A Microtubule-associated Protein Antigen Spindle Microtubules in PtKt Cells Unique to Mitotic

Microtubule-associated proteins (MAPs) that copurify with tubulin through multip•e cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the celt cycle. N2B5 antibody microinjected into living PtK~ cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network. Microtubules purified by cell fractionation or by cycles of assembly/disassembly contain, in addition to tubulin, a heterogeneous population of accessory proteins called microtubuleassociated proteins (MAPs). Many of the MAPs that have been isolated are capable of stimulating microtubule assembly in vitro (6, 26, 29, 31, 34). Immunocytochemistry has shown that MAPs from brain tissue associate with microtubules in situ (8, 9, 13, 28), consistent with the hypothesis that MAPs have tubulin modulating function(s) in living cells as well as promoting tubulin polymerization in vitro. MAP cofactors may therefore mediate the cytoskeletal and motility functions of microtubules in vivo, and this has fostered our interest in the identification and characterization of those MAPs that are involved in the formation and function of the mitotic apparatus. Brain is commonly used for the preparation of microtubule protein because it contains a high density of microtubules competent to polymerize in vitro as well as in vivo. Brain is, 424 however, a poor source of material for the investigation of mitotic spindle components because after neurogenesis the tissue has a low mitotic index. Thus any mitotic apparatusspecific factors would be expected to be present at low concentrations. Microtubule protein prepared from cultured cells contains a different complement of assembly MAPs than neurotubule protein prepared from brain tissue (4, 27, 30, 32, 36). As first suggested by Nagle and colleagues (27), this may indicate that some brain MAPs have neuron-specific functions. Indeed, MAPs from brain either do not appear to be associated with the microtubules of nonneuronal ceils (13, 24) or are found in association with the interphase microtubule network as well as the mitotic apparatus and thus are not spindlespecific components (8). There are many polypeptides that associate with HeLa cell microtubules purified by cycles of assembly/disassembly (4, 30). Such material is potentially a plentiful source of mitotic apparatus MAPs. We are producing a catalogue of specific lymphocyte hybridoma monoclonal TH~ JOURNAL OF CELL BIOLOGY • VOLUMf 96 FEBRUARY 1983 424-434 © The Rockefel ler Univers i ty Press 0021-9525/83/02/0424/11 $1.00 on O cber 9, 2017 jcb.rress.org D ow nladed fom antibodies against the mixture of HeLa MAPs to serve as biochemical, cytological, and pharmaceutical probes for MAP function. This report presents observations on two of the hybridoma clones that produce IgGs directed against determinants on a high molecular weight MAP from HeLa cells. While both immunoglobulins stain cytoplasmic and mitotic microtubules in human cells, only one of the antibodies cross-reacts with PtKI cells in which the staining is restricted to mitotic spindle microtubules. Microinjection of this antibody into living PtK1 cells interferes with normal mitosis, suggesting that this MAP contributes to the formation and/or function of the mitotic spindle. MATERIALS AND METHODS Antigen and Antibodies: HeLa ceils were grown in suspension culture in modified Eagle's medium (MEM) supplemented with 10°7o fetal calf serum. Ceils were collected by centrifugation, washed in 100 mM PIPES, pH 6.9, 1.0 mM MgC12, 1.0 mM EGTA (PME buffer) and lysed by sonication at 0°C. Microtubules were polymerized and purified by cycles of assembly/disassembly as described in detail elsewhere (30). The microtubule-associated protein (MAP) fraction was isolated by ion exchange chromatography (31). BALB/c mice were inoculated intraperitoneaUy with 100/~g of HeLa MAPs in complete Freund's adjuvant and boosted intravenously 21 d later with 50/~g of HeLa MAPs in phosphate-buffered saline (PBS). On day twenty-four, the mice were sacrificed, their spleens excised, and 1.25 x 10 a spleen lymphocytes were fused to 1.25 × 107 SP2/0 myeloma cells as described previously (13). Hybridomas were screened by a solid phase radioimmunoassay using the original HeLa MAP antigen as ligand, and antibody reacting with the immunogen was detected with r2SI-Staphylococcus aureus protein A (SAP) labeled with diiodinated Bolton-Hunter reagent (New England Nuclear, Boston, MA). Two of the hybrids (N-2B5.11.2 and N-3B4.3.10) that secreted antibody that bound to the HeLa MAP mixture in solid phase radioimmunoassay and stained HeLa cell microtubnles by immunoftuorescance microscopy were cloned twice for use in subsequent experiments. Antitubulin antiserum was the generous gift of Dr. Joanna Olmsted (University of Rochester). Immunochemical Characterization: The specificity of the antibodies from the resultant clones was tested by staining gel slices containing electrophoretically separated cell proteins with [3SS]methionine-labeled antibody (7). 3SS-monoclonal antibody was prepared by growing the hybridomas overnight in MEM lacking methionine (Flow Laboratories, Rockville, MD) supplemented with pyruvate, glutaraine, nonessential amino acids (Gibco Laboratories, Grand Island Biological Co., Grand Island, NY), and l0/~Ci/ml [aSS]methionine (New England Nuclear). Labeled antibody was harvested by ammonium sulfate precipitation and purified by chromatography on SAP-Sepharose A-50 (Pharmacia) (25). Cell homogenates were prepared by removing HeLa and 3T3 cell raonolayers from their culture vessels with a rubber policeman, washing the cells twice in PBS containing aprotinin and p-tosyl-arginine methyl ester (Sigma Chemical Co., St. Louis, MO), and then spraying the cells into a 10-fold excess of acetone at -20°C. After sonication for 1 rain at setting 5 of a Branson sonifier, the acetone precipitate was collected by centrifugation at 3,000 g for 10 min at 4°C and washed once with acetone at -20°C. The final pellet was resuspended directly in sample buffer, heated to 100°C for 2 rain, and run on 7.5% SDSacrylamide gels (18). Gel slices were fixed, stained with 3SS-antibody, washed, and autoradiographed by the method of Burridge (7). The HeLa MAP antigen was covalently linked to CNBr-activated cellulose disks for antibody competition experiments. The CNBr disks (23) were the generous gift of Dr. Susan Strome (University of Colorado). They had been prepared by soaking Whatman 50 paper (Whatman Inc., Clifton, N J) in distilled water for 1 h and then in a freshly made 5% solution of CNBr, pH 10-10.5 at 19°C for 3 rain. The reaction was stopped by the addition of four volumes of 5 raM NaHCO3, and the paper was washed five times with cold 5 mM NaHCO3. Following three rinses in cold acetone, the paper was dried, and 4-ram disks were cut with a paper punch. The disks were activated by a rinse in 1.0 raM HCI and then were washed in distilled water. Next, the disks were incubated with ~ 10 ~tg/ disk of HeLa MAP antigen on a rotary shaker. All of the antigen appeared to bind. The reaction was quenched by the addition of 0.1 M ethanolamine. For competition experiments 0.1 ml of a~S-monoclonal antibody diluted 1:50 in PBS containing 1.0 rag/ml ovalbumin(PBS/O) with or without a 10-fold excess of unlabeled antibody was added to the disks in a "Seal-a-Meal bag" (Sears, Roebuck & Co., Chicago, IL) and incubated for 1 h at 37°C on a rotary shaker. The disks were washed five times in PBS/O and once in distilled water, dried, and then counted in a liquid scintillation counter. As diagrammed in Fig. 3, additional competition experiments were done using monoclonal antibody bound to Sepharose by an hydroxysuccinimide linkage. An excess of monoclonal antibody in PBS was incubated with activated affi-gel l0 (Bio-Rad Laboratories, Richmond, CA) overnight at 4°C. After quenching with ethanolamine, the resin was washed in PBS containing l0 mg/ml bovine serum albumin (BSA) and l0 mg/ml ovalbumin and then washed three times in PBS/ O. 0.05-ml aliquots of a 50% slurry of resin in PBS/O were added to 0.1 ml of PBS/O containing 4.0 #g of HeLa MAP antigen and 10 a cpm complementary 35S-labeled monoclonat antibody. In control samples the antigen was omitted or excess unbound antibody was added to assess the degree of nonspecific adsorption of radioactive label to the beads. The resin was then washed five times in PBS/ O and counted in a liquid scintillation counter. Immunofluorescence Microscopy: Human (Va90 and IMR), african green monkey kidney (BSC), and rat kangaroo (PtK1) cells were grown in Ham's F-12 (Gibco Laboratories) supplemented with 10% fetal calf serum. Mouse 3T3 and human HeLa cells were grown in modified Eagle's medium (Gibco Laboratories) with 1% nonessential amino acids supplemented with 10% fetal calf and 10% calf serum, respectively. Cells were subcultured for iramunofluorescence microscopy on [2-mm polylysine-coated coverslips. Cells were fixed in paraformaldehyde/glutaraldehyde as previously described (14) or, alternatively, the cells were luted and permeabilized directly in anhydrous methanol at -20°C for 4 min followed by postfixation in acetone at -20°C for 1 min. After preincubation in 50% goat serum, the coverslips were incubated in a 1:25 dilution of primary mouse antibody in 20@0 goat serum for 60 rain at 37°C. After three 10-min washes in PBS the cells were incubated in secondary tetramethylrhodamine-labeled goat anti-mouse immunoglobulin (U.S.A. Biochemicals) diluted 1:30 in 20% goat serum. After another set of PBS washes, the coverslips were mounted in polyvinylalcohol for observation. For some studies, primary antibody was absorbed with art excess of protein at 37°C for 4 h and then clarified at 8,000 g for 15 min at 4°C. The antibody-stained cell preparations were observed on a Zeiss photomicroscope III equipped with epi-fluorescence optics. Images were recorded on Plus-X film and developed in HC-110. Microinjection: For microinjection experiments the PtK, cells were subcultured on 12-mm glass coverslips for 2 d, and mounted in 35-mm diameter observation chambers with 3 ml of warm medium. During prolonged observation fresh 5% CO2-equiLibrated medium was circulated through the chamber at the rate of 10 ml/h. Temperature was maintained with an air curtain incubator (Sage Instruments, Cambridge, MA) and monitored with a needle probe thermister placed directly in the culture medium (Yellow Springs Instrument Co., Yellow Springs, OH). Cells were injected with glass microneedles (W-P Instruments, Inc., New Haven, CT) with an outside tip diameter of 0.25 #m (as determined by scanning electron microscopy) pulled on a P-77 Brown-Flaming micropipet puller (Sutter Instruments, San Francisco, CA). Needles were backfiUed by capillary action and then inserted into the microtool of a Leitz micromanipulator (E. Leitz, Inc., Rockleigh, N J). Monoclonal antibody at a concentration of 1.0-2.5 mg/ml containing 0.5 mg/ ml of fluorescein-isothiocyanate-labeled BSA was dialyzed into the injection buffer (0.1 M glutamic Acid, 0.039 M citric Acid, 0.14 M KOH, 1.0 mM MgCI~, 0.1 mM dithiothreitol, pH 7.0) before use. Antibody was injected by initiating a gentle flow of antibody using air pressure developed by a 50-ml disposable syringe connected to the needle by silastic tubing and then briefly touching the needle to the cell. Injection volume was -1.4 x 10 -~z 1 as determined by the injection of t25I-BSA (Zavortink, M., and J. R. Mclntosh, unpublished results) or by calculation using the size of the vesicle that forms transiently during microinjection (Izant, L, and J. R. Mclntosh, unpublished observations). The cells were observed with a 40x lens immersed in the tissue culture medium on a Zeiss Universal microscope equipped with phase, epi-fluorescence, differential interference contrast (DIC), and polarization optics. Images were captured with a Venus DV2/9022 video intensification camera and recorded on an NEC #VC-9507 video tape recorder using a Panasonic # W J-810 time-date generator. This produced high quality images at low incident light intensities. Photographic records were recorded on Pan-X film from a Panasonic #WV-5361U monitor.