Abstract
Fluorescence live microscopy is a powerful technique to study complex cellular dynamics such as cell division. The availability of fluorescent markers based on GFP fusion proteins for virtually any cellular structure allows efficient visualization of specific processes, and the combination of different fluorophores can be used to study their coordination. In this chapter, we present methods for automated live cell microscopy to study mitotic gene function systematically and in high throughput. In particular, we provide protocols for efficient generation of fluorescent reporter cell lines stably expressing combinations of cellular markers, and provide detailed guidelines for optimizing imaging protocols for automated long-term live microscopy.
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References
Gerlich, D., Beaudouin, J., Gebhard, M., Ellenberg, J., and Eils, R. (2001) Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells. Nat Cell Biol 3, 852–5.
Gerlich, D. and Ellenberg, J. (2003) 4D imaging to assay complex dynamics in live specimens. Nat Cell Biol Suppl, S14–9.
Dailey, M.E., Manders, E., Soll, D.R., and Terasaki, M. (2006) Confocal Microscopy of Living Cells, in Handbook of Biological Confocal Microscopy (Pawley, J.B., ed.), Springer, New York, NY, pp. 381–403.
Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., and Wright, W.E. (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–52.
Neumann, B., Held, M., Liebel, U., Erfle, H., Rogers, P., Pepperkok, R., and Ellenberg, J. (2006) High-throughput RNAi screening by time-lapse imaging of live human cells. Nat Methods 3, 385–90.
Kanda, T., Sullivan, K.F., and Wahl, G.M. (1998) Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol 8, 377–85.
Snapp, E.L., Hegde, R.S., Francolini, M., Lombardo, F., Colombo, S., Pedrazzini, E., Borgese, N., and Lippincott-Schwartz, J. (2003) Formation of stacked ER cisternae by low affinity protein interactions. J Cell Biol 163, 257–69.
Shu, X., Shaner, N.C., Yarbrough, C.A., Tsien, R.Y., and Remington, S.J. (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–47.
Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., and Tsien, R.Y. (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99, 7877–82.
Stepanova, T., Slemmer, J., Hoogenraad, C.C., Lansbergen, G., Dortland, B., De Zeeuw, C.I., Grosveld, F., van Cappellen, G., Akhmanova, A., and Galjart, N. (2003) Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein). J Neurosci 23, 2655–64.
Piel, M., Meyer, P., Khodjakov, A., Rieder, C.L., and Bornens, M. (2000) The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J Cell Biol 149, 317–30.
Sugimoto, K., Fukuda, R., and Himeno, M. (2000) Centromere/kinetochore localization of human centromere protein A (CENP-A) exogenously expressed as a fusion to green fluorescent protein. Cell Struct Funct 25, 253–61.
Zacharias, D.A., Violin, J.D., Newton, A.C., and Tsien, R.Y. (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–6.
Leonhardt, H., Rahn, H.P., Weinzierl, P., Sporbert, A., Cremer, T., Zink, D., and Cardoso, M.C. (2000) Dynamics of DNA replication factories in living cells. J Cell Biol 149, 271–80.
Zaal, K.J., Smith, C.L., Polishchuk, R.S., Altan, N., Cole, N.B., Ellenberg, J., Hirschberg, K., Presley, J.F., Roberts, T.H., Siggia, E., Phair, R.D., and Lippincott-Schwartz, J. (1999) Golgi membranes are absorbed into and reemerge from the ER during mitosis. Cell 99, 589–601.
Terasaki, M., Jaffe, L.A., Hunnicutt, G.R., and Hammer, J.A., 3rd (1996) Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein. Dev Biol 179, 320–8.
Rizzuto, R., Brini, M., De Giorgi, F., Rossi, R., Heim, R., Tsien, R.Y., and Pozzan, T., et al. (1996) Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr Biol 6, 183–8.
Muhlhausser, P. and Kutay, U. (2007) An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown. J Cell Biol 178, 595–610.
Rao, P.N. and Engelberg, J. (1965) Hela cells: Effects of temperature on the life cycle. Science 148, 1092–4.
Rieder, C.L. and Cole, R.W. (2002) Cold-shock and the Mammalian cell cycle. Cell Cycle 1, 169–75.
Waters, J.C. (2007) Live-cell fluorescence imaging. Methods Cell Biol 81, 115–40.
Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G.H. (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat Cell Biol Suppl, S7–14.
Swedlow, J.R. (2007) Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol 81, 447–65.
Abramowitz, M., Spring, K.R., Keller, H.E., and Davidson, M.W. (2002) Basic principles of microscope objectives. Biotechniques 33, 772–4, 776–8, 780–1.
Keller, H.E. (2006) Objective lenses for confocal microscopy, in Handbook of Biological Confocal Microscopy (Pawley, J.B., ed.), Springer, New York, pp. 145–161.
Inoué, S. and Spring, K. (1986) Video Microscopy. Plenum Press, New York.
Rabut, G. and Ellenberg, J. (2004) Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J Microsc 216, 131–7.
Acknowledgments
We thank P. Steigemann for helpful discussions and critical reading of this manuscript. We thank M. Held for software programming and S. Maar for technical help. We thank G. Csucs and J. Kusch from the light microscopy center for technical support; Toni Lehmann for making LabTek stage holders, and technical assistance; R. Y. Tsien for mRFP, mCherry, and MyrPalm-EYFP; J. Lippincott-Schwartz for mEGFP; J. Ellenberg for H2B-mRFP; and S. Narumiya for HeLa “Kyoto” cells. This work was supported by a European Young Investigator (EURYI) award of the European Science Foundation to D.G., and a Roche Ph.D. fellowship to M.S. M. S. is a fellow of the Life Science Zurich Graduate School, Zurich, Switzerland.
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Schmitz, M.H., Gerlich, D.W. (2009). Automated Live Microscopy to Study Mitotic Gene Function in Fluorescent Reporter Cell Lines. In: McAinsh, A. (eds) Mitosis. Methods in Molecular Biology, vol 545. Humana Press. https://doi.org/10.1007/978-1-60327-993-2_7
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