Abstract
This protocol describes how to perform comparative measurements of the permeability of the nuclear envelope in adherent cells. The plasma membrane is permeabilized at low digitonin concentrations, leaving the nuclear membrane intact. These semi-permeabilized cells are incubated with cytosolic extracts prepared in advance and with a fluorescent reporter molecule whose molecular weight exceeds the size-exclusion limit of the nuclear envelope. Images are taken with a confocal microscope and subsequently analyzed using a custom-made software program that recognizes the nuclei automatically and calculates the mean nuclear fluorescence signal. Here, we measure the increase in nuclear permeability triggered by cytosolic extracts from cells dying by apoptosis. This method can be employed for the study of processes that affect the nucleocytoplasmic distribution of fluorescent molecules in cell populations. The large size of the samples means that subtle fluctuations in nuclear fluorescence can be detected with a high confidence level. Isolation of cell extracts takes 5–6 h, and the preparation and imaging of 15 or so specimens takes 4–5 h.
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References
Fried, H. & Kutay, U. Nucleocytoplasmic transport: taking an inventory. Cell Mol. Life Sci. 60, 1659–1688 (2003).
Tran, E.J. & Wente, S.R. Dynamic nuclear pore complexes: life on the edge. Cell 125, 1041–1053 (2006).
Ferrando-May, E. Nucleocytoplasmic transport in apoptosis. Cell Death Differ. 12, 1263–1276 (2005).
Kodiha, M., Chu, A., Matusiewicz, N. & Stochaj, U. Multiple mechanisms promote the inhibition of classical nuclear import upon exposure to severe oxidative stress. Cell Death Differ. 11, 862–874 (2004).
Faleiro, L. & Lazebnik, Y. Caspases disrupt the nuclear-cytoplasmic barrier. J. Cell Biol. 151, 951–959 (2000).
Patre, M. et al. Caspases target only two architectural components within the core structure of the nuclear pore complex. J. Biol. Chem. 281, 1296–1304 (2006).
Belov, G.A. et al. Bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores. J. Virol. 78, 10166–10177 (2004).
Makhnevych, T., Lusk, C.P., Anderson, A.M., Aitchison, J.D. & Wozniak, R.W. Cell cycle regulated transport controlled by alterations in the nuclear pore complex. Cell 115, 813–823 (2003).
Jiang, L.W. & Schindler, M. Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape. J. Cell Biol. 106, 13–19 (1988).
Feldherr, C.M. & Akin, D. Regulation of nuclear transport in proliferating and quiescent cells. Exp. Cell Res. 205, 179–186 (1993).
Adam, S.A., Marr, R.S. & Gerace, L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111, 807–816 (1990).
Ribbeck, K. & Gorlich, D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20, 1320–1330 (2001).
Ribbeck, K. & Gorlich, D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. EMBO J. 21, 2664–2671 (2002).
Ferrando-May, E. et al. Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis. Cell Death Differ. 8, 495–505 (2001).
Roehrig, S., Tabbert, A. & Ferrando-May, E. In vitro measurement of nuclear permeability changes in apoptosis. Anal. Biochem. 318, 244–253 (2003).
Lenart, P. & Ellenberg, J. Monitoring the permeability of the nuclear envelope during the cell cycle. Methods 38, 17–24 (2006).
Acknowledgements
We thank Marijke Baldock for technical assistance and DatInf GmbH for software development. This work is supported by a grant from the Deutsche Forschungsgemeinschaft to E.F.-M. (MA 2385/4).
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Grote, P., Ferrando-May, E. In vitro assay for the quantitation of apoptosis-induced alterations of nuclear envelope permeability. Nat Protoc 1, 3034–3040 (2006). https://doi.org/10.1038/nprot.2006.460
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DOI: https://doi.org/10.1038/nprot.2006.460
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