Abstract
Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope breakdown, one of the major morphological changes of mitosis. Here, we describe such a strategy in which the plasma membrane of cells expressing fluorescently tagged nucleoporin POM121 and Histone H2B is permeabilized with digitonin. These cells are then incubated with mitotic Xenopus egg extract to create conditions that recapitulate the major events of mitotic nuclear remodeling seen in live-cell imaging, providing the opportunity to probe mechanisms and pathways that coordinate nuclear disassembly.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Guttinger S, Laurell E, Kutay U (2009) Orchestrating nuclear envelope disassembly and reassembly during mitosis. Nat Rev Mol Cell Biol 10:178–191
Prunuske AJ, Ullman KS (2006) The nuclear envelope: form and reformation. Curr Opin Cell Biol 18:108–116
Cotter L, Allen TD, Kiseleva E, Goldberg MW (2007) Nuclear membrane disassembly and rupture. J Mol Biol 369:683–695
Georgatos SD, Pyrpasopoulou A, Theodoropoulos PA (1997) Nuclear envelope breakdown in mammalian cells involves stepwise lamina disassembly and microtubule-drive deformation of the nuclear membrane. J Cell Sci 110(pt 17):2129–2140
Beaudouin J, Gerlich D, Daigle N, Eils R, Ellenberg J (2002) Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108:83–96
Salina D, Bodoor K, Eckley DM, Schroer TA, Rattner JB, Burke B (2002) Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell 108:97–107
Muhlhausser P, 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
Dultz E, Zanin E, Wurzenberger C, Braun M, Rabut G, Sironi L, Ellenberg J (2008) Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J Cell Biol 180:857–865
Lenart P, Rabut G, Daigle N, Hand AR, Terasaki M, Ellenberg J (2003) Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes. J Cell Biol 160:1055–1068
Liu J, Prunuske AJ, Fager AM, Ullman KS (2003) The COPI complex functions in nuclear envelope breakdown and is recruited by the nucleoporin Nup153. Dev Cell 5:487–498
Murray AW, Kirschner MW (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339:275–280
Blow JJ, Laskey RA (1988) A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature 332:546–548
Newmeyer DD, Forbes DJ (1988) Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell 52:641–653
Hetzer M, Meyer HH, Walther TC, Bilbao-Cortes D, Warren G, Mattaj IW (2001) Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly. Nat Cell Biol 3:1086–1091
Volkova EG, Kurchashova SY, Polyakov VY, Sheval EV (2011) Self-organization of cellular structures induced by the overexpression of nuclear envelope proteins: a correlative light and electron microscopy study. J Electron Microsc (Tokyo) 60:57–71
Polychronidou M, Hellwig A, Grosshans J (2010) Farnesylated nuclear proteins Kugelkern and lamin Dm0 affect nuclear morphology by directly interacting with the nuclear membrane. Mol Biol Cell 21:3409–3420
Higa MM, Ullman KS, Prunuske AJ (2006) Studying nuclear disassembly in vitro using Xenopus egg extract. Methods 39:284–290
Powers M, Evans EK, Yang J, Kornbluth S. (2001) Preparation and use of interphase Xenopus egg extracts. Curr Protoc Cell Biol 11:11.10
Kihlmark M, Imreh G, Hallberg E (2001) Sequential degradation of proteins from the nuclear envelope during apoptosis. J Cell Sci 114:3643–3653
Mackay DR, Makise M, Ullman KS (2010) Defects in nuclear pore assembly lead to activation of an Aurora B-mediated abscission checkpoint. J Cell Biol 191:923–931
Lenart P, Ellenberg J (2006) Monitoring the permeability of the nuclear envelope during the cell cycle. Methods 38:17–24
Acknowledgements
We thank Drs. Don Ayer, Einar Hallberg, Mary Dasso, and Maureen Powers for reagents and Drs. Mark Smith and Chris Rodesch and the University of Utah Fluorescence Microscopy Core Facility for assistance. Core facilities are supported in part by a grant (P30 CA042014) awarded to the Huntsman Cancer Institute. This work was funded by the National Institute of Health (grant R01 GM61275) and the Huntsman Cancer Foundation (to K.S.U.). D.R.M. is supported by an NIH Developmental Biology Training Grant (5T32 HD07491).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Shankaran, S.S., Mackay, D.R., Ullman, K.S. (2012). A Time-Lapse Imaging Assay to Study Nuclear Envelope Breakdown. In: Taatjes, D., Roth, J. (eds) Cell Imaging Techniques. Methods in Molecular Biology, vol 931. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-056-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-62703-056-4_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-055-7
Online ISBN: 978-1-62703-056-4
eBook Packages: Springer Protocols