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Mammalian Mad2 and Bub1 Bubr1 Recognize Distinct Spindle

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Mammalian Mad2 and Bub1 Bubr1 Recognize Distinct Spindle Mammalian mad2 and bub1͞bubR1 recognize distinct spindle-attachment and kinetochore- tension checkpoints Dimitrios A. Skoufias*†, Paul R. Andreassen*†, Franc¸oise B. Lacroix*, Leslie Wilson‡, and Robert L. Margolis*§ *Institut de Biologie Structurale J.-P. Ebel (Commissariat a`l’Energie Atomique–Centre National de la Recherche Scientifique), 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1, France; and ‡Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93120 Communicated by J. Richard McIntosh, University of Colorado, Boulder, CO, February 15, 2001 (received for review September 17, 2000) Metaphase checkpoint controls sense abnormalities of chromo- sponse (15–17). HeLa, human cervical cancer cells, arrest at some alignment during mitosis and prevent progression to an- metaphase for prolonged periods of time when exposed to low aphase until proper alignment has been attained. A number of concentrations of microtubule inhibitors that do not sever the proteins, including mad2, bub1, and bubR1, have been implicated kinetochore–microtubule association (18). Here we demon- in the metaphase checkpoint control in mammalian cells. Meta- strate that the metaphase arrest arising in HeLa in response to phase checkpoints have been shown, in various systems, to read nanomolar levels of vinblastine correlates with a loss of spindle loss of either spindle tension or microtubule attachment at the tension. Because of their apparent checkpoint response to loss of kinetochore. Characteristically, HeLa cells arrest in metaphase in tension, HeLa cells are ideal for determining the molecular response to low levels of microtubule inhibitors that leave an intact requirements particular to a loss of tension checkpoint response. spindle and a metaphase plate. Here we show that the arrest We find that, in this circumstance, bub1 and bubR1 return to the induced by nanomolar vinblastine correlates with loss of tension at kinetochores, whereas mad2 does not. Correlating with this the kinetochore, and that in response the checkpoint proteins bub1 distinction in response, we also find that bub1 and bubR1 do not and bubR1 are recruited to the kinetochore but mad2 is not. mad2 coimmunoprecipitate with mad2, and that mad2 but not bub1 remains competent to respond and is recruited at higher drug clearly associates with cdc20 in cell extracts. We conclude that doses that disrupt spindle association with the kinetochores. Fur- mammalian mad2 and bub1͞bubR1 operate as elements of ther, although mad2 forms a complex with cdc20, it does not distinct pathways in HeLa, sensing attachment and tension, associate with bub1 or bubR1. We conclude that mammalian respectively. bub1͞bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively. Materials and Methods Cell Culture. HeLa cells were grown as monolayers in DMEM ͞ he proper segregation of chromosomes in anaphase is es- (GIBCO BRL) supplemented with 10% FBS (HyClone) Tsential to maintain the integrity of the genome. Metaphase and maintained in a humid incubator at 37°C in a 5% CO2 checkpoint controls at the kinetochore can sense failure of environment. spindle attachment or of spindle tension and thereby assure Cells were grown for a minimum of 48 h to ensure good accurate chromosome segregation. The proteins mad1, mad2, adherence to the substrate before any treatment. Vinblastine, a mad3, bub1, bub3, and mps1 are linked to the spindle checkpoint generous gift from Eli Lilly Research Laboratories, was kept as in budding yeast (1, 2). Genetic analysis in yeast suggests a a frozen stock in H20. Nocodazole (Sigma) was dissolved in hierarchy among the different checkpoint control proteins, with DMSO and kept frozen until used. bub1 and mps1 upstream of mad1 and mad2, which in turn are upstream of mad3; however, data do not rule out the existence Immunofluorescence Microscopy. HeLa cells were grown on polyD- of multiple pathways (3). Homologues of yeast checkpoint lysine-coated glass coverslips for immunofluorescence micros- proteins, such as mad2 (4, 5), bub1 (6), and bubR1 (7), are copy. Cells were fixed with 1% paraformaldehyde-PBS for 20 present in vertebrates. mad2, bub1, and bubR1 are all required min, washed 5 min with PBS, permeabilized with 0.2% Triton for checkpoint control in higher eukaryotes (4–8). mad2 appears X-100 in PBS for 3 min, and washed again for 5 min with PBS, to sense microtubule attachment to kinetochores in mammalian then processed with primary and secondary antibodies and cells (9). It is unknown, however, whether the different elements counterstained with propidium iodide, where utilized, as de- of metaphase checkpoint control are parts of a single cascade of scribed (19). Rabbit mad2 antiserum, purchased from Babco response or represent independent and parallel sensing mech- (Richmond, CA), was diluted 100-fold. Rabbit anti-bub1 and anisms after distinct failures. anti-bubR1 antisera, a kind gift from T. Yen (Fox Chase Cancer There are known associations among the mammalian kineto- Institute, Philadelphia), were used at dilutions of 1,000- and chore-associated checkpoint proteins. mad1, a dimer with a 2,000-fold, respectively. Results for bub1 were confirmed with an coiled-coil motif, binds to mad2 at the kinetochore, forming an independent antibody (data not shown) obtained from F. association essential to mad2 function (10). mad2 binds to cdc20, McKeon (Harvard Medical School, Boston) and used at a 200-fold dilution after fixation with absolute methanol for 10 min one of the proteins required for activation of the anaphase- Ϫ promoting complex (APC), and indirectly associates with other at 20°C. Secondary antibodies, from Jackson ImmunoRe- search, included FITC-conjugated affinity purified goat anti- APC elements through cdc20 (11). Additionally, bub1 and bubR1 appear to require bub3 for kinetochore binding (12). bubR1 appears to be associated in turn with the kinetochore- Abbreviations: APC, anaphase promoting complex; VBL, vinblastine; L-VBL, low (6.7 nM) associated motor protein CENP-E (13, 14) and with APC VBL; GFP, green fluorescent protein. elements cdc16, cdc27, and APC7 (7). †D.A.S. and P.R.A. contributed equally to this work. In addition to the known requirement for mad2 in sensing §To whom reprint requests should be addressed. E-mail: [email protected]. microtubule attachment at the kinetochore, there is evidence The publication costs of this article were defrayed in part by page charge payment. This from different nonmammalian sources that tension of microtu- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. bules at the kinetochores can also generate a checkpoint re- §1734 solely to indicate this fact. 4492–4497 ͉ PNAS ͉ April 10, 2001 ͉ vol. 98 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.081076898 Downloaded by guest on September 30, 2021 rabbit IgG, anti-mouse IgG, and anti-human IgG; and Texas fluorescence signal (high VBL treatment). Twenty scans were red-conjugated sheep anti-mouse IgG antibodies. All antibodies made in accumulation mode of different cells under identical were applied at 2.5 ␮g͞ml. Images were collected with a MRC- machine settings for the mad2 antigen and 10 scans for bub1 and 600 Laser Scanning Confocal Apparatus (Bio-Rad) coupled to bubR1. Pixel values were obtained for invariant square areas that a Nikon Optiphot microscope. encompassed kinetochores visualized with the different specific antibodies, and these values were collected as data sets for each Measurement of Distances Between Paired Sister Kinetochores. Cells of the conditions from at least 10 different cells. For mad2 were grown on glass coverslips. Control metaphases were se- measurements, a mad2 polyclonal antibody (9), kindly provided lected from random cultures. Experimental conditions included by Ted Salmon (University of North Carolina), was used, and treatment with either 6.7 nM vinblastine (VBL) for 20 min to these cells were first permeabilized then fixed to reduce back- suppress microtubule dynamics or with 0.5 ␮M VBL for 20 min ground before analysis, by published protocols (9). For mad2, to depolymerize microtubules. Cells were fixed with 2% para- collected at metaphase or in low drug, a diffuse background was formaldehyde–PBS for immunofluorescence microscopy, per- present at the position of the chromosomes, with no discrete meabilized as above, then incubated with scleroderma CREST concentrations of antigen. This background, which was consis- (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, tent for all conditions, was subtracted from all kinetochore pixel sclerodactyly, telangiectasia) autoimmune antiserum (M. values for mad2 plots. Wener, University of Washington) to identify centromeres and with antitubulin antibodies (Sigma) to identify metaphase fig- Microinjection. HeLa cells were grown on polyD-lysine-coated ures (19). Optical sections were imaged by confocal microscopy, coverslips for at least 3 days before microinjection. Affinity and the distance between paired kinetochores was measured by purified and concentrated anti-XMAD2 antibodies at concen- Ϫ using COMOS software (Bio-Rad). For each treatment condition, trations suitable for microinjection (9) (0.4 mg ml 1 in 50 mM all kinetochore pairs resolvable in an optical section were K-glutamate, 0.5 mM MgCl2) were kindly provided by Ted measured from the outer edge of each kinetochore in a pair, and Salmon. Antibodies were microinjected into untreated meta- data were accumulated for 15
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