Monoclonal antibody against the centrosome. POTU N. RAO*, JI-YING ZHAO, RAMESH K. GANJU and CHERYL L. ASHORN. Department of Medical Oncology, ...
Monoclonal antibody against the centrosome
POTU N. RAO*, JI-YING ZHAO, RAMESH K. GANJU and CHERYL L. ASHORN Department of Medical Oncology, The University of Texas M. D. Atiderson Cancer Center, Houston, TX 77030, USA •Author for correspondence
A monoclonal antibody, MPM-13, raised against mitotic HeLa cell extracts recognized a perinuclear area in interphase cells and spindle poles in mitotic cells of human, mouse, Chinese hamster and sea urchin. On immunoblots MPM-13 recognized a major protein band at 43xlO3Mr and a variable minor band at 56 X103 Af, in both mitotic and interphase HeLa cells. These antigens were detectable in a variety of mammalian cells as 'well as in the unicellular ciliate Tetrahymena. In cells arrested in mitosis by colcemid and stained with MPM-13 by indirect immunofluorescence, numerous fluorescent speckles were seen throughout the cytoplasm. Reversal of colcemid block in Chinese hamster ovary (CHO) cells by washing resulted in the
reappearance of a fluorescent patch at the poles of the re-formed spindles. In HeLa cells arrested in mitosis by the microtubule stabilizing drug taxol, MPM-13 stained a large fluorescent patch encircled by dark metaphase chromosomes. This pattern indicated the failure of centrioles to move to the opposite poles in the presence of taxol. These data indicate that the MPM-13 antigens are associated with the colcemid-sensitive pericentriolar material from which microtubules originate but not with the centrioles themselves. It is also clear that these antigens are highly conserved during evolution.
specificity, recognizes a major polypeptide of about 43K (K = 10 3 M r ) that is present in a variety of cell types, ranging from Tetrahymena to human cells.
The centrosome, consisting of a pair of centrioles surrounded by the pericentriolar material (Gould & Borisy, 1977) or the microtubule-organizing center (MTOC) (Pickett-Heaps, 1971), is an important organelle in eukaryotic cells and especially distinct in animal cells. In interphase cells the centrosome is located in a perinuclear area from which microtubules emanate. Just before mitosis the centrioles separate to opposite poles from which the mitotic spindle microtubules arise (Robbins & Gonatas, 1964; Robbins et al. 1968; Roos, 1973; Kuriyama & Borisy, 1981; Verobjev & Chentsov, 1982). Using isolated centrosomes in vitro from lysed Chinese hamster ovary (CHO) cells Gould & Borisy (1977) demonstrated that microtubules arise from the fibrous pericentriolar material but not from the centrioles themselves. The nature of this electron-dense pericentriolar material and how it generates the microtubules remain to be elucidated. A number of human autoantibodies against the centrosome have been reported (Brenner et al. 1980; McCarty et al. 1981; Osborn et al. 1982; Moroi et al. 1983; Calarco-Gillam et al. 1983; Tuffanelli et al. 1983; Sagerefa/. 1986). In this study we describe a monoclonal antibody that recognizes the centrosome in both interphase and mitotic cells. This antibody, which does not exhibit species Journal of Cell Science 93, 63-69 (1989) Printed in Great Britain © The Company of Biologists Limited 1989
Key words: monoclonal antibodies, centrosome, pericentriolar material, mammalian cells, Tetrahymena.
Materials and methods Cell cultures HeLa cells and CHO cells were maintained in nionolayer culture in McCoy's 5A modified medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated bovine fetal calf serum (KC Biological, Inc., Lenexa, KS) and a penicillin-streptomycin mixture (Gibco). SV40-transformed mouse 3T3 cells were grown as monolayers in Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), non-essential amino acids, sodium pyruvate, and a penicillin-streptomycin mixture. Mouse lymphoid L5178 cells were grown as suspension culture in Fischer's medium with 10 % FCS. Mouse neuroblastoma N115D cells were grown in Dulbeco's MEM supplemented with 15% FCS. All the culture media were from Gibco.
Chemicals The compound LY 195448 was a gift from Ely Lilly Co., Indianapolis, IN. Taxol was dissolved in dimethyl sulfoxide (DMSO) and then diluted to the desired concentration by adding medium without serum.
Cell synchrony HeLa cells were partially synchronized in S phase by a single thymidine (2-5 HIM) block for 20 h. After reversal of the block by washing with medium cells were arrested in metaphase using N 2 O blockade as described (Rao, 1968). Mitotic cells of 98% purity were obtained by selective detachment. Production of hybridomas Synchronized mitotic HeLa cells were homogenized and extracts made for immunization. The procedures for making mitotic cell extracts, immunization of female Balb/c mice, fusion of spleen cells from immunized mice with mouse myeloma cells (SP2/0-Agl4) and screening of the hybridomas have been described (Davis et al. 1983). Ascites fluid was prepared by injecting 107 cells of the MPM-13 clone in 0-5 ml saline into the peritoneal cavity of each mouse and harvesting the ascites tumor 20-25 days later. Immunofluorescence Indirect immunofluorescence was performed as described (Davis et al. 1978). Cells were grown on 10-well slides for 2 days. Slides were washed in cold Hanks' balanced salt solution (HBSS) without NaHCO 3 (Gibco), fixed in methanol (100%) for lOmin at 22°C, and air dried. Cells in each well were overlaid with 15 /il of culture fluid or appropriately diluted ascites fluid as primary antibody. Fluorescein isothiocyanateconjugated rabbit anti-mouse IgG heavy and light chains (Miles Laboratories, Naperville, IL) was used as the indicator antibody. Cell surface and intracellular antigens are stained by this method. Cell extracts for immunoblots Trypsinized cells were collected by centrifugation at 600 g for 5 min at 4°C. After three to five washings with MEM without serum at 4°C, the extracts were prepared as described (Adlakha et al. 1982, 1983). Briefly, mitotic cells (4X107 cells ml" 1 ) were suspended in a buffer containing 10mM-Na2HPO4/NaH2PO4, 200mM-NaCl, 2mM-EGTA, 10mM-MgSO4, 1 mM-ATP, 1 mMphenylmethylsulfonyl fluoride, 5mM-NaF, 5 mM-sodium /3glycerolphosphate, and 10% glycerol at pH6-5. Cells were disrupted by sonication with an ultrasonic cell disrupter (Heat Systems Ultrasonics, Farmingdale, NY) at a setting of 6 at 4°C. Three 20-s pulses were used for sonication, each separated by a 30-s interval. Phase-contrast microscopy indicated that sonication invariably disrupted 98% of the cells. The sonicated cells were then centrifuged at 100 000 # for 60 min at 4°C in an ultracentrifuge (L5-50 Beckman Instruments, Palo Alto, CA). The supernatants were stored at —70°C until further analysis. The procedures for culturing Tetrahymena and the preparation of whole cell extracts for electrophoresis have been described (Guttman et al. 1980). The extracts for these studies were kindly provided by Dr David Allis and Mr Pat Colini, Baylor College of Medicine, Houston, TX. Immunoblots A 100 ^1 sample of the cell extract was mixed with 50/il of 3x sample buffer containing 3 % sodium dodecyl sulfate and polypeptides were separated by electrophoresis in 8 % sodium dodecyl sulfate-polyacrylamide gels as described (Laemmli, 1970). The following prestained molecular weight markers (Sigma, St Louis, MO) were used: a^-macroglobulin (180K), /3-galactosidase (116K), fructose-6-phosphate kinase (84K), pyruvate kinase (58K), fumarase (48-5K), lactic dehydrogenase (36-5K) and triosephosphate isomerase (266K). The polypeptides in the gel were then transferred electrophoretically to nitrocellulose paper as described (Towbine/a/. 1979; Burnette, 64
P. N. Rao et al.
1981). The nitrocellulose was rinsed in 0-15 M-NaCl/l0mMTris-HCl, pH7-4 (Tris/saline buffer) and then incubated with blocking solution (Tris/saline buffer containing 0-05 % Tween 20 and 10% bovine serum albumin (BSA)) for 2h at 25°C on a shaker bath. Then the nitrocellulose was transferred to a tray containing the primary antibody (i.e. MPM-13 ascites diluted 1:1000 with the blocking solution), and incubation was continued for another hour while shaking. After the nitrocellulose was washed three times with Tris/saline buffer containing 0-05 % Tween 20 (TBS/TW20), it was incubated for 30 min at 25°C while being shaken with alkaline phosphatase-conjugated, affinity-purified goat anti-mouse IgG heavy and light chains (Bio-Rad, Richmond, CA) diluted 1:5000 with the blocking solution. After three washes with TBS/TW20 the color was developed by adding a mixture of 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP) and p-nitro blue tetrazolium chloride (NBT) stock solutions in alkaline phosphatase buffer (Bio-Rad). These substrates develop a purple color on the membrane where alkaline phosphatase-conjugated antibodies are bound.
Results Specificity of MPM-13 antibody to spindle poles in mitotic cells and perinuclear area in inteiphase HeLa cells In our earlier studies, we showed that cells undergoing mitosis possess certain activities or factors that are absent in interphase cells. A cell in mitosis upon fusion with an interphase cell can induce premature chromosome condensation in the interphase nucleus (Johnson & Rao, 1970). Extracts of mitotic HeLa cells microinjected into immature oocytes of Xenopus laevis can induce maturation, i.e. chromosome condensation and germinal vesicle breakdown (Sunkara et al. 1979). Extracts from interphase HeLa cells do not have this activity. On the basis of these observations, we have decided to identify and characterize the mitosis-specific proteins using the hybridoma technique (Kohler & Milstein, 1975). Using crude or partially purified mitotic HeLa cell extracts as the immunogen, we have generated a panel of monoclonal antibodies that specifically stain cells in mitosis (Davis & Rao, 1987). One of the antibodies thus generated is MPM-13, which is specific to the centrosome. MPM-13 is an IgM antibody with K light chains. When HeLa cells grown on slides were stained with MPM-13 by indirect immunofluorescence, we observed a bright fluorescent patch in the perinuclear area of interphase cells and staining at the spindle poles in cells undergoing metaphase or anaphase (Fig. 1). The identical staining patterns seen in mitotic cells of sea-urchin (Lytechinus pictus) after staining with MPM-13 and other centrosome-specific antibodies confirm the conclusion that MPM-13 is specific to centrosomal structures (Fig. 2). MPM-13 recognizes a major protein of 43K The polypeptides from the extracts of synchronized mitotic and random populations of HeLa cells were electrophoretically separated on 8 % sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, electrophoretically transferred to nitrocellulose paper and incubated with antibodies. Fig. 3 shows that MPM-13 is specific to
Fig. 1. Specificity of MPM-13 antibody to centrosomes. Random populations of HeLa cells were stained by indirect immunofluorescence with MPM-13. Fluorescence is present in the perinuclear area in interphase cells (A) and spindle poles in metaphase cells (B). The chromosomes on the metaphase plate appear as a dark band between the two fluorescent patches at the spindle poles. A metaphase cell at higher magnification exhibits a bright single patch of fluorescence at each pole (C).
Fig. 2. A comparison of MPM-13 staining of mitotic sea-urchin cells with other centrosome-specific antibodies. Mitotic cells of sea urchin (Lytechinus pictus) were extracted as described (Balczon & Schatten, 1983). The 5051 human autoimmune antibody was used 1: 100 with FITC-conjugated second human antibody at 1:40. The Ah-6 antibody (Schatten et al. 1987) was used in a 1: 10 dilution, with FITC-conjugated mouse IgM at a dilution of 1:40 as the second antibody. The MPM-13 was diluted 1:50, with a rhodamine-conjugated second mouse IgG antibody in a 1:40 dilution. PBS rinses followed each antibody step with 2-5/tM-Hoechst 22358 added during the last rinse. All the antibodies stained the spindle poles while Hoechst 22358 stained the chromosomes.
a protein of approximately 43K among the numerous proteins present in mitotic and interphase HeLa cell extracts as revealed by Coomassie Blue staining. MPM-13 exhibits no species specificity To determine whether MPM-13 exhibits any species specificity, we tested a number of cell types by using indirect immunofluorescence and immunoblots. In mouse (SV40-transferred 3T3, lymphoma line L5178, neuroblastoma N115D), hamster (CHO) and human
(diploid fibroblast 1508, HeLa, and the breast cancer cell line MCF-7) cells MPM-13 stained spindle poles and recognized a major band at 43K on immunoblots. A very faint band of approximately 56K was sometimes observed (Fig. 4A). In Tetrahymena some low molecular weight bands were seen in addition to the 43K band (Fig. 4B). Since some reactive peptides appear to be running at the ion front in 8% SDS-PAGE (Fig. 4) we decided to separate these proteins on 6% to 20% gradient SDS-PAGE gels. The results shown in Fig. 5 confirm the specificity of MPM-13 to the 43K band. However, Monoclonal antibody to centmsomes
X10" 3 180
116 116 . 84 58
58 48-5 36-5 26-6
Fig. 3. MPM-13 recognizes an antigen of approximately 43K. A. Polypeptides from a random population of HeLa cells or cells synchronized in mitosis were separated in 8 % sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose sheets, and stained with MPM-13 by an indirect immunoalkaline phosphatase procedure. Approximately 100 jug of protein per lane was loaded. Lane 1, molecular weight markers; lane 2, synchronized mitotic HeLa cells; lane 3, a random population of HeLa cells. The arrow indicates the protein band of approximately 43K present both in mitotic and interphase cells. B. Another gel prepared in a similar manner was stained with Coomassie Blue to detect the various proteins present in the extracts of a random population (lane 4) and a synchronized mitotic population (lane 5) of HeLa cells. Lane 6 contains molecular weight markers.