Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase

doi:10.1083/jcb.129.5.1195

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Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase

MS Campbell and GJ Gorbsky
Department of Cell Biology, University of Virginia Health Sciences Center, Charlottsville 22908, USA.

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.
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Wong, O. K., Fang, G. (2007). Cdk1 phosphorylation of BubR1 controls spindle checkpoint arrest and Plk1-mediated formation of the 3F3/2 epitope. JCB 179: 611-617 [Abstract] [Full Text]
Yang, F., Camp, D. G. II, Gritsenko, M. A., Luo, Q., Kelly, R. T., Clauss, T. R. W., Brinkley, W. R., Smith, R. D., Stenoien, D. L. (2007). Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus. J. Cell Sci. 120: 4060-4070 [Abstract] [Full Text]
Wong, O. K., Fang, G. (2006). Loading of the 3F3/2 Antigen onto Kinetochores Is Dependent on the Ordered Assembly of the Spindle Checkpoint Proteins. Mol. Biol. Cell 17: 4390-4399 [Abstract] [Full Text]
Wong, O. K., Fang, G. (2005). Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores. JCB 170: 709-719 [Abstract] [Full Text]
Morrow, C. J., Tighe, A., Johnson, V. L., Scott, M. I.F., Ditchfield, C., Taylor, S. S. (2005). Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/CCdc20. J. Cell Sci. 118: 3639-3652 [Abstract] [Full Text]
Logarinho, E., Bousbaa, H., Dias, J. M., Lopes, C., Amorim, I., Antunes-Martins, A., Sunkel, C. E. (2004). Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells. J. Cell Sci. 117: 1757-1771 [Abstract] [Full Text]
Rodriguez, M., Li, S. S.-C., Harper, J. W., Songyang, Z. (2004). An Oriented Peptide Array Library (OPAL) Strategy to Study Protein-Protein Interactions. J. Biol. Chem. 279: 8802-8807 [Abstract] [Full Text]
Yu, R., Lu, W., Chen, J., McCabe, C. J., Melmed, S. (2003). Overexpressed Pituitary Tumor-Transforming Gene Causes Aneuploidy in Live Human Cells. Endocrinology 144: 4991-4998 [Abstract] [Full Text]
Wilson, P. J., Forer, A., Wise, D. (2003). Microtubule distribution during meiosis I in flea-beetle [Alagoasa (Oedionychus)] spermatocytes: evidence for direct connections between unpaired sex chromosomes. J. Cell Sci. 116: 1235-1247 [Abstract] [Full Text]
Shannon, K. B., Canman, J. C., Salmon, E. D. (2002). Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension. Mol. Biol. Cell 13: 3706-3719 [Abstract] [Full Text]
Zhou, J., Yao, J., Joshi, H. C. (2002). Attachment and tension in the spindle assembly checkpoint. J. Cell Sci. 115: 3547-3555 [Abstract] [Full Text]
Taylor, S. S., Hussein, D., Wang, Y., Elderkin, S., Morrow, C. J. (2002). Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells. J. Cell Sci. 114: 4385-4395 [Abstract] [Full Text]
Canman, J. C., Sharma, N., Straight, A., Shannon, K. B., Fang, G., Salmon, E. D. (2002). Anaphase onset does not require the microtubule-dependent depletion of kinetochore and centromere-binding proteins. J. Cell Sci. 115: 3787-3795 [Abstract] [Full Text]
Cimini, D., Fioravanti, D., Salmon, E. D., Degrassi, F. (2002). Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells. J. Cell Sci. 115: 507-515 [Abstract] [Full Text]
Biggins, S., Murray, A. W. (2001). The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint. Genes Dev. 15: 3118-3129 [Abstract] [Full Text]
Hoffman, D. B., Pearson, C. G., Yen, T. J., Howell, B. J., Salmon, E.D. (2001). Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores. Mol. Biol. Cell 12: 1995-2009 [Abstract] [Full Text]
Nicklas, R. B., Waters, J. C., Salmon, E. D., Ward, S. C. (2001). Checkpoint signals in grasshopper meiosis are sensitive to microtubule attachment, but tension is still essential. J. Cell Sci. 114: 4173-4183 [Abstract] [Full Text]
Sugata, N., Li, S., Earnshaw, W. C., Yen, T. J., Yoda, K., Masumoto, H., Munekata, E., Warburton, P. E., Todokoro, K. (2000). Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere-kinetochore complexes. Hum Mol Genet 9: 2919-2926 [Abstract] [Full Text]
Pluta, A., Earnshaw, W., Goldberg, I. (2000). Interphase-specific association of intrinsic centromere protein CENP-C with HDaxx, a death domain-binding protein implicated in Fas-mediated cell death. J. Cell Sci. 111: 2029-2041 [Abstract]
Saffery, R., Irvine, D. V., Griffiths, B., Kalitsis, P., Wordeman, L., Choo, K.H. A. (2000). Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins.. Hum Mol Genet 9: 175-185 [Abstract] [Full Text]
Saavedra, H. I., Fukasawa, K., Conn, C. W., Stambrook, P. J. (1999). MAPK Mediates RAS-induced Chromosome Instability. J. Biol. Chem. 274: 38083-38090 [Abstract] [Full Text]
GORBSKY, G. J., KALLIO, M., DAUM, J. R., TOPPER, L. M. (1999). Protein dynamics at the kinetochore: cell cycle regulation of the metaphase to anaphase transition. FASEB J. 13: 231S-234S [Abstract] [Full Text]
Sugata, N., Munekata, E., Todokoro, K. (1999). Characterization of a Novel Kinetochore Protein, CENP-H. J. Biol. Chem. 274: 27343-27346 [Abstract] [Full Text]
Basu, J., Bousbaa, H., Logarinho, E., Li, Z., Williams, B. C., Lopes, C., Sunkel, C. E., Goldberg, M. L. (1999). Mutations in the Essential Spindle Checkpoint Gene bub1 Cause Chromosome Missegregation and Fail to Block Apoptosis in Drosophila. JCB 146: 13-28 [Abstract] [Full Text]
Dawe, R. K., Reed, L. M., Yu, H.-G., Muszynski, M. G., Hiatt, E. N. (1999). A Maize Homolog of Mammalian CENPC Is a Constitutive Component of the Inner Kinetochore. Plant Cell 11: 1227-1238 [Abstract] [Full Text]
Fraschini, R., Formenti, E., Lucchini, G., Piatti, S. (1999). Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2. JCB 145: 979-991 [Abstract] [Full Text]
Yu, H.-G., Muszynski, M. G., Kelly Dawe, R. (1999). The Maize Homologue of the Cell Cycle Checkpoint Protein MAD2 Reveals Kinetochore Substructure and Contrasting Mitotic and Meiotic Localization Patterns. JCB 145: 425-435 [Abstract] [Full Text]
Ainsztein, A. M., Kandels-Lewis, S. E., Mackay, A. M., Earnshaw, W. C. (1998). INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1. JCB 143: 1763-1774 [Abstract] [Full Text]
Daum, J. R., Gorbsky, G. J. (1998). Casein Kinase II Catalyzes a Mitotic Phosphorylation on Threonine 1342�of Human DNA Topoisomerase IIalpha , Which Is Recognized by the 3F3/2 Phosphoepitope Antibody. J. Biol. Chem. 273: 30622-30629 [Abstract] [Full Text]
Shapiro, P. S., Vaisberg, E., Hunt, A. J., Tolwinski, N. S., Whalen, A. M., McIntosh, J. R., Ahn, N. G. (1998). Activation of the MKK/ERK Pathway during Somatic Cell Mitosis: Direct Interactions of Active ERK with Kinetochores and Regulation of the Mitotic 3F3/2 Phosphoantigen. JCB 142: 1533-1545 [Abstract] [Full Text]
Taylor, S. S., Ha, E., McKeon, F. (1998). The Human Homologue of Bub3 Is Required for Kinetochore Localization of Bub1 and a Mad3/Bub1-related Protein Kinase. JCB 142: 1-11 [Abstract] [Full Text]
Kallio, M., Weinstein, J., Daum, J. R., Burke, D. J., Gorbsky, G. J. (1998). Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events. JCB 141: 1393-1406 [Abstract] [Full Text]
Waters, J. C., Chen, R.-H., Murray, A. W., Salmon, E.D. (1998). Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension. JCB 141: 1181-1191 [Abstract] [Full Text]
Gorbsky, G. J., Chen, R.-H., Murray, A. W. (1998). Microinjection of Antibody to Mad2 Protein into Mammalian Cells in Mitosis Induces Premature Anaphase. JCB 141: 1193-1205 [Abstract] [Full Text]
Vitrat, N., Cohen-Solal, K., Pique, C., LeCouedic, J. P., Norol, F., Larsen, A. K., Katz, A., Vainchenker, W., Debili, N. (1998). Endomitosis of Human Megakaryocytes Are Due to Abortive Mitosis. Blood 91: 3711-3723 [Abstract] [Full Text]
Tavormina, P. A., Burke, D. J. (1998). Cell Cycle Arrest in cdc20 Mutants of Saccharomyces cerevisiae Is Independent of Ndc10p and Kinetochore Function but Requires a Subset of Spindle Checkpoint Genes. Genetics 148: 1701-1713 [Abstract] [Full Text]
LeMaire-Adkins, R., Radke, K., Hunt, P. A. (1997). Lack of Checkpoint Control at the Metaphase/Anaphase Transition: A Mechanism of Meiotic Nondisjunction in Mammalian Females. JCB 139: 1611-1619 [Abstract] [Full Text]
Duesbery, N. S., Choi, T., Brown, K. D., Wood, K. W., Resau, J., Fukasawa, K., Cleveland, D. W., Vande Woude, G. F. (1997). CENP-E is an essential kinetochore motor in maturing oocytes and is masked during Mos-dependent, cell cycle arrest at metaphase�II. Proc. Natl. Acad. Sci. USA 94: 9165-9170 [Abstract] [Full Text]
Wang, X. M., Zhai, Y., Ferrell, J. E. Jr. (1997). A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells. JCB 137: 433-443 [Abstract] [Full Text]
Nicklas, R. B. (1997). How Cells Get the Right Chromosomes. Science 275: 632-637 [Abstract] [Full Text]
Bousbaa, H, Correia, L, Gorbsky, G., Sunkel, C. (1997). Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster. J. Cell Sci. 110: 1979-1988 [Abstract]
Li, X, Nicklas, R. (1997). Tension-sensitive kinetochore phosphorylation and the chromosome distribution checkpoint in praying mantid spermatocytes. J. Cell Sci. 110: 537-545 [Abstract]
Elledge, S. J. (1996). Cell Cycle Checkpoints: Preventing an Identity Crisis. Science 274: 1664-1672 [Abstract] [Full Text]
Li, Y., Benezra, R. (1996). Identification of a Human Mitotic Checkpoint Gene: hsMAD2. Science 274: 246-248 [Abstract] [Full Text]
Waters, J., Skibbens, R., Salmon, E. (1996). Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push. J. Cell Sci. 109: 2823-2831 [Abstract]
Pluta, A. F., Mackay, A. M., Ainsztein, A. M., Goldberg, I. G., Earnshaw, W. C. (1995). The Centromere: Hub of Chromosomal Activities. Science 270: 1591-1594 [Abstract]
McKim, K. S., Hawley, R. S. (1995). Chromosomal Control of Meiotic Cell Division. Science 270: 1595-1601 [Abstract]