Abstract
To follow the cell division cycle in the living state, certain biological activity or morphological changes must be monitored keeping the cells intact. Mitotic events from prophase to telophase are well defined by morphology or movement of chromatin, nuclear envelope, centrosomes, and/or spindles. To paint or simultaneously visualize these mitotic subcellular structures, we have been using ECFP-histone H3 for chromatin and chromosomes, EGFP-Aurora-A for centrosomes and kinetochore spindles and DsRed-fused truncated peptide of importin alpha for the outer surface of nuclear envelope as living cell markers. Time-lapse images from prophase through to early G1 phase can be obtained by constructing a triple-fluorescent cell line (Sugimoto et al., Cell Struct. Funct. 27, 457–467, 2002). Here, we describe the multicolor imaging of mitosis of a human breast cancer cell line, MDA435, and a further application to characterizing the apoptotic chromatin condensation process in isolated nuclei by simultaneously visualizing kinetochores with EGFP and chromatin with a fluorescent dye, SYTO 59.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sugimoto, K., Senda-Murata, K., and Oka, S. (2008) Construction of three quadruple-fluorescent MDA435 cell lines that enable monitoring of the whole chromosome segregation process in the living state. Mut Res 657, 56–62.
Brenner S., Pepper, D., Berns, M.W., Tan, E., and Brinkley, B. (1981) Kineochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients. J Cell Biol. 91, 95–102.
Rieder, C.L. (1982) The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber. Int Rev Cytol 79, 1–58.
Shaner N.C., Steinbach, P.A. and Tsien R.Y. (2004) A guide to choosing fluorescent proteins. Nat Methods 2, 905–909.
Wyllie, A.H., Morris, R.G., Smith, A.L., and Dunlop, D. (1984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular systheswis. J Pathol 142, 67–77.
Lazebnik, Y.A., Cole, S., Cooke, C.A., Nelson, W.G., and Earnshaw, W.C. (1993) Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis, J Cell Biol 123, 7–22.
Samejima, K., Tone, S., Kottke, T.J., Enari, M., Sakahira, H., Cooke, C.A., Durrieu, F., Martins, L.M., Nagata, S., Kaufmann, S.H. and Earnshaw, W.C. (1998) Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution, J Cell Biol 143, 225–239.
Liu, X., Kim, C.N., Yang, J., Jemmerson, R., and Wang, X. (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c, Cell 86, 147–157.
Widlak, P., Palyvoda, O., Kumala, S., and Garrard, W.T. (2002) Modeling apoptotic chromatin condensation in normal cell nuclei. Requirement for intranuclear mobility and actin involvement, J Biol Chem 277, 21683–21690.
Tone, S., Sugimoto, K., Tanda, K., Suda, T., Uehira, K., Kanouchi, H., Samejima, K., Minatogawa, Y. and Earnshaw, W.C. (2007) Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis, Exp Cell Res 313, 3635–3644.
Sugimoto, K., Fukuda, R. and Himeno, M. (2000) Centromere/kinetochore localization of human centromere protein A (CENP-A) exogeneously expressed as a fusion to green fluorescent protein. Cell Struct Funct 25, 253–261.
Sugimoto, K., Urano, T., Zushi, H., Inoue, K., Tasaka, H., Tachibana, M. and Dotsu, M. (2002) Molecular dynamics of Aurora-A kinase in living mitotic cells simultaneously visualized with histone H3 and nuclear membrane protein importin-alpha. Cell Struct Funct 27, 457–467.
Sugimoto, K. (2006) Setting up a fluorescent microscope for live cell imaging in three colors. Jikken Igaku (Expeimental Medicine, in Japanese), 24, 87–92.
Fukada, T., Inoue, K., Urano, T., and Sugimoto, K. (2004) Visualization of chromosomes and nuclear envelope in living cells for molecular dynamics studies. BioTechniques 37, 552–556.
Sugimoto, K. (2006) Live cell imaging analysis of three-color cell-lines. Jikken Igaku (Experimental Medicine, in Japanese), 24, 397–401.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sugimoto, K., Tone, S. (2010). Imaging of Mitotic Cell Division and Apoptotic Intra-Nuclear Processes in Multicolor. In: Papkovsky, D. (eds) Live Cell Imaging. Methods in Molecular Biology, vol 591. Humana Press. https://doi.org/10.1007/978-1-60761-404-3_8
Download citation
DOI: https://doi.org/10.1007/978-1-60761-404-3_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-403-6
Online ISBN: 978-1-60761-404-3
eBook Packages: Springer Protocols