MAP Kinase Regulation of the Mitotic Spindle Checkpoint
Maintaining the integrity of the cell cycle is critical for ensuring that cells only undergo DNA replication and proliferation under controlled conditions in response to discrete stimuli. One mechanism by which the fidelity of this process is guaranteed i
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1. Introduction The spindle checkpoint is activated during mitosis by defects in spindle tension or attachment, causing a delay in chromosome segregation until the problem is corrected by Aurora B kinase. A conserved kinase, Aurora B kinase is associated with chromosomal kinetochores in complex with other “chromosomal passenger” proteins including Borealin/Dasra B, Survivin, and INCENP (1). Increased mitogen-activated protein kinase (MAPK) activity resulting from loss or depletion of Raf Kinase Inhibitory Protein (RKIP) causes a reduction in the mitotic index due to a decrease in mitotic traversal time from nuclear envelope breakdown to anaphase (2). When cells are exposed to drugs that reduce spindle tension such as Taxol, the spindle checkpoint is normally activated.
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_31, © Springer Science+Business Media, LLC 2010
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However, RKIP depletion and enhanced MAPK activity causes override of the spindle checkpoint and increased chromosomal defects following Taxol treatment. Expression, activation, or localization of MAPK (ERK1,2) or its regulators can be monitored across mitosis by immunofluorescence. This methodology, as opposed to methods such as flow analysis, enables one to follow MAPK signaling in the context of specific stages of mitosis. Similarly, fluorescent DNA staining in fixed cells to show chromatin morphology and time-lapse microscopy of cells traversing mitosis reveal the effects of changes in ERK activity on mitotic progression and the spindle checkpoint. Finally, synchronization and/or arrest with drugs that disrupt the spindle enable biochemical analysis of mitotic cell populations in addition to single cells.
2. Materials1 2.1. Cell Culture, Synchronization, and Mitotic Arrest
1. Dulbecco’s Modified Eagle’s Medium with high glucose (Gibco/ Invitrogen, Bethesda, MD) (DMEM) supplemented with 10% fetal bovine serum, 50 u/ml penicillin, and 50 µg/ml streptomycin (100× stock, Gibco/Invitrogen, Bethesda, MD) and selection antibiotics appropriate for any ectopically expressed markers. 2. Thymidine is dissolved in DMEM at 100 mM and sterilized by passage through a 0.22 µm filter. 3. Taxol (paclitaxel) (cat. no. 1097, Tocris Bioscience, Ellisville, MD) stock, 5 mM in DMSO. 4. Nocodazole stock, 20 mM in DMSO. 5. 0.5% trypsin (BD Diagnostic Systems, Sparks, MD), 5 mM EDTA solution in PBS (see Subheading 2.2 below). 6. 15 mm round #1 glass coverslips. 7. Small beaker of acetone. 8. Small beaker of 100% ethanol. 9. Bunsen or alcohol burner. 10. Forceps.
2.2. Cell Fixation and Immunofluorescence
1. Freshly made 4% paraformaldehyde (cat. no. PX0055-3, EMD Chemicals, Gibbstown, NJ) in 0.1 M Na phosphate buffer, pH = 7.4. Store at 4°C for no more than 3 days. 2. Dulbecco’s Ca++-free, Mg++-free phosphate-buffered saline (PBS). 3. 2% sodium azide in H2O (Note 1) (100× stock). 4. 5.0% IgG-free bovine serum albumin (BSA) (cat. no. 001000-161, Jackson I
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