol. 7, 3527 (1987). 2 D. W. Maher, B. A. Lee, and D. Donoghue, Mol. Cell. Biol. 9, 2251 (1989). 3 S. J. Burwen and A. L. Jones, Trends Biochem. Sci. 12, 159 (1987). 4 G. Bouche, N. Gas, H. Prats, V. Baldin, J. P. Tauber, J. Teissi6, and F. Amalric, Proc. Natl. Acad. Sci. U.S.A. 84, 6770 (1987). 5 D. Gospodarowicz, G. Neufeld, and L. Schweigerer, Cell Differ. 19, 1 (1986). 6 S. lnou6, "Video Microscopy." Plenum, New York, 1986.
METHODS IN ENZYMOLOGY, VOL. 198
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or between different cells. Changes in both time and space can be readily determined by mathematical image processing. Cell System and Measurement of DNA Synthesis Adult bovine aortic endothelial (ABAE) cells are established from the aortic arch according to Gospodarowicz et al. 7 Quiescent sparse endothelial cells are obtained as follows: endothelial cells are seeded at low density [105 cells per 10 cm diameter plastic petri dishes (Nunc, Roskilde, Denmark)] in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% calf serum (Intermed, Roskilde, Denmark) and 1 ng/ml bFGF, and routinely cultured at 37 ° (10% CO2 atmosphere). After 72 hr, the cells are washed twice with serum-free DMEM supplemented with transferrin (10 tzg/ml) (Sigma, St. Louis, MO), and cultures are continued in the same medium for 48 hr. At this point, these primary cell cultures are arrested in the GI phase of the cell cycle. The GI --* mitosis transition is obtained by stimulation of quiescent cells with serum-free DMEM containing only bFGF (5 ng/ml). Synchronously growing ABAE cells provide a good system to study the influence of different bFGF preparations on cell growth, bFGF purified from bovine pituitaries 8 or human recombinant bFGF is used for the experiments. These two bFGF preparations correspond to the 146 amino acid form. For measurement of DNA synthesis, cells undergoing the Gj --~ mitosis transition are pulse-labeled for 15 rain with [methyl-3H]thymidine (Amersham; 10 ~Ci/ml; 47.5 Ci/mmol) at different times after bFGF stimulation. The rate of DNA synthesis is measured by cell counting and determination of the [3H]thymidine incorporated into trichloroacetic acid-insoluble material. 9 Indirect Immunofluorescence Preparation o f Antibodies Female rabbits (1.5 kg) are immunized with 100/~g of human recombinant bFGF in 1 ml of sterile phosphate-buffered saline (PBS; 0.15 M NaCI, 10 mM sodium phosphate buffer, pH 7.5) diluted 1 : 1 in Freund's complete adjuvant by dorsal subcutaneous injection at multiple sites.~° The rabbits 7 D. G. Gospodarowicz, J. Moran, D. Braun, and C. R. Birdwell, Proc. Natl. Acad. Sci. U.S.A. 73, 4120 (1976). 8 D. Gospodarowicz, this series, Vol, 147, p. 106. 9 F. Amalric, M. Nicoloso, and J. P. Zalta, FEBS Lett. 22, 62 (1972). to j. L. Vaitukaitis, this series, Vol. 73, p. 46.
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are boosted at 2- and 4-week intervals by intradermal injection of 50/xg of bFGF diluted in Freund's incomplete adjuvant. Two weeks after the last booster injection, rabbits are bled every 2 weeks. The serum is stored frozen at - 7 0 °. Preimmune serum is obtained before immunization of the rabbits.
Antibody Screening An enzyme-linked immunoabsorbent assay (ELISA) has been developed for detection of anti-human bFGF antibodies. A 96-well polystyrene microtiter plate (Nunc) is coated with 50/zl of purified human bFGF (0.5 txg/ml of PBS) and left for 16 hr at 4 °. Wells are washed 3 times with PBS containing 0.1% Tween 20 (PBS/Tween) and filled with PBS/Tween (2 hr at 20°) to saturate the remaining protein binding sites. Plates are then incubated for 2 hr at 37 ° with 100 ~1 of serial dilutions of antiserum in PBS/ Tween. Wells are washed with PBS/Tween (3 times) and incubated for an additional 2 hr at 37° with 100/xl alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega Biotec, Madison, WI). Following this incubation, wells are washed as above, and 100 t~l of I mg/ml p-nitrophenyl phosphate (Sigma) in I M Tris, pH 8.8, is added. Color development is measured after 30 min at 405 nm using a Titertek Multiscan ELISA plate photometer (Flow Lab., Puteaux, France).
Characterization of Affinity-Purified Anti-Human bFGF lgG The IgG fraction of the serum is obtained by chromatography on a protein A-Sepharose column (Pharmacia, Uppsala, Sweden). The bound immunoglobulins are eluted with 0.1 M glycine-HCl, pH 2.5, buffer, and the fractions are neutralized immediately with I M Tris and dialyzed overnight against PBS. Affinity-purified anti-human bFGF IgG is prepared by applying purified IgG to a column of human recombinant bFGF conjugated to agarose beads (Affi-Gei 10; Bio-Rad Laboratories, Richmond, CA) l' or to a column of human recombinant bFGF conjugated to AHSepharose (Pharmacia) according to the manufacturer's instructions. 4 In both cases, affinity-purified antibodies are then eluted with 0.1 M glycineHCI, pH 2.5, neutralized with 1 M K2HPO 4, and extensively dialyzed against PBS. The eluates from the affinity columns are collected and subjected to two additional rounds of chromatography on the bFGF affinity column to prepare anti-human bFGF depleted IgG for use in control studies. tl j. Joseph-Silverstein, S. A. Consigli, K. M. Lyser, and C. Verpault, J. Cell. Biol. 108, 2459 (1989).
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The evaluation of affinity-purified anti-human bFGF IgG is carried out by dot-blot analysis. For dot-blot analysis, purified human bFGF (from 10 to 20 ng) or other unrelated purified proteins are absorbed on a nitrocellulose membrane (0.1 ~m, Schleicher and Schuell, Dassel, Germany) using a dot-blot apparatus (Bio-Rad). The filters are washed in blocking buffer [3% bovine serum albumin (BSA fraction V, pH 7, IBF, Paris, France), 0.15 M NaCI, 10 mM Tris-HCl, pH 7.5] for 2 hr at 20° to saturate additional protein binding sites. These filters are then incubated at 4° overnight with affinity-purified anti-human bFGF IgG or preimmune rabbit lgG in blocking buffer. Subsequently, after extensive washing with blocking buffer, the antibody-treated sheets are incubated for 1 hr at 37° with ~25Ilabeled protein A (Amersham) to detect antigen-antibody complexes, then extensively washed in blocking buffer and put on film for autoradiography. ELISA and dot-blot analyses are two complementary techniques that enable a rapid determination of the sensitivity and the specificity of the antibodies. The antibodies must not recognize antigens other than bFGF in the total extract of the ABAE cells.
Subcellular Localization of bFGF by Immunofluorescence Microscopy Cells that are grown on glass coverslips are fixed by one of the two following methods before immunodetection. Fixation without Permeabilization. Cell monolayers are fixed with 3% paraformaldehyde-PBS for 15 min at 4°, washed with PBS-0.5% BSA (2 times, 5 min each), and then excess paraformaldehyde is removed by treatment with 50 mM NHaCI-PBS at 4° for 20 min. Fixation Followed by Methanol Permeabilization. The cell monolyaers are incubated with absolute methanol at 20° for 2 min, then washed twice with PBS and twice with PBS-0.5% BSA at 4°.
lmmunodetection Coverslips treated as above are then incubated at 37° for 1 hr with the affinity-purified anti-human bFGF lgG diluted to 60/~g/ml in PBS-0.5% BSA. In all cases, nonimmune rabbit lgG is included at the same dilution as controls. After 3 washes with PBS-0.5% BSA, cells are further incubated for 1 hr at 37° with fluorescein-conjugated goat anti-rabbit IgG (Nordic Immunological Laboratories, Lausanne, Switzerland) diluted 1 : 80 according to the manufacturer's instructions. Finally, the coverslips are extensively washed with PBS-0.5% BSA (2 times, 5 min each) and with PBS alone (2 times, 5 min), mounted on glass slides, and examined in a Leitz Ortholux II microscope equipped for epifluorescence with a 100-W mercury lamp. Micrographs are obtained after an exposure of 2-4
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min (Kodak Timax 400 ASA, Rochester, NY) (Fig. 1). In parallel, the nucleolar compartment is characterized using IgG raised against nucleolin, 4 a nucleolar-specific antigen. To rule out the possibility that reorganization of the nucleus and nucleolus after bFGF stimulation is responsible for nonspecific trapping of antibody within these organelles, controls are performed with an unrelated antibody (e.g., antitubulin) using an identical technique. 4 Fluorescence Localization and Quantification
Digitized Video Microscopy Figure 2 shows a diagram of the imaging system used in our laboratory. Cells are observed under a Leitz fluorescence inverted microscope. The light source for fluorescence observations is an HBO 100W2 (Osram, Munich, Germany), and wavelengths are selected by a Leitz H3 filter block. Two video monitoring setups are connected to the microscope, a charge coupled device (C. C.D.) camera (Panasonic, G saka, Japan) associated with a color monitor (Sony, Fellbach, Germany) for direct or phasecontrast observations (high light level) and a light-intensifying camera (Lhesa, Pontoise, France) associated with a black-and-white monitor (RCA, Lancaster, PA) for fluorescence observations (low light level). The two video setups are connected by means of a selector to a digitizer (Info'Rop, Toulouse, France) driven by a computer (CPU 68010, Motorola, Tempe, AZ). In this way, the video signal is converted to a matrix with a 8-bit gray scale, namely, 256 different light levels. The size of this matrix is selected to be 256 x 256 to shorten computer calculation times. The software library (Trimago, Ifremer, Paris, France) contains the major routines for digital image processing. The following peripherals are connected to the computer: a hard disk (85 Mo) to store the matrix, a color monitor (Sony) on which the resulting images and associated graphs are displayed, a color printer (Canon, Le Blanc-Mesnil, France) for printing color graphs, and another printer (Epson, Levallois-Perret, France) to print the data (subroutines used or numerical pixel values).
Digital Image Processing The first step of the analysis is the selection of cells and their digitalization. Images which quantify the dark level and illumination heterogeneity are digitalized. 1. Glass coverslips of fixed cells are mounted on glass slides and observed by fluorescence under oil immersion with a magnification of 63 (Leitz Wetzlar objective)
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~z
i
FIG. 1. Indirect immunofiuorescence staining of exponentially growing cells. (B, C, E, F) Staining with affinity-purified anti-bFGF lgG. (H) Control with preimmune rabbit IgG. (A, D, G) Corresponding phase-contrast photos. Magnifications: B, E, × 220; C, F, × 660. (B, C) Fixation without permeabilization. Exponentially growing ABAE cells in the presence of bFGF (5 ng/ml culture medium) show low diffuse cytoplasmic staining with intensive
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MICROSCOPE
i
i
CAMERA
CAMERA
i
i I
s~cro~ t
I
DIGITALIZER
8 bits MEMORY 128 x 256 x 256 x 8 bits
I
f] TR,MAOO68010ceuSOf [,,~ tware (Fortran)
I KEYBOARD
It
INTERACTIVE [ KEYBOARD
HARD DISK
!
color PRINTER
FiG. 2. Diagram of the digitalizing microscopy video station.
fluorescence located around the nucleus. Nuclei and nucleoli appear barely stained. (E, F) Fixation followed by methanol permeabilization. Under these conditions, most of the cytoplasmic label is lost during permeabilization, which allows the detection of bFGF in the nucleus and in the nucleolus.
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2. The gain of the light-intensifying camera is fixed with a manual adjustment. Thus, the light levels are directly comparable between each digitalized image after mathematical correction. The gain is set so as to avoid saturation of the camera and to keep the signal arising from unlabeled cell intrinsic fluorescence at the background level of detection. 3. Isolated cells are selected by microscope observation and then digitized. 4. For each selected cell, the same image is digitalized 12 times with a frequency of 1 image/sec. 5. All cell images are obtained by averaging the 12 uncorrected images in order to improve the signal-to-noise ratio (Wiener's law). 6. Several images of both labeled and unlabeled cells are stored. 7. In the same way, the image corresponding to the blank level of the camera (Ib), that is, fluorescence observation with no object between the light source and the camera, is digitalized and stored. 8. A reference illumination map (Im) is obtained by fluorescence observation of a slide with a fluorescein-conjugate dye solution and is digitalized by the same technique.
Mathematical Image Processing The aim of the mathematical treatment u is to compare fluorescence levels between each cell compartment or between different cells. Mathematical corrections are carried out in order to reduce or eliminate the contribution of noise to the light level. Just after the digitalization step, the encoded light level value (If) for a point M of coordinates x and y, is given by the following equation:
lf(x,y) = KC(x,y)lm(x,y) + lb(x,y) where K is a constant which depends on the apparatus and adjustments (manual gain value), C(x,y) the concentration of fluorescent dye at the point M, Im(x,y) the incident light level (illumination heterogeneity), and Ib(x,y) the blank light level (dark level). Thus, after correction for illumination and background noise, a direct relationship between light levels and dye concentration may be obtained. !. A mathematical enlargement with linear extrapolation or with duplication and suppression may be used if cells cover less than 50% of the image area. In this way, the computer transforms an image area of 128 × 128 to an image with a size of 256 x 256. The mathematical method effectively suppresses areas containing little information, such as regions where no cells are present. 12 K. R. Castleman, "'Digital Image Processing." Prentice-Hall, Englewood Cliffs, New Jersey, 1979.
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2. If a cell image is transformed by a mathematical enlargement, it is important to process the background image (Ib) and the illumination map (Im) in the same way. For each cell analysis, three equally enlarged images are produced which represent the cell (li), the background (Ibi), and the illumination map (lmi). 3. The image (Ibi) is subtracted from each image (li) and (lmi) to allow correction for the background: lil = Ii - Ibi
and
lmil = Imi - Ibi
4. Each corrected image (lid is divided by its associated illumination map (Imil) to correct for heterogeneity in incident light level and camera sensitivity Ii2 = l i l / I m i l 5. Contrast in images may be improved by spreading their pixel values from 0 to 255 using a Look Up Table (L.U.T.). The same L.U.T. must be used for all images. After such processing, all the pixel values are directly related to the local concentration of dye. Figure 3a-c shows differences between initial (li) and processed (Ii2) cell images. Image Analysis Fluorescence Localization. Several routines are used to analyze the fluorescence in the cells: (1) histograms which represent the distribution of pixel light levels characterizing the change in fluorescence in each cell; (2) horizontal or vertical line scans which depict unidirectional (1D) light level distributions allowing observation of the fluorescence compartmentation in the cell (Fig. 3d shows such a representation indicating relative fluorescence levels of the different cell compartments, e.g., cytoplasm, nucleus, nucleolus); (3) Pseudocolor fluorescence intensity maps which display dye distribution in the cells. Fluorescence Quantification. Using pseudocolor maps, it is also possible to quantify the fluorescence. The total fluorescence intensity F(r) of an area may be represented by F(r) = f ~ l(x,y) dx dy x
(1)
y
where l(x,y) is the fluorescence level of a point M of coordinates x and y. This equation assumes that digitalized fluorescence images are representative of a planar concentration of dyes, that is, the microscope field depth is null or independent of z. Equation (1) may be written as
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FIG. 3. Image contrast improvement through data processing. (a) Original image; (b) enlargement through pixel expansion of the region of interest; (c) image b processed through background subtraction, shading distorsion correction, and contrast linear enhancement; (d) ID light level distribution along the indicated horizontal line. Image d indicates a strong accumulation of the fluorescent antibodies in the nucleolus.
F
=
NI o
(2)
where N is the total number of pixels included in the area, and I o the average value of fluorescence in the area. This fluorescence quantification gives information on the number of dye molecules owing to the linear relationship between fluorescence level and dye concentration in the final corrected cell images. 1. The cells that are observed under fluorescence and digitalized are subsequently observed using phase contrast. An image of each cell is digitalized. 2. Each cell subcompartment surface is quantified by the corresponding number of pixels. 3. From fluorescence-corrected images, the average and the standard error of the pixel values are calculated for each fluorescent area.
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4. Quantification is then obtained by multiplying the number of pixels by the average pixel value for the different cell areas. The main advantage of this video technique is that pixel light levels, and thus the number of dye molecules, may be compared in the various domains of the cell or between different cells. Ultrastructural Immunocytochemistry Exponentially growing cells are fixed at 4 ° in 0.1% glutaraldehyde and 2% formaldehyde in PBS (pH 7.5, for 15 min). The cells are treated with 10 mM sodium borohydride, dehydrated in ethanol, and embedded in Lowicryl K4M, using the low temperature procedure.13 Ultrathin sections are placed on nickel grids and preincubated for 30 rain at 20 ° with normal goat serum at 10% to block the nonspecific sites of protein absorption. Excess liquid is removed, and the grids are incubated overnight at 4 ° with affinity-purified anti-bFGF IgG (diluted at 1 : 1000) in PBS (10% BSA). The grids are washed and incubated for 1 hr at 20° with goat anti-rabbit IgG coupled to colloidal gold (GAR 15, Jansen Life Science, Beerse, Belgium) diluted 1 : 20 in 20 mM Tris-HCl buffer, pH 8.2, 0.5% BSA. After several washes with Tris-HCl buffer, the grids are rinsed with doubledistilled water and dried. The sections are contrasted with uranyl acetate and viewed in a Jeol JEM 200 CX electron microscope at 80 KV. Control sections from the same resin block are incubated only with PBS and preimmune IgG (Fig. 4). Biochemical Localization of bFGF in ABAE Cells Iodinated bFGF is utilized to provide biochemical support for the microscopic observations and to quantify the amount of growth factor accumulated in each cellular compartment. lodination of bFGF Purified bFGF is radioiodinated using the chloramine-T method. 14 Three micrograms of bFGF in 10/zi of 50 mM sodium phosphate buffer, pH 7.2, is added to 1 mCi of NaJ25I (Amersham) and 10/zl of a freshly prepared solution of chloramine-T (Sigma) (5 mM in 50 mM sodium phosphate buffer, pH 7.2). After I min at 20° the reaction is stopped by adding 20/zl of 5 mM sodium bisulfite. Free Na125I is separated from ~25I-labeled bFGF by chromatography on Sephadex G-25 equilibrated with 50 mM 13 H, Yeh, G. F. Pierce, and T. M. Deuel, Proc. Natl. Acad. Sci. U.S.A. 84, 2317 11987). ~4 W. M. H u n t e r and F. C. G r e e n w o o d , Nature (London) 194, 495 (1962).
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TABLE 1 INTRACELLULAR DISTRIBUTION OF 125I-LABELED bFGF IN GROWING AND STIMULATED CELLS Nucleus
Cytoplasm
Cells
Amount (pg)"
Molecules ~
Amount (pg)"
Molecules ~
Growing c Stimulated c
55 _+ 10 70 -+ 12
1800 2400
610 _+ 53 706 -+ 45
20,400 24,000
" Values (picograms per 106 cells) calculated from the specific activity of radiolabeled bFGF are the means -+ S.E.M. from four experiments. b The number of bFGF molecules per subcellular fraction corresponding to one cell was calculated using Avogadro's number and the specific activity of bFGF. " Growing cells refer to an asynchronous primary culture of sparse cells; stimulated cells. G1-arrested cells stimulated to growth (G~ --~ S) by the addition of bFGF.
sodium phosphate buffer, pH 7.2, containing 0,25% BSA. The analysis of 125I-labeled bFGF by SDS-PAGE (15% acrylamide) followed by autoradiography reveals a single band at 18.4 kDa. The specific activity of ~zsIlabeled bFGF in this experiment is 50,000 to 150,000 counts/min (cpm)/ ng. ~25I-Labeled bFGF has the same mitogenic activity as unlabeled bFGF. Cell Fractionation
Exponentially growing ABAE cells are stimulated for 2 hr with ~25Ilabeled bFGF, 5 ng/ml of culture medium. At this time, cells are washed 3-5 times with PBS and harvested by trypsinization, with 0.05% trypsin (Intermed), 0.025% EDTA in PBS at 4° over 10 min. The trypsin solution is discarded, and the cells are harvested in 5 ml of DMEM-10% calf serum (Intermed) and then centrifuged for 10 min (400 g at 4°). All the cell fractionation steps are carried out at 4 °. Cells (2 × l06) are disrupted for 15-30 sec (Ultra Turrax, Janke and Hunkel, Staufen, Germany) in 10 ml of medium A (0.3 M sucrose, 60 mM KCI, 15 mM NaC1, 1.5 mM spermine, 0.5 mM spermidine, 14 mM 2-mercaptoethanol, 5 mM EGTA, 2 mM
FIG. 4. Ultrastructural localization of bFGF in the nucleolus of exponentially growing ABAE cells by immunogold staining. (A) Immunocytochemical localization of bFGF with affinity-purified anti-bFGF lgG. (B) Control with preimmune rabbit lgG. Nu, Nucleolus; Np, nucleoplasm; Cy, cytoplasm; FC, fibrillar center; F and G are, respectively, fibrillar and granular components. (A) The gold particles show some enrichment around the fibrillar center in dense fibrillar components and are also scattered throughout the granular components. Signifcant labeling is also observed in the nucleoplasmic network. (13) Low background is observed with preimmune IgG.
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2 b a
3 ba
4 ba
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5 b
FIG. 5. Nuclear and cytoplasmic localization of 1251-1abeled bFGF in ABAE cells. Two hours after the addition of i251-1abeled bFGF (5 ng/ml), ABAE cells were harvested and fractionated into nuclear (a) and cytoplasmic (b) fractions. Total nuclear proteins corresponding to 106 cells and 1/10 of the cytoplasmic proteins were then analyzed on a 15% SDS-polyacrylamide slab gel. After protein fixation and Coomassie blue staining (lanes 1), gels were dried and exposed for 24 hr for autoradiography. Lanes 2 show Gi-arrested ABAE cells stimulated by t251-1abeled bFGF; lanes 3 show exponentially growing cells. For lanes 4, a control experiment, t25I-labeled bFGF (5 × 104 cpm) was added to disrupted ABAE cells. Cellular fractionation and analysis of proteins were carried out as described in the text. For lanes 5, an aliquot of J251-1abeledbFGF recovered in the culture medium after stimulation of the cells was analyzed as described above, i25I-labeled bFGF is detected in nucleus and cytoplasm both in exponentially growing cells and in synchronized cells undergoing the G~ S transition, lz51-Labeled bFGF is detected in essentially full length form (18.4 kDa) in the nucleus, whereas in the cytoplasm two molecular forms of the growth factor (18.4 and 16.5 kDa) are found in an identical molar ratio. As shown in lanes 4, J251-1abeled bFGF is not detected in nuclei, and little degradation of growth factor occurs during cell fractionation when the growth factor is added to disrupted cells.
EDTA, 20 mM HEPES, pH 7.5) containing a cocktail of protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF), 10 /~g/ml of leupeptin, 0.02% NPG (Cemulsol SFOS, Persan, France), and Trasylol] and centrifuged at 800 g for 5 min. The supernatant represents the crude cytoplasmic fraction. The nuclear pellet is washed twice in medium A. The purity and integrity of nuclei preparations are monitored by microscopy under phase contrast. The radioactivity of ~25I-labeled growth factor bound to the particular fractions is measured in a y counter, or using y-vials and a liquid scintilla-
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tion counter. The number of bFGF molecules bound is calculated from Avogadro's number and the specific activity of the bFGF (Table I). Aliquots of crude cytoplasmic fraction and nuclear pellets are precipitated by 15% trichloroacetic acid (30 min, 4 °) and centrifuged at 15,000 g for 30 min. The pellets are then dissolved under highly reducing conditions (1 mM Tris-HC1, pH 8, 10 mM EDTA, 2% SDS, 1.5 M 2-mercaptoethanol, 30% glycerol) and analyzed by 15% SDS-PAGE and autoradiography (Fig. 5). In control experiments, the same amount of 125I-labeled bFGF is added to untreated cells in the cell fractionation mixture. When intact nuclei are prepared from control cells, no ~25Ilabeling should be recovered in the nuclear fraction (Fig. 5). Concluding Comments A combination of three methods provides an unambiguous technique for nuclear and subnuclear localization of growth factors in cell cultures. Using synchronously growing ABAE cells stimulated with bFGF it is possible to study the cellular uptake of growth factor during the cell cycle and to quantify the accumulation of growth factor in subcellular compartments.
[46] A n t i p h o s p h o t y r o s i n e A n t i b o d i e s in O n c o g e n e a n d Receptor Research B y D A V I D F . STERN
Introduction Protein-tyrosine kinases regulate cell proliferation and differentiation. These kinases include receptors for several peptide growth factors and many of the oncogene products. The original, and most direct, method for detecting tyrosine phosphorylation of proteins was separation of 3Zp_ labeled amino acids from partial protein hydrolyzates on thin-layer plates. J More recently, antibodies have been developed that react specifically with phosphotyrosine, z-4 Use of these antibodies for immunoprecipitation or i j. A. Cooper, B. M. Sefton, and T. Hunter, this series, Vol. 99, p. 387. 2 A. H. Ross, D. Baltimore, and H. Eisen, Nature (London) 294, 654 (1981). 3 M. Ohtsuka, S. lhara, R. Ogawa, T. Watanabe, and Y. Watanabe, Int. J. Cancer 34, 855 (1984). 4 M. P. Kamps and B. M. Seflon, Oncogene 2, 305 (1988).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[45] L o c a l i z a t i o n o f P e p t i d e G r o w t h F a c t o r s in t h e N u c l e u s By
BRUNO GABRIEL, V~RONIQUE BALDIN, ANNA MARIA ROMAN, ISABELLE BOSC-BIERNE, JACQUELINE NOAILLAC-DEPEYRE, HERV~ PRATS, JUSTIN TEISSIi~, GI~RARD BOUCHE, a n d FRAN¢~OIS AMALRIC
Introduction Work in several laboratories has shown that various peptide hormones and growth factors are associated with nuclei of target cells. This has usually been demonstrated by biochemical or immunocytologicai detection of the hormone or growth factor in the nucleus. For platelet-derived growth factor (PDGF) and related molecules, ~'2 the signal for nuclear targeting has also been identified. The accumulated data have been largely ignored because they do not fit with the current concept that all endocytosed polypeptides are destroyed by lysosomes, while the signal transduction is carried out by second messengers. Nuclear localization of peptide hormones and growth factors requires the existence of an alternative transport pathway, resulting in delivery of the polypeptide to the nucleus rather than to lysosomes. In addition, nuclear localization suggests that these polypeptides may exert some of their biological effects directly at the nuclear level. 3'4 In this chapter, we describe the procedures used to demonstrate the localization of basic fibroblast growth factor (bFGF) in the nucleus and the nucleolus of proliferative primary cultures of adult bovine aortic arch endothelial (ABAE) cells; b F G F is mitogenic for a wide variety of mesoderm- and neuroectoderm-derived cells) The methodologies consist of the use of radioiodinated growth factors followed by cellular fractionation and utilization of immunocytochemistry and indirect immunofluorescence techniques. Because of the low level of fluorescence, a video-enhanced fluorescence microscope coupled to a digitized image processor 6 is used to quantify fluorescein-conjugated anti-FGF antibodies at the single cell level and to compare fluorescence intensities in the various domains of the cell I B. A. Lee, D. W. Maher, M. Hannink, and D. Donoghue, Mol. Cell. Biol. 7, 3527 (1987). 2 D. W. Maher, B. A. Lee, and D. Donoghue, Mol. Cell. Biol. 9, 2251 (1989). 3 S. J. Burwen and A. L. Jones, Trends Biochem. Sci. 12, 159 (1987). 4 G. Bouche, N. Gas, H. Prats, V. Baldin, J. P. Tauber, J. Teissi6, and F. Amalric, Proc. Natl. Acad. Sci. U.S.A. 84, 6770 (1987). 5 D. Gospodarowicz, G. Neufeld, and L. Schweigerer, Cell Differ. 19, 1 (1986). 6 S. lnou6, "Video Microscopy." Plenum, New York, 1986.
METHODS IN ENZYMOLOGY, VOL. 198
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or between different cells. Changes in both time and space can be readily determined by mathematical image processing. Cell System and Measurement of DNA Synthesis Adult bovine aortic endothelial (ABAE) cells are established from the aortic arch according to Gospodarowicz et al. 7 Quiescent sparse endothelial cells are obtained as follows: endothelial cells are seeded at low density [105 cells per 10 cm diameter plastic petri dishes (Nunc, Roskilde, Denmark)] in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% calf serum (Intermed, Roskilde, Denmark) and 1 ng/ml bFGF, and routinely cultured at 37 ° (10% CO2 atmosphere). After 72 hr, the cells are washed twice with serum-free DMEM supplemented with transferrin (10 tzg/ml) (Sigma, St. Louis, MO), and cultures are continued in the same medium for 48 hr. At this point, these primary cell cultures are arrested in the GI phase of the cell cycle. The GI --* mitosis transition is obtained by stimulation of quiescent cells with serum-free DMEM containing only bFGF (5 ng/ml). Synchronously growing ABAE cells provide a good system to study the influence of different bFGF preparations on cell growth, bFGF purified from bovine pituitaries 8 or human recombinant bFGF is used for the experiments. These two bFGF preparations correspond to the 146 amino acid form. For measurement of DNA synthesis, cells undergoing the Gj --~ mitosis transition are pulse-labeled for 15 rain with [methyl-3H]thymidine (Amersham; 10 ~Ci/ml; 47.5 Ci/mmol) at different times after bFGF stimulation. The rate of DNA synthesis is measured by cell counting and determination of the [3H]thymidine incorporated into trichloroacetic acid-insoluble material. 9 Indirect Immunofluorescence Preparation o f Antibodies Female rabbits (1.5 kg) are immunized with 100/~g of human recombinant bFGF in 1 ml of sterile phosphate-buffered saline (PBS; 0.15 M NaCI, 10 mM sodium phosphate buffer, pH 7.5) diluted 1 : 1 in Freund's complete adjuvant by dorsal subcutaneous injection at multiple sites.~° The rabbits 7 D. G. Gospodarowicz, J. Moran, D. Braun, and C. R. Birdwell, Proc. Natl. Acad. Sci. U.S.A. 73, 4120 (1976). 8 D. Gospodarowicz, this series, Vol, 147, p. 106. 9 F. Amalric, M. Nicoloso, and J. P. Zalta, FEBS Lett. 22, 62 (1972). to j. L. Vaitukaitis, this series, Vol. 73, p. 46.
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are boosted at 2- and 4-week intervals by intradermal injection of 50/xg of bFGF diluted in Freund's incomplete adjuvant. Two weeks after the last booster injection, rabbits are bled every 2 weeks. The serum is stored frozen at - 7 0 °. Preimmune serum is obtained before immunization of the rabbits.
Antibody Screening An enzyme-linked immunoabsorbent assay (ELISA) has been developed for detection of anti-human bFGF antibodies. A 96-well polystyrene microtiter plate (Nunc) is coated with 50/zl of purified human bFGF (0.5 txg/ml of PBS) and left for 16 hr at 4 °. Wells are washed 3 times with PBS containing 0.1% Tween 20 (PBS/Tween) and filled with PBS/Tween (2 hr at 20°) to saturate the remaining protein binding sites. Plates are then incubated for 2 hr at 37 ° with 100 ~1 of serial dilutions of antiserum in PBS/ Tween. Wells are washed with PBS/Tween (3 times) and incubated for an additional 2 hr at 37° with 100/xl alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega Biotec, Madison, WI). Following this incubation, wells are washed as above, and 100 t~l of I mg/ml p-nitrophenyl phosphate (Sigma) in I M Tris, pH 8.8, is added. Color development is measured after 30 min at 405 nm using a Titertek Multiscan ELISA plate photometer (Flow Lab., Puteaux, France).
Characterization of Affinity-Purified Anti-Human bFGF lgG The IgG fraction of the serum is obtained by chromatography on a protein A-Sepharose column (Pharmacia, Uppsala, Sweden). The bound immunoglobulins are eluted with 0.1 M glycine-HCl, pH 2.5, buffer, and the fractions are neutralized immediately with I M Tris and dialyzed overnight against PBS. Affinity-purified anti-human bFGF IgG is prepared by applying purified IgG to a column of human recombinant bFGF conjugated to agarose beads (Affi-Gei 10; Bio-Rad Laboratories, Richmond, CA) l' or to a column of human recombinant bFGF conjugated to AHSepharose (Pharmacia) according to the manufacturer's instructions. 4 In both cases, affinity-purified antibodies are then eluted with 0.1 M glycineHCI, pH 2.5, neutralized with 1 M K2HPO 4, and extensively dialyzed against PBS. The eluates from the affinity columns are collected and subjected to two additional rounds of chromatography on the bFGF affinity column to prepare anti-human bFGF depleted IgG for use in control studies. tl j. Joseph-Silverstein, S. A. Consigli, K. M. Lyser, and C. Verpault, J. Cell. Biol. 108, 2459 (1989).
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The evaluation of affinity-purified anti-human bFGF IgG is carried out by dot-blot analysis. For dot-blot analysis, purified human bFGF (from 10 to 20 ng) or other unrelated purified proteins are absorbed on a nitrocellulose membrane (0.1 ~m, Schleicher and Schuell, Dassel, Germany) using a dot-blot apparatus (Bio-Rad). The filters are washed in blocking buffer [3% bovine serum albumin (BSA fraction V, pH 7, IBF, Paris, France), 0.15 M NaCI, 10 mM Tris-HCl, pH 7.5] for 2 hr at 20° to saturate additional protein binding sites. These filters are then incubated at 4° overnight with affinity-purified anti-human bFGF IgG or preimmune rabbit lgG in blocking buffer. Subsequently, after extensive washing with blocking buffer, the antibody-treated sheets are incubated for 1 hr at 37° with ~25Ilabeled protein A (Amersham) to detect antigen-antibody complexes, then extensively washed in blocking buffer and put on film for autoradiography. ELISA and dot-blot analyses are two complementary techniques that enable a rapid determination of the sensitivity and the specificity of the antibodies. The antibodies must not recognize antigens other than bFGF in the total extract of the ABAE cells.
Subcellular Localization of bFGF by Immunofluorescence Microscopy Cells that are grown on glass coverslips are fixed by one of the two following methods before immunodetection. Fixation without Permeabilization. Cell monolayers are fixed with 3% paraformaldehyde-PBS for 15 min at 4°, washed with PBS-0.5% BSA (2 times, 5 min each), and then excess paraformaldehyde is removed by treatment with 50 mM NHaCI-PBS at 4° for 20 min. Fixation Followed by Methanol Permeabilization. The cell monolyaers are incubated with absolute methanol at 20° for 2 min, then washed twice with PBS and twice with PBS-0.5% BSA at 4°.
lmmunodetection Coverslips treated as above are then incubated at 37° for 1 hr with the affinity-purified anti-human bFGF lgG diluted to 60/~g/ml in PBS-0.5% BSA. In all cases, nonimmune rabbit lgG is included at the same dilution as controls. After 3 washes with PBS-0.5% BSA, cells are further incubated for 1 hr at 37° with fluorescein-conjugated goat anti-rabbit IgG (Nordic Immunological Laboratories, Lausanne, Switzerland) diluted 1 : 80 according to the manufacturer's instructions. Finally, the coverslips are extensively washed with PBS-0.5% BSA (2 times, 5 min each) and with PBS alone (2 times, 5 min), mounted on glass slides, and examined in a Leitz Ortholux II microscope equipped for epifluorescence with a 100-W mercury lamp. Micrographs are obtained after an exposure of 2-4
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min (Kodak Timax 400 ASA, Rochester, NY) (Fig. 1). In parallel, the nucleolar compartment is characterized using IgG raised against nucleolin, 4 a nucleolar-specific antigen. To rule out the possibility that reorganization of the nucleus and nucleolus after bFGF stimulation is responsible for nonspecific trapping of antibody within these organelles, controls are performed with an unrelated antibody (e.g., antitubulin) using an identical technique. 4 Fluorescence Localization and Quantification
Digitized Video Microscopy Figure 2 shows a diagram of the imaging system used in our laboratory. Cells are observed under a Leitz fluorescence inverted microscope. The light source for fluorescence observations is an HBO 100W2 (Osram, Munich, Germany), and wavelengths are selected by a Leitz H3 filter block. Two video monitoring setups are connected to the microscope, a charge coupled device (C. C.D.) camera (Panasonic, G saka, Japan) associated with a color monitor (Sony, Fellbach, Germany) for direct or phasecontrast observations (high light level) and a light-intensifying camera (Lhesa, Pontoise, France) associated with a black-and-white monitor (RCA, Lancaster, PA) for fluorescence observations (low light level). The two video setups are connected by means of a selector to a digitizer (Info'Rop, Toulouse, France) driven by a computer (CPU 68010, Motorola, Tempe, AZ). In this way, the video signal is converted to a matrix with a 8-bit gray scale, namely, 256 different light levels. The size of this matrix is selected to be 256 x 256 to shorten computer calculation times. The software library (Trimago, Ifremer, Paris, France) contains the major routines for digital image processing. The following peripherals are connected to the computer: a hard disk (85 Mo) to store the matrix, a color monitor (Sony) on which the resulting images and associated graphs are displayed, a color printer (Canon, Le Blanc-Mesnil, France) for printing color graphs, and another printer (Epson, Levallois-Perret, France) to print the data (subroutines used or numerical pixel values).
Digital Image Processing The first step of the analysis is the selection of cells and their digitalization. Images which quantify the dark level and illumination heterogeneity are digitalized. 1. Glass coverslips of fixed cells are mounted on glass slides and observed by fluorescence under oil immersion with a magnification of 63 (Leitz Wetzlar objective)
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~z
i
FIG. 1. Indirect immunofiuorescence staining of exponentially growing cells. (B, C, E, F) Staining with affinity-purified anti-bFGF lgG. (H) Control with preimmune rabbit IgG. (A, D, G) Corresponding phase-contrast photos. Magnifications: B, E, × 220; C, F, × 660. (B, C) Fixation without permeabilization. Exponentially growing ABAE cells in the presence of bFGF (5 ng/ml culture medium) show low diffuse cytoplasmic staining with intensive
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I
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MICROSCOPE
i
i
CAMERA
CAMERA
i
i I
s~cro~ t
I
DIGITALIZER
8 bits MEMORY 128 x 256 x 256 x 8 bits
I
f] TR,MAOO68010ceuSOf [,,~ tware (Fortran)
I KEYBOARD
It
INTERACTIVE [ KEYBOARD
HARD DISK
!
color PRINTER
FiG. 2. Diagram of the digitalizing microscopy video station.
fluorescence located around the nucleus. Nuclei and nucleoli appear barely stained. (E, F) Fixation followed by methanol permeabilization. Under these conditions, most of the cytoplasmic label is lost during permeabilization, which allows the detection of bFGF in the nucleus and in the nucleolus.
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2. The gain of the light-intensifying camera is fixed with a manual adjustment. Thus, the light levels are directly comparable between each digitalized image after mathematical correction. The gain is set so as to avoid saturation of the camera and to keep the signal arising from unlabeled cell intrinsic fluorescence at the background level of detection. 3. Isolated cells are selected by microscope observation and then digitized. 4. For each selected cell, the same image is digitalized 12 times with a frequency of 1 image/sec. 5. All cell images are obtained by averaging the 12 uncorrected images in order to improve the signal-to-noise ratio (Wiener's law). 6. Several images of both labeled and unlabeled cells are stored. 7. In the same way, the image corresponding to the blank level of the camera (Ib), that is, fluorescence observation with no object between the light source and the camera, is digitalized and stored. 8. A reference illumination map (Im) is obtained by fluorescence observation of a slide with a fluorescein-conjugate dye solution and is digitalized by the same technique.
Mathematical Image Processing The aim of the mathematical treatment u is to compare fluorescence levels between each cell compartment or between different cells. Mathematical corrections are carried out in order to reduce or eliminate the contribution of noise to the light level. Just after the digitalization step, the encoded light level value (If) for a point M of coordinates x and y, is given by the following equation:
lf(x,y) = KC(x,y)lm(x,y) + lb(x,y) where K is a constant which depends on the apparatus and adjustments (manual gain value), C(x,y) the concentration of fluorescent dye at the point M, Im(x,y) the incident light level (illumination heterogeneity), and Ib(x,y) the blank light level (dark level). Thus, after correction for illumination and background noise, a direct relationship between light levels and dye concentration may be obtained. !. A mathematical enlargement with linear extrapolation or with duplication and suppression may be used if cells cover less than 50% of the image area. In this way, the computer transforms an image area of 128 × 128 to an image with a size of 256 x 256. The mathematical method effectively suppresses areas containing little information, such as regions where no cells are present. 12 K. R. Castleman, "'Digital Image Processing." Prentice-Hall, Englewood Cliffs, New Jersey, 1979.
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2. If a cell image is transformed by a mathematical enlargement, it is important to process the background image (Ib) and the illumination map (Im) in the same way. For each cell analysis, three equally enlarged images are produced which represent the cell (li), the background (Ibi), and the illumination map (lmi). 3. The image (Ibi) is subtracted from each image (li) and (lmi) to allow correction for the background: lil = Ii - Ibi
and
lmil = Imi - Ibi
4. Each corrected image (lid is divided by its associated illumination map (Imil) to correct for heterogeneity in incident light level and camera sensitivity Ii2 = l i l / I m i l 5. Contrast in images may be improved by spreading their pixel values from 0 to 255 using a Look Up Table (L.U.T.). The same L.U.T. must be used for all images. After such processing, all the pixel values are directly related to the local concentration of dye. Figure 3a-c shows differences between initial (li) and processed (Ii2) cell images. Image Analysis Fluorescence Localization. Several routines are used to analyze the fluorescence in the cells: (1) histograms which represent the distribution of pixel light levels characterizing the change in fluorescence in each cell; (2) horizontal or vertical line scans which depict unidirectional (1D) light level distributions allowing observation of the fluorescence compartmentation in the cell (Fig. 3d shows such a representation indicating relative fluorescence levels of the different cell compartments, e.g., cytoplasm, nucleus, nucleolus); (3) Pseudocolor fluorescence intensity maps which display dye distribution in the cells. Fluorescence Quantification. Using pseudocolor maps, it is also possible to quantify the fluorescence. The total fluorescence intensity F(r) of an area may be represented by F(r) = f ~ l(x,y) dx dy x
(1)
y
where l(x,y) is the fluorescence level of a point M of coordinates x and y. This equation assumes that digitalized fluorescence images are representative of a planar concentration of dyes, that is, the microscope field depth is null or independent of z. Equation (1) may be written as
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FIG. 3. Image contrast improvement through data processing. (a) Original image; (b) enlargement through pixel expansion of the region of interest; (c) image b processed through background subtraction, shading distorsion correction, and contrast linear enhancement; (d) ID light level distribution along the indicated horizontal line. Image d indicates a strong accumulation of the fluorescent antibodies in the nucleolus.
F
=
NI o
(2)
where N is the total number of pixels included in the area, and I o the average value of fluorescence in the area. This fluorescence quantification gives information on the number of dye molecules owing to the linear relationship between fluorescence level and dye concentration in the final corrected cell images. 1. The cells that are observed under fluorescence and digitalized are subsequently observed using phase contrast. An image of each cell is digitalized. 2. Each cell subcompartment surface is quantified by the corresponding number of pixels. 3. From fluorescence-corrected images, the average and the standard error of the pixel values are calculated for each fluorescent area.
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4. Quantification is then obtained by multiplying the number of pixels by the average pixel value for the different cell areas. The main advantage of this video technique is that pixel light levels, and thus the number of dye molecules, may be compared in the various domains of the cell or between different cells. Ultrastructural Immunocytochemistry Exponentially growing cells are fixed at 4 ° in 0.1% glutaraldehyde and 2% formaldehyde in PBS (pH 7.5, for 15 min). The cells are treated with 10 mM sodium borohydride, dehydrated in ethanol, and embedded in Lowicryl K4M, using the low temperature procedure.13 Ultrathin sections are placed on nickel grids and preincubated for 30 rain at 20 ° with normal goat serum at 10% to block the nonspecific sites of protein absorption. Excess liquid is removed, and the grids are incubated overnight at 4 ° with affinity-purified anti-bFGF IgG (diluted at 1 : 1000) in PBS (10% BSA). The grids are washed and incubated for 1 hr at 20° with goat anti-rabbit IgG coupled to colloidal gold (GAR 15, Jansen Life Science, Beerse, Belgium) diluted 1 : 20 in 20 mM Tris-HCl buffer, pH 8.2, 0.5% BSA. After several washes with Tris-HCl buffer, the grids are rinsed with doubledistilled water and dried. The sections are contrasted with uranyl acetate and viewed in a Jeol JEM 200 CX electron microscope at 80 KV. Control sections from the same resin block are incubated only with PBS and preimmune IgG (Fig. 4). Biochemical Localization of bFGF in ABAE Cells Iodinated bFGF is utilized to provide biochemical support for the microscopic observations and to quantify the amount of growth factor accumulated in each cellular compartment. lodination of bFGF Purified bFGF is radioiodinated using the chloramine-T method. 14 Three micrograms of bFGF in 10/zi of 50 mM sodium phosphate buffer, pH 7.2, is added to 1 mCi of NaJ25I (Amersham) and 10/zl of a freshly prepared solution of chloramine-T (Sigma) (5 mM in 50 mM sodium phosphate buffer, pH 7.2). After I min at 20° the reaction is stopped by adding 20/zl of 5 mM sodium bisulfite. Free Na125I is separated from ~25I-labeled bFGF by chromatography on Sephadex G-25 equilibrated with 50 mM 13 H, Yeh, G. F. Pierce, and T. M. Deuel, Proc. Natl. Acad. Sci. U.S.A. 84, 2317 11987). ~4 W. M. H u n t e r and F. C. G r e e n w o o d , Nature (London) 194, 495 (1962).
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TABLE 1 INTRACELLULAR DISTRIBUTION OF 125I-LABELED bFGF IN GROWING AND STIMULATED CELLS Nucleus
Cytoplasm
Cells
Amount (pg)"
Molecules ~
Amount (pg)"
Molecules ~
Growing c Stimulated c
55 _+ 10 70 -+ 12
1800 2400
610 _+ 53 706 -+ 45
20,400 24,000
" Values (picograms per 106 cells) calculated from the specific activity of radiolabeled bFGF are the means -+ S.E.M. from four experiments. b The number of bFGF molecules per subcellular fraction corresponding to one cell was calculated using Avogadro's number and the specific activity of bFGF. " Growing cells refer to an asynchronous primary culture of sparse cells; stimulated cells. G1-arrested cells stimulated to growth (G~ --~ S) by the addition of bFGF.
sodium phosphate buffer, pH 7.2, containing 0,25% BSA. The analysis of 125I-labeled bFGF by SDS-PAGE (15% acrylamide) followed by autoradiography reveals a single band at 18.4 kDa. The specific activity of ~zsIlabeled bFGF in this experiment is 50,000 to 150,000 counts/min (cpm)/ ng. ~25I-Labeled bFGF has the same mitogenic activity as unlabeled bFGF. Cell Fractionation
Exponentially growing ABAE cells are stimulated for 2 hr with ~25Ilabeled bFGF, 5 ng/ml of culture medium. At this time, cells are washed 3-5 times with PBS and harvested by trypsinization, with 0.05% trypsin (Intermed), 0.025% EDTA in PBS at 4° over 10 min. The trypsin solution is discarded, and the cells are harvested in 5 ml of DMEM-10% calf serum (Intermed) and then centrifuged for 10 min (400 g at 4°). All the cell fractionation steps are carried out at 4 °. Cells (2 × l06) are disrupted for 15-30 sec (Ultra Turrax, Janke and Hunkel, Staufen, Germany) in 10 ml of medium A (0.3 M sucrose, 60 mM KCI, 15 mM NaC1, 1.5 mM spermine, 0.5 mM spermidine, 14 mM 2-mercaptoethanol, 5 mM EGTA, 2 mM
FIG. 4. Ultrastructural localization of bFGF in the nucleolus of exponentially growing ABAE cells by immunogold staining. (A) Immunocytochemical localization of bFGF with affinity-purified anti-bFGF lgG. (B) Control with preimmune rabbit lgG. Nu, Nucleolus; Np, nucleoplasm; Cy, cytoplasm; FC, fibrillar center; F and G are, respectively, fibrillar and granular components. (A) The gold particles show some enrichment around the fibrillar center in dense fibrillar components and are also scattered throughout the granular components. Signifcant labeling is also observed in the nucleoplasmic network. (13) Low background is observed with preimmune IgG.
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1 a
2 b a
3 ba
4 ba
493
5 b
FIG. 5. Nuclear and cytoplasmic localization of 1251-1abeled bFGF in ABAE cells. Two hours after the addition of i251-1abeled bFGF (5 ng/ml), ABAE cells were harvested and fractionated into nuclear (a) and cytoplasmic (b) fractions. Total nuclear proteins corresponding to 106 cells and 1/10 of the cytoplasmic proteins were then analyzed on a 15% SDS-polyacrylamide slab gel. After protein fixation and Coomassie blue staining (lanes 1), gels were dried and exposed for 24 hr for autoradiography. Lanes 2 show Gi-arrested ABAE cells stimulated by t251-1abeled bFGF; lanes 3 show exponentially growing cells. For lanes 4, a control experiment, t25I-labeled bFGF (5 × 104 cpm) was added to disrupted ABAE cells. Cellular fractionation and analysis of proteins were carried out as described in the text. For lanes 5, an aliquot of J251-1abeledbFGF recovered in the culture medium after stimulation of the cells was analyzed as described above, i25I-labeled bFGF is detected in nucleus and cytoplasm both in exponentially growing cells and in synchronized cells undergoing the G~ S transition, lz51-Labeled bFGF is detected in essentially full length form (18.4 kDa) in the nucleus, whereas in the cytoplasm two molecular forms of the growth factor (18.4 and 16.5 kDa) are found in an identical molar ratio. As shown in lanes 4, J251-1abeled bFGF is not detected in nuclei, and little degradation of growth factor occurs during cell fractionation when the growth factor is added to disrupted cells.
EDTA, 20 mM HEPES, pH 7.5) containing a cocktail of protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF), 10 /~g/ml of leupeptin, 0.02% NPG (Cemulsol SFOS, Persan, France), and Trasylol] and centrifuged at 800 g for 5 min. The supernatant represents the crude cytoplasmic fraction. The nuclear pellet is washed twice in medium A. The purity and integrity of nuclei preparations are monitored by microscopy under phase contrast. The radioactivity of ~25I-labeled growth factor bound to the particular fractions is measured in a y counter, or using y-vials and a liquid scintilla-
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tion counter. The number of bFGF molecules bound is calculated from Avogadro's number and the specific activity of the bFGF (Table I). Aliquots of crude cytoplasmic fraction and nuclear pellets are precipitated by 15% trichloroacetic acid (30 min, 4 °) and centrifuged at 15,000 g for 30 min. The pellets are then dissolved under highly reducing conditions (1 mM Tris-HC1, pH 8, 10 mM EDTA, 2% SDS, 1.5 M 2-mercaptoethanol, 30% glycerol) and analyzed by 15% SDS-PAGE and autoradiography (Fig. 5). In control experiments, the same amount of 125I-labeled bFGF is added to untreated cells in the cell fractionation mixture. When intact nuclei are prepared from control cells, no ~25Ilabeling should be recovered in the nuclear fraction (Fig. 5). Concluding Comments A combination of three methods provides an unambiguous technique for nuclear and subnuclear localization of growth factors in cell cultures. Using synchronously growing ABAE cells stimulated with bFGF it is possible to study the cellular uptake of growth factor during the cell cycle and to quantify the accumulation of growth factor in subcellular compartments.
[46] A n t i p h o s p h o t y r o s i n e A n t i b o d i e s in O n c o g e n e a n d Receptor Research B y D A V I D F . STERN
Introduction Protein-tyrosine kinases regulate cell proliferation and differentiation. These kinases include receptors for several peptide growth factors and many of the oncogene products. The original, and most direct, method for detecting tyrosine phosphorylation of proteins was separation of 3Zp_ labeled amino acids from partial protein hydrolyzates on thin-layer plates. J More recently, antibodies have been developed that react specifically with phosphotyrosine, z-4 Use of these antibodies for immunoprecipitation or i j. A. Cooper, B. M. Sefton, and T. Hunter, this series, Vol. 99, p. 387. 2 A. H. Ross, D. Baltimore, and H. Eisen, Nature (London) 294, 654 (1981). 3 M. Ohtsuka, S. lhara, R. Ogawa, T. Watanabe, and Y. Watanabe, Int. J. Cancer 34, 855 (1984). 4 M. P. Kamps and B. M. Seflon, Oncogene 2, 305 (1988).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
