Mitosis Methods and Protocols
With the advent of modern methodology, the phenomenal complexity of the protein components and regulatory steps involved in mitosis has become approachable. In Mitosis: Methods and Protocols, experts in the field provide an up-to-date collection of method
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Introduction During mitosis a cell divides into two daughter cells, each of them inheriting the same set of chromosomes. To achieve chromosome segregation, chromosomes bind to microtubules emanating from the spindle poles via kinetochores, multiprotein complexes located on centromeric DNA. Kinetochores have three fundamental functions during mitosis: (1) They act as the microtubule attachment platform, (2) they generate the forces necessary for chromosome movement during chromosome segregation, and (3) they regulate the spindle checkpoint (1–4). Bipolar kinetochore–microtubule attachment is a fundamental step not only for correct chromosome alignment on the Andrew D. McAinsh (ed.), Mitosis: Methods and Protocols, Methods in Molecular Biology 545, DOI 10.1007/978-1-60327-993-2 12, © Humana Press, a part of Springer Science+Business Media, LLC 2009
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metaphase plate but also for chromosome segregation during anaphase. In the case of attachment defects (e.g., unattached chromosomes, syntelic attachment), the spindle checkpoint is engaged and arrests the cell cycle at the metaphase/anaphase transition. After bipolar attachment of the chromosomes and alignment on the metaphase plate, the checkpoint is silenced and anaphase proceeds (4). The spindle checkpoint depends on a set of conserved proteins such as Mad1, Mad2, Bub1, Bub3, Mad3/BubR1, and Mps1. Several studies have shown that failure of the spindle checkpoint can lead to aneuploidy, as segregation errors result in daughter cells with additional or missing chromosomes (5–10). As the majority of cancers show aneuploidy this suggests that spindle checkpoint failure could be an early event in carcinogenesis (11–14). Based on biochemical and functional studies during the last few years, human kinetochore proteins have been clustered into several subcomplexes representing different functions (3, 4, 15). The method of choice to study the function of human kinetochores is RNA-interference-mediated protein depletion (16). Although this method is very powerful, there is an important caveat: the phenotype resulted from RNAi treatment does not necessarily reveal the function of the specific protein, but shows to which degree whole kinetochore function is impaired during cell division. Here we describe several assays to analyze RNAi phenotypes according to kinetochore–microtubule attachment and the efficiency of the spindle checkpoint.
2. Materials 2.1. Cell Culture
1. Cells: Any adherent human cell line is appropriate to analyze kinetochore function (see Note 1). For live cell imaging, cells have to stably express a fluorescent marker, for example, a histone or kinetochore protein, to render chromosome or kinetochore movements visible by fluorescence microscopy. 2. Growth medium: For normal cell culture use standard mammalian medium appropriate for the cells. For live cell imaging, the cells either have to be cultured in CO2 -independent medium, for example, Leibovitz’s L-15 medium (GIBCO) containing 10% FCS, or the microscope has to be equipped with
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