Abstract
During cell division microtubules of the mitotic spindle segregate the duplicated chromosomes into the two daughter cells. Chromosome–microtubule attachment is mediated by kinetochores, multiprotein complexes assembled on specialized regions of the DNA. Kinetochores modulate microtubule dynamics to generate the forces necessary to power chromosome movement and regulate the spindle checkpoint. Errors in kinetochore function can cause aneuploidy, a hallmark of 80% of solid tumors in humans, suggesting a fundamental link to tumorigenesis. Human kinetochores are complex protein machines with over 100 different proteins. Here we present fixed- and live-cell-based assays used to functionally categorize kinetochore proteins with regard to spindle checkpoint activity and kinetochore–microtubule attachment.
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References
Cleveland, D. W., Mao, Y., and Sullivan, K. F. (2003) Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, 407–21.
Chan, G. K., Liu, S. T., and Yen, T. J. (2005) Kinetochore structure and function. Trends Cell. Biol. 15, 589–98.
Westermann, S., Drubin, D. G., and Barnes, G. (2007) Structures and functions of yeast kinetochore complexes. Annu. Rev. Biochem. 76, 563–91.
Musacchio, A., and Salmon, E. D. (2007) The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–93.
Michel, L. S., Liberal, V., Chatterjee, A., Kirchwegger, R., Pasche, B., Gerald, W., Dobles, M., Sorger, P. K., Murty, V. V., and Benezra, R. (2001) MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 409, 355–9.
Hernando, E., Nahle, Z., Juan, G., Diaz-Rodriguez, E., Alaminos, M., Hermann, M., Michel, L., Mittal, V., Gerald, W., Benezra, R., Lowe, S. W., and Cordon-Cardo, C. (2004) Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature 430: 797–802.
Warren, C. D., Brady, D. M., Johnston, R. C., Hanna, J. S., Hardwick, K. G., and Spencer, F.A. (2002) Distinct chromosome segregation roles for spindle checkpoint proteins. Mol. Biol. Cell 13, 3029–41.
Draviam, V. M., Xie, S., and Sorger, P. K. (2004) Chromosome segregation and genomic stability. Curr. Opin. Genet. Dev. 14, 120–5.
Dai, W., Wang, Q., Liu, T., Swamy, M., Fang, Y., Xie, S., Mahmood, R., Yang, Y. M., Xu, M., and Rao, C. V. (2004) Slippage of mitotis arrest and enhanced tumor development in mice with BubR1 haploinsuffiency. Cancer Res. 64: 440–5.
Meraldi, P., and Sorger, P. K. (2005) A dual role of Bub1 in the spindle checkpoint and chromosome congression. EMBO J. 24, 1621–33.
Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1997) Genetic instability in colorectal cancers. Nature 386, 623–7.
Masuda, A., and Takahashi, T. (2002) Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis. Oncogene 21, 6884–97.
Lengauer, C., and Wang, Z. (2004) From spindle chechpoint to cancer. Nature Genet. 36, 1144–5.
Yuen, K. W., Montpetit, B., and Hieter, P. (2005) The kinetochore and cancer: what’s the connection? Curr. Opin. Cell Biol. 17, 576–82.
Meraldi, P., McAinsh, A. D., Rheinbay, E., and Sorger P. K. (2006) Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins. Genome Biol. 7, R23.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–8.
McAinsh, A. D., Meraldi, P., Draviam, V. M., Toso, A., Sorger, P.K. (2006) The human kinetochore proteins Nnf1R and Mcm21R are required for accurate chromosome segregation. EMBO J. 25, 4033–49.
Harper, J. V. (2005) Synchronization of cell populations in G1/S and G2/M phases of the cell cycle. Methods Mol. Biol. 296, 157–66.
Uzbekov, R., Chartrain, I., Philippe, M., and Arlot-Bonnemains, Y. (1998) Cell cycle analysis and synchronization of the Xenopus cell line XL2. Exp. Cell. Res. 242, 60–8.
Tanudji, M., Shoemaker, J., L’Italien, L., Russell, L., Chin, G., and Schebye, X. M. (2004) Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay. Mol. Biol. Cell. 15, 3771–81.
De Brabander, M. J., Van de Veire, R. M. L., Aerts, F. E. M., Borgers, M., and Janssen, P. A. J. (1976) The effects of methyl [5-(2-Thienylcarbonyl)-1H-benzimindazol-2-yl]carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultures in vitro. Cancer Res. 36: 905–916.
De Brabander, M., Geuens, G., Nuydens, R., Willebrords, R., and De Mey, J. (1981) Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores. Proc. Natl. Acad. Sci. USA 78: 5608–12.
Mayer, T. U., Kapoor, T. M., Haggarty, S. J., King, R. W., Schreiber, S. L., and Mitchison, T. J. (1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–4.
Tapia, C., Kutzner, H., Mentzel, T., Savic, S., Baumhoer, D., and Glatz, K. (2006) Two mitosis-specific antibodies, MPM-2 and phospho-histone H3 (Ser28), allow rapid and precise determination of mitotic activity. Am. J. Surg. Pathol. 30, 83–9.
Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A. L. (1994) Inhibitors of the proteasome block the degradation of most proteins and the generation of peptides presented on MHC class I molecules. Cell 78: 761–71.
Gergely, F., Draviam, V. M., and Raff, J. W. (2003) The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells. Genes Dev. 17, 336–41.
Cassimeris, L., and Morabito, J. (2004) TOGp, the human homolog of XMAP215/ Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly. Mol. Biol. Cell. 15, 1580–90.
DeLuca, J. G., Moree, B., Hickey, J. M., Kilmartin, J. V., and Salmon, E.D. (2002) hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells. J. Cell. Biol. 159, 549–55.
Hauf, S., Cole, R. W., LaTerra, S., Zimmer, C., Schnapp, G., Walter, R., Heckel, A., van Meel, J., Rieder, C. L., and Peters, J. M. (2003) The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J. Cell. Biol. 161, 281–94.
Salmon, E. D., Cimini, D., Cameron, L. A., and DeLuca, J. G. (2005) Merotelic kinetochores in mammalian tissue cells. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 360: 553–68.
Salmon, E. D., and Begg, D. A. (1980) Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase. J Cell Biol. 85, 853–65.
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Klebig, C., Toso, A., Borusu, S., Meraldi, P. (2009). Analysing Kinetochore Function in Human Cells: Spindle Checkpoint and Chromosome Congression. In: McAinsh, A. (eds) Mitosis. Methods in Molecular Biology, vol 545. Humana Press. https://doi.org/10.1007/978-1-60327-993-2_12
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DOI: https://doi.org/10.1007/978-1-60327-993-2_12
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