Nuclear Membrane Disassembly and Rupture Laura Cotter 1 , Terence D. Allen 1 , Elena Kiseleva 2 and Martin W. Goldberg 3 ⁎ 1 Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK 2 Laboratory of Morphology and Function of Cell Structure, Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, 630090, Russia 3 School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK The nuclear envelope consists of two membranes traversed by nuclear pore complexes. The outer membrane is continuous with the endoplasmic reticu- lum. At mitosis nuclear pore complexes are dismantled and membranes disperse. The mechanism of dispersal is controversial: one view is that membranes feed into the endoplasmic reticulum, another is that they vesiculate. Using Xenopus egg extracts, nuclei have been assembled and then induced to breakdown by addition of metaphase extract. Field emission scanning electron microscopy was used to study disassembly. Strikingly, endoplasmic reticulum-like membrane tubules form from the nuclear surface after the addition of metaphase extracts, but vesicles were also observed. Microtubule inhibitors slowed but did not prevent membrane removal, whereas Brefeldin A, which inhibits vesicle formation, stops mem- brane disassembly, suggesting that vesiculation is necessary. Structures that looked like coated buds were observed and buds were labelled for β-COP. We show that nuclear pore complexes are dismantled and the pore closed prior to membrane rupturing, suggesting that rupturing is an active process rather than a result of enlargement of nuclear pores. © 2007 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: nuclear envelope; pore complex; disassembly Introduction The nuclear envelope (NE) breaks down and reforms each cell cycle. The NE consists of the inner nuclear membrane (INM), the outer nuclear membrane (ONM), which are separated by the peri- nuclear space and punctuated by nuclear pore complexes (NPCs). The ONM shares proteins and properties with the endoplasmic reticulum (ER), whose lumen is continuous with it. The INM con- tains a distinct set of proteins, such as lamin B receptor (LBR) and LAP2 and the nuclear lamina. 1 The lamina is a protein network thought to pro- vide a structural framework for the NE and an an- choring site for interphase chromosomes but may also have a more active role in controlling nuclear organisation. 2 Movement of macromolecules across the NE is regulated through the NPCs. NPCs are channels of ∼120 MDa and ∼120 nm diameter in vertebrates 3 and may be smaller in yeast, 4,5 but in many respects are conserved. The NPC has an 8-fold rotational symmetry and comprises multiple copies of ∼30 or more 6,7 nucleoporins. The struc- tural framework of the NPC consists of the spoke- ring complex, the central transporter, 8 cytoplasmic and nucleoplasmic rings, attached cytoplasmic filaments and the nuclear basket. 9–14 At mitosis, the NE membranes, NPCs and lamina are disassembled. They are then recycled to form new NEs around the chromatin in telophase and early G1 phase. NE assembly has been characterised in a variety of model systems including Xenopus egg extracts. 15 When sperm chromatin is added to interphase extracts it decondenses, membrane vesi- cles bind 16–18 in a cell-cycle regulated manner. 18,19 Once bound, vesicles fuse to form a double membrane, NPCs assemble in flattened patches, and the membranes undergo “smoothing” which requires nuclear transport. 17,20 NPC assembly is a stepwise process via intermediates such as dimples, pores, star-rings, thin rings and the cytoplasmic ring with cytoplasmic filaments. 10,21,22 Older electron microscopy data suggested the formation of NPC- like structures (“pre-pores”) on the chromosome Abbreviations used: NE, nuclear envelope; INM, inner nuclear membrane; ONM, outer nuclear membrane; NPC, nuclear pore complexe; ER, endoplasmic reticulum; LBR, lamin B receptor; ARF, ADP-ribosylation factor; GFP, green fluorescent protein; feSEM, field emission scanning electron microscopy. E-mail address of the corresponding author: m.w.goldberg@durham.ac.uk doi:10.1016/j.jmb.2007.03.051 J. Mol. Biol. (2007) 369, 683–695 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.