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Published online
doi:10.1083/jcb.200703002
The Journal of Cell Biology, Vol. 178, No. 4, 595-610
The Rockefeller University Press, 0021-9525 $30.00
© Mühlhäusser et al.
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Article

An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown



Petra Mühlhäusser and Ulrike Kutay

Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland

Correspondence to Ulrike Kutay: ulrike.kutay{at}bc.biol.ethz.ch

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein–tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.

Abbreviations used in this paper: CSF, cytostatic factor; IBB, importin ß–binding domain; INM, inner nuclear membrane; LAP2ß, lamina-associated polypeptide 2ß; NE, nuclear envelope; NEBD, NE breakdown; NPC, nuclear pore complex; PB, permeabilization buffer; RanGAP, RanGTPase-activating protein.


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