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The nuclear envelope (NE) acts as a selective barrier around the genome and as a ... during mitosis indicates new mechanisms by which nuclear membrane ...
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REMODELLING THE WALLS OF THE NUCLEUS Brian Burke* and Jan Ellenberg ‡ The nuclear envelope (NE) acts as a selective barrier around the genome and as a scaffold to organize DNA in the nucleus. During cell division, the NE is broken down and chromosome confinement is taken over by microtubules. After chromosome segregation, a new NE is reassembled in each daughter cell. In this complex cycle of disassembly and reassembly, the fate of the NE is intimately linked to the activity of the mitotic spindle. The finding that components of the nuclear membrane become distributed throughout a continuous endoplasmic reticulum during mitosis indicates new mechanisms by which nuclear membrane domains are established, and highlights unique problems in the establishment of NE topology. LAMINS

Lamins are rod-shaped proteins of the intermediate filament class. They consist of a head and tail domain that flank a conserved α-helical rod domain. Lamins form parallel homoand probably heterodimers which, in turn, can polymerize in a head-to-tail fashion. These linear polymers are thought to associate laterally into 10-nm lamin fibres, which form the fibrous lamina meshwork in the nuclear periphery.

*Department of Anatomy and Cell Biology, The University of Florida, Gainesville, Florida 32610, USA. ‡Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory, D-69117, Heidelberg, Germany. Correspondence to J.E. e-mail: jan.ellenberg@ embl-heidelberg.de doi:10.1038/nrm860

Intracellular compartmentalization has provided eukaryotes with unique opportunities for the regulation of many cellular processes. Nowhere is this more obvious than in the enclosure of the chromosomes in a nuclear envelope (NE) This effectively separates — both temporally and spatially — the nuclear processes of gene transcription and replication from translation, which is generally a cytoplasmic event. Clearly, control of communication between the nucleus and cytoplasm is a prerequisite for normal eukaryotic cell function. This enhanced regulatory potential of eukaryotes, however, comes at the price of greatly complicating the mechanics of cell division. To complete mitosis successfully, microtubules of the mitotic spindle must gain access to the chromosomes. To accomplish this, the spindle can either be assembled in the nucleus, as occurs in yeast, or the NE can be partially or completely broken down to allow the chromosomes to engage with a cytoplasmic spindle. This second option is referred to as ‘open mitosis’ and, in vertebrate cells, involves the disassembly and dispersal of all the main elements of the NE. On completion of mitosis, components of the NE are reused in the formation of nuclei within each daughter cell. The breakdown and subsequent reassembly of the NE is remarkably complex, and involves coordination of activities as diverse as membrane fusion and microtubule-based movement. In recent years, a consensus

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model of nuclear dynamics has been formulated that accounts for virtually all changes in NE architecture that occur during mitosis1–5. This article provides a synopsis of this new view of NE dynamics in M phase and the molecular mechanisms that underlie them, and focuses mainly on results from vertebrate systems. The nuclear envelope protein network

A complete catalogue of the protein composition of the NE in vertebrates is not yet available, but proteomics approaches are starting to fill the gaps6,7. It is now clear that we should add chromosomes to the three classic NE components (BOX 1), and we can then define four main classes of proteins that organize the nuclear periphery: LAMINS, inner nuclear membrane (INM) proteins, nuclear pore complex (NPC) proteins and peripheral chromatin proteins (see TABLE 1 and FIG. 1). Class I. Class I comprises the nuclear lamins, which are type-V INTERMEDIATE-FILAMENT PROTEINS that can be subdivided into B-type (ubiquitous) and A/C-type lamins (found only in differentiated cells) on the basis of their primary sequence8,9. These proteins have a well-established function. They form a peripheral fibrous meshwork — the nuclear lamina — that supports the INM10. Intranuclear lamins have proposed functions in DNA replication11,12, RNA polymerase II transcription13 and nuclear organization14.

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Box 1 | Architectural concepts of the nuclear envelope over the past 100 years

INTERMEDIATE FILAMENT PROTEINS

Intermediate filaments are protein fibres with a diameter of ~10 nm that represent the third class of cytoskeletal polymers, after microtubules and actin. Intermediate filaments can be subdivided into five classes. Classes I–IV are found in the cytoplasm and contain, for example, keratins, neurofilaments and vimentin. Class V is nuclear and is comprised exclusively of nuclear lamins. METAZOANS

The nuclear envelope (NE) has been defined to consist of three distinct structural components — the nuclear membranes, the nuclear pores and the underlying nuclear lamina111 (FIG. 1). In the 1960s, fractionation and purification of the NE from cells became possible112, and we have learned much about its molecular composition since then. This understanding began with biochemical studies on lamins9, nuclear membrane proteins later 2,113 and, in the case of yeast, has culminated in an almost complete inventory of the nuclear pore complex (NPC) proteins7,25. The first insight about the fate of the NE in dividing cells came from transmitted-light-microscopy observations in the early days of cell biology114, when fading and re-emergence of membranous material — ‘Kernbläschen’ (or nuclear bubbles) — on the chromosome surface was observed in dividing cells of echinoderm embryos115. Six decades later, pioneering electron-microscopic studies on mitotic plant and animal cells57,58,116 started to address the fate of NE components during the open mitosis of METAZOANS at the ultrastructural level. Those studies indicated an intimate relationship of nuclear membranes to membrane tubules and CISTERNAE of the endoplasmic reticulum (ER) in dividing cells. In addition, the duplicated CENTROSOMES were noted to be closely associated with the NE and often buried in an invagination or ‘Hof ’ in prophase58,116. The next big step in mechanistic understanding came in the 1980s, with the finding that NE proteins are phosphorylated specifically in mitosis40. Nuclear assembly and breakdown were reconstituted in amphibian oocyte extracts68,117 — a system that led to a wealth of biochemical data. The fragmented membrane homogenates of this system led to the conclusion that membrane vesicles function as the natural precursors for nuclear assembly. Curiously, the studies in oocyte extracts largely ignored the earlier electron-microscope observations in somatic cells, except for a more contemporary study118, which indicated that, in rat thyroid gland cells, the ER-membrane network is broken down into small, round fragments. On this basis, such membrane vesiculation and fusion of vesicles to reform larger membrane surfaces was proposed as the primary mechanism of nuclear remodelling33. Only recently have studies in intact somatic cells revisited the fate of NE components in mitosis, and shown that the ER serves as the reservoir for nuclear membrane proteins in M phase19,30,44. Given this turbulent history, we are quite likely to witness the emergence of new concepts or rediscoveries of long-forgotten ones for other aspects of nuclear dynamics in the future.

Organisms that consist of more than one cell. CISTERNAE

Flat sheets of endoplasmic reticulum that enclose a lumen like a hollow pancake. CENTROSOMES

The microtubule-organizing centres in animal cells. NUCLEAR BASKET

A fishtrap-like structure on the nuclear side of the nuclear pore that is made up of eight fibrils joined by a distal ring. FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING

(FRAP). A technique in which a pool of fluorescent molecules is destroyed locally by highintensity laser irradiation. After this ‘photobleach’, the exchange of the non-fluorescent molecules with the surrounding fluorescent molecules is monitored and measured as ‘recovery’ of fluorescence in the bleached area. M-PHASE PROMOTING FACTOR

(MPF). The complex of a B-type cyclin and cyclin-dependent kinase 1, which is also referred to as cdc2 or p34, depending on the species. Only the complex of both proteins in a specific state of phosphorylation is active, and this is the main enzyme that is responsible for entry into M phase in both meiosis and mitosis.

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Class II. Class II NE components consist of a growing list of INM proteins and protein families (TABLE 1; FIG. 1). These specialized integral membrane proteins are found specifically in the INM and not (or only at low levels) in the endoplasmic reticulum (ER) and the secretory pathway. Most of these proteins act as adaptors that link the INM to the lamina and/or chromatin by direct interactions through their nucleoplasmic domains2,15. The nucleoplasmic domains contain several sequence motifs that mediate these interactions. The nuclear magnetic resonance (NMR) structure of one of these domains, the LEM domain (found in LAP2, emerin and MAN1; TABLE 1), shows that variations of this domain can bind directly to either DNA or a chromatin protein16,17. The adaptor function of these proteins provides a mechanism for their appropriate localization on the basis of selective retention. In this scheme, proteins that are mobile within the ER can access the INM through the membrane continuities at the periphery of each NPC — this is a signal-independent process. However, only those proteins that can specifically interact with nuclear components, lamins or chromatin, for example, are retained and concentrated. This model predicts that integral INM proteins should show a reduced mobility on localization to the NE and, indeed, this has been confirmed in dynamic imaging studies of live cells that express several green-fluorescent-protein (GFP)-tagged INM proteins18–20. Further support for the model is provided by the finding that localization of emerin to the INM depends, in part, on the expression of A-type lamins21,22 and that it can be inhibited by cytoplasmic domains that are too bulky to fit through the periphery of the NPC23. The concept of selective retention is also

important in nuclear membrane dynamics in mitotic cells (see below and BOX 2). Class III. Class III comprises NPC proteins, also known as nucleoporins. This group contains two transmembrane, and more than 20 soluble proteins that constitute the NPC (FIG. 1). This very large protein complex forms an aqueous transport channel through the NE, and thereby connects the inner and outer nuclear membranes (ONM) with a domain that is also referred to as the pore membrane (POM). The NPC has an estimated maximum mass of 125 MDa (REF. 24). It resembles a flat, hollow cylinder that is embedded in the NE (termed the ‘spoke-ring complex’) and is ~120 nm wide and ~40 nm long. This cylinder contains a central ~40-nm-wide channel, which contains proteinaceous material that is sometimes referred to as the ‘central plug’. From the walls of the cylinder emanate eight cytoplasmic and nuclear filaments, and the latter are joined by a distal ring to form the NUCLEAR 25 BASKET . The function of the NPC is nucleo-cytoplasmic transport, but it is also an important structural unit of the NE, and is probably involved in nuclear organization in general26,27. Unlike the INM proteins, the two transmembrane nucleoporins gp210 and POM121 interact only with other nucleoporins, and not with nuclear lamins or chromosomes. However, soluble nucleoporins of the nuclear basket bind to both lamins and chromatin26,28, and the lamina directly connects to the nucleoplasmic face of the spoke-ring complex29. Class IV. Class IV consists of chromatin proteins that mediate interactions of peripheral chromatin with members of the lamina and/or INM-protein classes.

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Table 1 | Proteins that organize the nuclear envelope Protein class

Name

Interacts with

Lamins

Lamin A and B Lamin A Lamin B

LAP1 and 2 families, histones, DNA Emerin, pRB LBR, LAP2β

Reference

Integral INM

LBR LAP1α, β and γ LAP2 β LAP2 γ, δ and ε Emerin MAN1 Nurim LUMA RFBP Nesprins

Lamin B, DNA, HP1 Lamins Lamin B, DNA, BAF Lamins Lamin A, Lamin B, BAF BAF? ? ? ? Actin?

NPC

Nup153 gp210 POM121

Lamin B Nuclear membranes Nuclear membranes

Chromatin

BAF HP1 α, β and γ LAP2α H2A/H2B Methyl H3

*(DNA), emerin, MAN1, LAP2β (Methyl H3), LBR (DNA), lamin A (DNA), lamins LBR?

15,119 –121 15,122,123 15 15 15 15 15 15,123 15 6 124 125,126 28,102 97 127 15,123 15,128 15 15 129, but see 130,131

*Parentheses denote interactions that group a protein into one of the four classes of nuclear envelope protein. These interactions do not link two structural elements of the nuclear envelope.

this organization is that the NE is a complex crosslinked structural protein network. Recently, the dynamic and mechanical properties of this network have been addressed by time-lapse imaging and FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP) of GFP-tagged proteins in living cells. This approach found the lamina and pores to be immobile and stably bound for many hours in interphase NEs30,31, and could show that the NE behaves as an elastic, mechanically quite stable entity30,32. The stable structure of the NE, which encloses and is connected to the chromosomes, poses a fundamental problem for cell division in metazoan cells, in which the microtubules remain exclusively cytoplasmic throughout the cell cycle. For spindle microtubules to gain access to chromosomes, these cells have to undergo an open mitosis, and break down their NE at the onset of M phase. After the spindle has accomplished chromosome segregation, new NEs have to be assembled quickly in the daughter cells (FIG. 2). Although our insight into NE assembly has grown considerably over the past five years, we are only starting to work out the mechanism of NE breakdown (NEBD). Tearing down the walls: NE breakdown

This class is small, and comprises three isoforms of heterochromatin protein 1, two other peripheral chromatin proteins, and core histones (TABLE 1; FIG. 1). In summary, a network of direct and indirect protein–protein interactions connects the four structural units of the nuclear periphery — the lamina, INM, nuclear pores and chromosomes. The prediction from

In dividing cells, the NE is ruptured during NEBD, which defines the transition from prophase to prometaphase in the cell-division cycle33 (FIGS 2b,c). According to the textbook model33, the NE vesiculates after the lamina has depolymerized as a result of mitotic phosphorylation. However, studies in several systems point to a different mechanism that involves mechanical interactions of the NE, with spindle microtubules and disassembly of the NPC being key steps in NEBD 32,34–37.

Cytoplasm

a Nuclear pore proteins

b INM proteins c Lamins d Chromatin proteins

Nucleus

50 nm

Figure 1 | Topology of the nuclear envelope. The nuclear envelope (NE) consists of four structural units. The inner and outer nuclear membranes (light yellow) are joined at the nuclear pore complex (light purple). The inner nuclear membrane (INM) is anchored by transmembrane proteins to the underlying layer of lamina (light green) and to the peripheral chromatin (dark red). The right side shows examples from each of the four classes of NE protein (labelled a–d). Nucleoporins are represented by the soluble protein Nup358 (RanBP2, dark purple), a main part of the cytoplasmic filaments, and gp210 (dark purple), a transmembrane protein with a large lumenal domain in the perinuclear space. LAP2 (yellow) represents INM proteins, and has a transmembrane domain, a lamin-interacting region, and two LEM domains that interact with DNA and with the chromatin protein, which is represented by BAF (light red). These (and further) mutual interactions between all four structural units form the highly crosslinked structural protein network of the NE. The figure is drawn approximately to the known molecular scale of the structures and molecules that are shown.

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Mitotic phosphorylation of NE proteins. Many biochemical studies have shown that NE proteins are subject to mitotic phosphorylation by the M-PHASE PROMOTING cdc2 FACTOR (MPF) kinase p34 . This is believed to abolish the structural protein–protein interactions that are necessary for NE integrity, and lead to its disassembly. Nuclear lamins are phosphorylated by protein kinase C and, subsequently, by cdc2, which results in their depolymerization38,39 and dispersal in mitotic cells30,40,41. Nucleoporins42,43 and several INM proteins2 are also substrates of cdc2. In the case of the nucleoporins, this presumably causes their release from, and subsequently the disassembly of, the NPC, which explains the dispersed localization of nucleoporins that is observed in mitotic cells30,44,45. The effect of phosphorylation is less clear for INM proteins, but it is also assumed to abolish their ability to interact with lamins and/or chromatin, which allows the INM to detach from chromosomes. Vesiculation versus ER absorption. The fate of nuclear membrane proteins has been controversial, based on results in different experimental systems1,2. Studies that used fractions of Xenopus laevis oocytes have shown that vesicles that are enriched in NE proteins can be isolated and are competent to assemble the NE in vitro46. These studies gave rise to the model that vesiculation is

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Box 2 | Selective retention Selective retention is a mechanism for establishing the steady-state localization of a protein in a subcellular compartment. A protein that has no interactions is free to diffuse and will be distributed homogenously in its compartment, whether this is the cytoplasm or a membrane system, such as the endoplasmic reticulum (ER)–nuclear envelope (NE). However, a protein that can bind to a cellular structure that is accessible from its compartment will be retained at the site of binding. Its concentration there will be determined by the number of binding sites, the strength of the interaction and the ability of the protein to reach the binding site, typically by diffusion. This happens, for example, for cytoplasmic proteins that interact with the cell surface, or for ER membrane proteins that can interact with chromatin. In the latter case, in interphase cells, the transmembrane protein can freely diffuse between the ER and the inner nuclear membrane (INM). In the INM, it has access to its binding site on chromatin, and is therefore retained and concentrated. This mechanism underlies ‘targeting’ of INM proteins, although the movement of individual molecules is driven by non-directional Brownian motion between the ER and INM. In mitosis, the binding to chromatin is switched off by phosphorylation of the membrane protein, which then quickly equilibrates with the ER. At the end of mitosis, dephosphorylation activates the binding again, and selective retention leads to the attachment of ER cisternae that contain the chromatin-binding protein to chromosomes.

FLUORESCENCE RESONANCE ENERGY TRANSFER

(FRET). A technique to measure the proximity of two fluorophores, a donor and an acceptor. If these are within 2–10 nm of each other and if the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, energy can be transferred nonradiatively from acceptor to donor by dipole–dipole coupling or ‘resonance’. The efficiency of the transfer is extremely distance sensitive, so FRET is often referred to as a molecular ruler. DYNEIN

A large, cytoplasmic microtubule-dependent motor protein that converts the energy of ATP into motion towards the minus end of microtubules.

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the fate of nuclear membranes in vivo. But studies in intact mammalian cells have shown that nuclear membrane proteins reside in the ER in metaphase19,30,44, and that the ER itself remains an intact network in dividing somatic cells19,47. This apparent contradiction between vesiculation and ER dispersion has been proposed to reflect the difference between embryonic (Xenopus oocytes) and somatic (mammalian culture cells) systems. However, evidence for vesiculation that was obtained in fractionated HeLa cells48, and for intact ER in echinoderm embryos49, argues against cell-type-specific differences alone. A more plausible explanation is that the extraction procedures that are required to obtain pure membrane fractions in vitro inevitably result in vesiculation of the ER network that is present in the intact cell. This experimentally induced fragmentation of a continuous membrane network would cause transmembrane proteins and lipids with different properties to partition into distinct membrane fragments. This probably reflects their organization into ER subdomains in intact cells — a concept that is quite well accepted for the plasma membrane50. The idea that a membrane network is the precursor for NE assembly is strengthened by the recent finding that, even in Xenopus extracts, membrane homogenates form networks before NE formation51–53. To gain more insight into mitotic and meiotic ER organization, it will be interesting to investigate the properties of INM proteins in the mitotic ER using biophysical methods such as FRAP54 and FLUORESCENCE 55 RESONANCE ENERGY TRANSFER (FRET) . It would also be useful to immunolocalize nuclear membrane proteins in M-phase cells by immunoelectron microscopy, especially in Xenopus eggs. So, in summary, nuclearmembrane vesiculation does not seem to be involved in NEBD in the intact cell systems that have been examined so far.

Does NPC disassembly trigger NEBD? NEBD has recently been studied by imaging germinal vesicle breakdown in live starfish oocytes34, and by electron microscopy of prophase nuclei from syncytial Drosophila melanogaster embryos35. In starfish, there are two phases of NE permeabilization, as measured by the influx of inert fluorescent cytoplasmic molecules: a slow gradual increase followed by a sharp and localized entry wave34. The gradual increase in the permeability coefficient of the NE would be consistent with the slowly progressing disassembly of a pore complex, such as by removal of the central plug. This would make each individual pore leaky, yet leave the NE intact. However, the wave-like entry of material can be explained only by large discontinuities in the NE, such as a fenestration that would result from the complete removal of NPCs. The crescent shape of the wave that enters the nucleus further argues for a rapid expansion or spreading of such fenestrations once they are formed. Curiously, this occurs at only a few sites on the NE surface34. In Drosophila, scanning and transmission electron microscopy of prophase nuclei showed that NPCs lose their cytoplasmic fibrils in prophase (this is possibly preceded by removal of the central plug), whereas the nuclear membrane remains intact35. The data from echinoderms and flies indicate that disassembly of the pore complex could be an initial step and serve as a trigger of NEBD. It will be interesting to see whether a similar process occurs in vertebrate cells, in addition to the dramatic mechanical effects that are observed in this system (see below). Microtubules tear the nuclear lamina. The structure of the NE is affected by mitotic microtubules, which are nucleated by the separating centrosomes in early prophase. Early electron-microscopy studies observed an intimate connection of spindle microtubules and the NE in HeLa cells56,57 and plant cells58, as well as deep, pocket-like distortions of the NE in the centrosomal regions before NEBD 57 (FIG. 2b). A pioneering correlation of video light microscopy and electron microscopy in lily cells58 proposed that pulling and pushing of the spindle apparatus on the NE led to ‘undulations’. A more recent reinvestigation of these structures in mammalian cells found microtubule bundles deep within NE invaginations, and proposed piercing of the NE by microtubules as the mechanism for NEBD37. From studies of microtubule motors, we know that cytoplasmic DYNEIN localizes to the NE of mammalian cells in prophase 59, and that it is required to attach centrosomes to the NE in Caenorhabditis elegans and Drosophila 60–62. The NE–dynein interaction has been interpreted in the context of centrosome positioning and separation, as well as nuclear movement. Regarding the latter, it has been shown that dynein is responsible for nuclear movements in Xenopus extracts, which could be equivalent to female pronuclear movements after fertilization in many oocytes63. Two recent studies have now clarified the involvement of both dynein and microtubules in NEBD32,36. These studies, which used either a combination of live-cell

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DYNACTIN

A complex of several proteins that are associated with dynein, which links the motor to cargo and regulates its activity. MINUS END

The slower growing end of microtubules.

a

imaging and photobleaching, or immunofluorescence and electron microscopy, showed that microtubules facilitate NEBD by literally tearing the NE open (FIG. 3). This mechanism proceeds by the binding of dynein and DYNACTIN to the outer face of the NE in prophase, and subsequent massive distortion of the NE near the centrosomes that are dependent on dynein and microtubules. The MINUS-END-directed force of the dynein–dynactin complex, which is crosslinked to the NE but moves along microtubules towards the spindle poles, generates tension in the NE. As a result, it is

d

b

e

c

f

Figure 2 | Nuclear envelope, microtubules and chromosomes through the cell cycle. Immunofluorescence images of normal rat kidney cells stained for DNA (blue), microtubules (red) and the nuclear envelope (NE) marker POM121 (green). a | Interphase with smooth nuclear rim. b | Prophase with dramatic invaginations of the NE around the centrosomes. c | Prometaphase with fragmented NE. d | Metaphase with dispersed NE. e | Late anaphase with partially assembled NE. f | Cytokinesis with mature daughter nuclei. Scale bar in f, 10 µm.

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folded massively at the centrosomes and stretched further away from the ASTERS, where it finally tears when the tension reaches a critical point (FIGS 3b,c). This tearing site represents a physical discontinuity where NPCs, INM proteins and lamins are absent, and through which cytoplasmic molecules can now rush unhindered into the nucleus. The dispersion of most NE proteins starts only after this tearing event in a stepwise disassembly, with B-type lamins one of the last components to be completely solubilized in late prometaphase (FIGS 3d,e). Interestingly, the interactions between microtubules and the NE do not stop after the tearing, but continue to pull NE fragments away from chromosomes towards the centrosome. This virtually clears the chromosome surface for spindle attachment. In conclusion, recent findings in mammalian cells have arrived at a model that elegantly explains NE deformations and emphasizes that spindle formation and NEBD are by no means separate. Instead, they are intimately related processes that work hand in hand to transfer the safeguarding of chromosomes from membranes to microtubules4 (FIG. 3). What lies ahead? In the future, it will be interesting to see whether — and how — NPC disassembly is coordinated with NE tearing in mammalian cells. In addition, it will be important to investigate how widespread the tearing mechanism of NEBD is in other species. It is interesting to speculate that a microtubule-based mechanical system might also be involved in separating other organelles, such as the ER, mitochondria and the Golgi apparatus64. Interestingly, NEBD is not always complete, and there are different degrees of ‘openness’ of mitosis in metazoans. In Drosophila, the NE is broken down only partially35,65, whereas NEBD in C. elegans is delayed until anaphase, but is eventually completed66. By contrast, in sea urchin49 and mammalian cells32,36, NEBD is complete and occurs early in prometaphase. At the same time, the complexity of the NE protein composition increases systematically through evolution67. This could point to more extensive and ‘antimitotic’ connections between NE and chromosomes in vertebrate cells, and might explain the need for a completely open mitosis by mechanical rupture and pulling of the NE to clear the chromosomes and make them available for microtubule attachment. In cells in which the chromosomes are easily detached from the NE by condensation, partial disassembly of the NE would be enough to allow the spindle microtubules efficient access to chromosomes. It is conceivable that, in this way, a mechanism for microtubule–NE interaction, based on dynein–dynactin that initially functioned in centrosome positioning and separation using the NE as a traction surface, evolved into a means of disrupting the NE barrier as it became increasingly rigid with the complex organization of vertebrate genomes. The dynamics of nuclear envelope biogenesis

Until the early 1980s, studies on NE re-formation had focused largely on the ultrastructure of mitotic cells.

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REVIEWS However, in 1983, Lohka and Masui published a landmark paper68 that described the in vitro assembly of authentic NEs around chromatin templates in Xenopus egg extracts. Nuclei that are formed in this way are transport competent and will replicate DNA68–70. These studies spurred the development of other in vitro assembly systems that are based on various cell types71–74, and laid the foundations for a biochemical dissection of assembly.

ASTERS

Many microtubules that are nucleated from one point (such as a centrosome) in a radial manner.

a

b

Tearing

c

d

e

Figure 3 | Model of nuclear envelope breakdown in mammalian cells. The schematic shows a ‘half-cut-open’ nucleus cleared from most of the surrounding endoplasmic reticulum (ER) for clarity. Depicted are chromosomes (purple), lamina (green), nuclear membranes (yellow), microtubules (red) and centrosomes (orange). Nuclear pores are omitted for simplicity. a | Interphase. Decondensed chromosomes are surrounded by the smooth and elastic lamina and the double membrane layer of the nuclear envelope (NE). Connections to the ER are truncated. Centrosomes nucleate long and stable interphase microtubules. b | Prophase. About 30 minutes before NE breakdown, microtubule-filled folds appear in the NE close to the centrosomes, followed by the appearance of condensed chromosomes along the NE. As prophase proceeds, folds in the NE increase in size. At this time, the NE becomes stretched on the side that is opposite to the centrosomes due to pulling forces of minus-end-directed microtubule motor proteins on its surface. c | NE breakdown occurs when the tension has reached a critical point and tears the lamina and the associated membranes, which produces a hole in the NE. Hole formation is followed by nuclear collapse as the tension is released. d | Prometaphase. The initial hole spreads rapidly and fragments the NE. However, large pieces of NE are still attached to chromosomes. e | Metaphase. NE-associated minus-end-directed motors remove NE remnants from the chromosomes towards the centrosomes.

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Forming a sphere from a network. Fractionation of Xenopus extracts has uncovered two biochemically distinct vesicle populations that together are required for nuclear formation46,75,76. Related fractionation studies of mammalian cells also show a heterogeneous microsomal population that is enriched in NE components over ER-specific proteins48. We would argue that these vesicles arise during extract preparation, through fragmentation of an ER network in which NE- and ER-specific proteins might be distributed in a mosaic of microdomains. Nevertheless, they provide a valuable means of examining the contributions of different membrane components to the NE assembly process. Indeed, Vigers and Lohka46 altered the frequency of NPC formation simply by changing the relative concentrations of different membrane fractions in their in vitro reactions. Observations on both fixed and live mammalian mitotic cells indicate that nuclear membrane re-formation involves the coating of newly segregated chromatids with ER-like cisternae — a process that begins during anaphase19,57. The emerging consensus is that membrane attachment is mediated by INM proteins77. In this way, increasing availability of chromatin and/or laminbinding sites leads to the immobilization of more INM proteins and establishment of the INM domain. Differences in the relative timing of binding of various nuclear membrane components, although often cited as evidence for NE-specific vesicles in vivo, would simply reflect the asynchronous appearance of different INMprotein-specific binding sites at the nuclear periphery. In addition, differential affinities of INM proteins for their specific receptors on chromatin, as well as differences in the ability of INM proteins to diffuse rapidly across the ER network, could underlie the ordered assembly. It is important to point out that this view of nuclear re-formation is simply another manifestation of the selectiveretention mechanism for INM protein localization during interphase (BOX 2). Focus on fusion. Even in the absence of NE precursor vesicles in vivo, nuclear re-formation from a membrane network at the end of mitosis must involve extensive membrane fusion (BOX 3). Recent studies from Mattaj, Warren and colleagues53,78 indicate the involvement of several fusion mechanisms. By following NE formation around demembranated sperm chromatin in Xenopus egg extracts, they documented several biochemically separable phases in the establishment of sealed nuclear membranes. Early stages in NE formation involve the appearance of an ER-like membrane network across the

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AAA ATPASE

A family of enzymes that hydrolyse ATP and have a common ATPase module. They typically form ring-shaped oligomers and are involved in diverse cellular functions, such as membrane fusion (for example, p97 and NSF), DNA replication and proteolysis.

surface of the sperm chromatin. This finding is consistent with the idea that, in vivo, the ER forms a mitotic reservoir of nuclear membrane components. The appearance of this network depends on GTP hydrolysis by Ran52,53, a small Ras-related protein that has an important regulatory function in nucleo-cytoplasmic transport, as well as in mitotic spindle assembly. The precise function of Ran in the formation of the chromatin-associated membrane network is not clear. However, it is intriguing that Ran-coated beads can substitute for chromatin as a template for NE assembly in vitro 79. After the appearance of the membrane network, two further steps can be recognized53. The first of these is the formation of a sealed NE; the second involves NE expansion to accommodate chromatin decondensation. Both steps are mediated by an AAA ATPASE called p97. This is closely related to the N-ethyl-maleimide-sensitive fusion protein (NSF), which regulates membranefusion events and is involved in many aspects of vesicular transport80. Originally identified as the product of the CDC48 gene in yeast, p97 and an associated protein, p47, mediate re-formation of the Golgi apparatus at the end of mitosis81, and are also involved in the establishment of transitional ER82,83. Hetzer et al.53 have shown that the final expansion phase of nuclear re-formation in vitro is mediated by p97/47, presumably in association with a receptor protein on the NE. However, for the initial closure or sealing of the NE, the fusion event is mediated by p97 in association with two other proteins, called Ufd1 and Npl4 (REFS 53,78). In yeast, Ufd1, in combination with cdc48, is involved in ubiquitin-dependent protein degradation84,85. Together with Npl4, which was originally identified as an NPC component that is essential for nuclear protein import86, these proteins have been implicated in the ubiquitin-dependent cleavage and activation of membrane-associated transcription factors 87,88. Whether p97–Ufd1–Npl4-mediated nuclear membrane fusion occurs through interactions with receptor molecules on the target membrane or through ubiquitin-dependent activation of another fusion molecule remains to be seen. Why use two separate fusion systems in sealing and expansion of the NE? This question has yet to be resolved, but one suggestion is that the geometry of the fusion reactions dictates two different mechanisms89. To form a sealed NE, it is necessary to close holes or gaps in the double nuclear membranes — a process that demands annular or circular fusion events. At some point, this must involve constriction of holes that span the nuclear membranes, and it is conceptually quite different from the familiar NSF model of vesicle fusion in membrane transport80 (BOX 3). By contrast, during NE expansion in vitro, new membrane components must be incorporated, and it is easy to see how this could be accomplished by p97/47-mediated homotypic fusion of ER elements with the ONM. In this way, p97/47 would catalyse what are basically point fusion events that are equivalent to those that occur in re-formation of the Golgi apparatus81. It is less

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY

Box 3 | Membrane fusion a Point fusion

b Annular fusion

Membrane fusion is central to many cellular activities. It is a tightly regulated process, and it is easy to see why — promiscuous fusion would lead to an intermixing of cellular membranes that would ultimately result in loss of organelle identities. Most intracellular fusion events are controlled by the AAA ATPase family members N-ethyl-maleimidesensitive fusion protein (NSF) or p97, and mainly involve point fusion. This would be exemplified by the fusion of a vesicle with a target organelle, as occurs in the secretory and endocytic pathways. Point fusion could also represent the mechanism of nuclear membrane expansion during nuclear re-formation in vitro (figure, part a). A second type of fusion process — annular fusion — is required for the closure of gaps in the double nuclear membranes. This is fundamentally different to point fusion — it requires the constriction of a membrane ring followed by resolution of the inner nuclear membrane and outer nuclear membrane, as the ring is eliminated by fusion at its cytoplasmic surface (figure, part b). Annular fusion also probably occurs in the fenestrated membranes of the endoplasmic reticulum and Golgi apparatus, as well as in the final step of cytokinesis. Although molecules that are involved in point fusion have been extensively characterized, little is known of the mechanisms of annular fusion.

clear whether this expansion mechanism would operate in vivo. Given their interconnections, the ER could, in principle, feed membrane components directly into the ONM, and thereby eliminate the requirement for p97/p47 in nuclear growth. In addition, the situation is more complex in vivo than the simple assembly of NEs around sperm heads. In frog oocytes and embyros (but also in Drosophila and sea urchin), NEs initially assemble and seal around individual chromosomes to form a set of ‘mini’ nuclei90. These karyomeres are already import and replication competent91; that is, they contain functional nuclear pores. Karyomeres then fuse together in a second step to form one homogeneous nucleus that contains all the chromosomes. It will be interesting to see whether karyomere fusion is

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a Seeding

and that it must involve fusion between the INM and ONM94–96. Although the identity of the fusion apparatus is still unknown, the large NPC membrane protein gp210 is one candidate97. This is because of its large lumenal domain, and the presence of hydrophobic sequences that are similar to the fusogenic peptides that are seen in certain viral-envelope glycoproteins.

b Fusion

NPC seed assembly

Membrane recruitment

Intralumenal fusion

NPC assembly

Figure 4 | Different modes of nuclear pore assembly. a | Nucleoporin-seeded assembly on the chromatin surface (blue) followed by membrane recruitment. b | Pore insertion into a continuous double membrane. Seeding can be explained simply by binding interactions; however, it cannot easily explain the pore insertion into an intact NE, as it occurs in S phase. Insertion requires a sophisticated, as-yet-unidentified fusion machinery. The two models are not mutually exclusive, and both mechanisms could conceivably function simultaneously during nuclear assembly. Yellow denotes endoplasmic reticulum; light/dark purple denotes components of the nuclear pore complex.

recapitulated in the in vitro assembly systems, or whether it requires its own specific fusion mechanism. How to make a channel through two membranes. NPC reassembly within the double nuclear membranes represents an intriguing topological problem (FIG. 4). There are two main ways in which this reassembly might be accomplished. The simplest would be to assemble the central region of the spoke-ring complex on the chromatin surface, then surround this by flattened membrane cisternae. These would then form the INM and ONM, as well as the POM. All that would be required is fusion to form sealed membranes around each NPC (FIG. 4a). This mechanism could operate during nuclear assembly before the chromatin surface is completely sealed by a continuous double membrane. The alternative approach would be to create a largely continuous double membrane using the appropriate p97-fusion apparatus, then to insert the NPC, or NPC subunits (FIG. 4b). Certainly, during S phase, when NPC numbers double92,93, de novo NPC assembly must occur in intact membranes. This mechanism demands a further fusion event between the lumenal faces of the INM and ONM to create an aqueous channel between the nucleus and cytoplasm, as well as to form the POM (FIG. 4b). Studies on artificial nuclei in Xenopus extracts used inhibitors of NPC assembly to show that NPC re-formation can indeed occur in regions of intact membranes,

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Which comes first? Sequence of nuclear assembly. Detailed observations in mammalian systems show that binding of proteins to the nuclear periphery after chromatid segregation occurs in a stepwise fashion19,30,45,98–100 (FIG. 5). The earliest events that have been recognized in NE reassembly begin in mid-anaphase, and involve the association of certain soluble NPC proteins with the chromatid surfaces. These include Nup153, which is a component of the nuclear basket, and Nup133, which is associated with the NPC spoke-ring complex30,45,98. Soon after the association of these soluble nucleoporins, membranes begin to associate with the lateral and polar margins of the chromatids. At this time, INM proteins such as LAP2, LBR and emerin concentrate at the membrane–chromatin interface19,98,100. This presumably reflects the appearance of specific chromatin-associated binding sites for these proteins. The NPC membrane protein POM121 is also bound during this period30. Surprisingly, the other vertebrate NPC membrane protein, gp210, does not begin to concentrate at the nuclear periphery until late telophase or early G1, when the NE is sealed98,99. The implication is that POM121, rather than gp210, is required for the establishment of early membraneassociated NPC assembly intermediates. This is consistent with findings that, in interphase, POM121 is an extremely stable component of the NPC (once incorporated into an NPC, it essentially never leaves)30, whereas gp210 seems to exchange between NPCs within minutes based on photobleaching experiments and heterokaryon analyses (B.B. and K. Bodoor; J.E. and G. Rabut, unpublished observations). The late enrichment of gp210 at the nuclear periphery is, at first sight, inconsistent with a role in fusion between the INM and ONM. However, given the continuity of the ER-membrane system, some gp210 will be present at the nuclear periphery, even at the earliest times during NE assembly (albeit at a low concentration). This, combined with its ability to exchange between NPCs, might allow gp210 to carry out early functions in NPC formation. Taking this thought one step further, gp210 might function more as an NPC assembly factor than as a bona fide structural nucleoporin. The early association of soluble nucleoporins with chromatin is reminiscent of the ‘pre-pores’ that Sheehan et al. observed101 on sperm chromatin after extended incubation in Xenopus egg extracts. A reasonable view is that these proteins could function in the immobilization of POM121 after the association of membranes with the chromatin surfaces. In this way, chromatin-attached nucleoporins would define assembly sites for NPCs. Indeed, Nup153 is required for the

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Anaphase Nup153

Nup98

Nup133

Telophase Nup93

Cytokinesis/G1

Nup214

Nup358

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Nup62

Pom121 LAP2 LBR Emerin

Nuclear membrane

gp210

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Lamin A

Lamins

Steen and Collas107 have devised a method of interfering with the binding of PP1 to AKAP149 in mitotic cells in vivo, which inhibits the reassembly of B-type lamins. Remarkably, the cells could still complete mitosis, but they underwent apoptosis about 6 h later. So it seems that NE assembly in vivo can occur independently of lamins. Consistent with this, B-type lamins can assemble only after the formation of closed NEs30,99 — although there is some debate about their timing31 and lamin fragments can interfere with the assembly of artificial nuclei in Xenopus extracts108. In mature nuclei, reduced lamin content is associated with extreme mechanical fragility of the NEs109,110, as well as mislocalization of certain INM proteins such as emerin21. Ultimately, this is not compatible with cell survival. Conclusions

Figure 5 | The sequence of events during nuclear envelope assembly. The time of nuclear envelope (NE) protein localization to the surface of post-mitotic chromatin is indicated by arrows for soluble nucleoporins (purple arrow), integral membrane proteins (yellow arrow) of the inner nuclear membrane (INM; yellow) and nuclear pore complex (NPC; purple), as well as for lamins (green arrow). The lower panel presents a schematic view of the re-forming NE by filling in the four structural units of the NE at the appropriate location when a component is bound (compare with FIG. 1). The first NE proteins bind to the chromatin surface during anaphase. Early NE proteins include nucleoporins and INM proteins, and both soluble cytoplasmic and transmembrane proteins that are dispersed in the ER. Early proteins, such as the Nup133 complex and Nup153, could potentially form the core of the NPC, which then sequentially recruits other nucleoporins. As can be seen by the localization mapping in the bottom panel, this core probably corresponds to part of the spoke-ring complex, to which the central plug and the cytoplasmic and nuclear filaments are assembled in subsequent steps. Compared with nucleoporins and INM proteins, lamins (green) are late components; they are imported through the already sealed NE.

immobilization of NPCs in the NE, and it is essential for the assembly of other nucleoporins, including Nup98 and Nup93 (REF. 102). Last but not least: the lamina. There is broad agreement that the nuclear lamina is essential in the maintenance of NE integrity. However, the function of individual lamins in NE reassembly is less clear. Certainly, A-type lamins cannot be required for nuclear re-formation, given that not all cell types express these proteins103,104. Furthermore, in cells in which A-type lamins are expressed, the bulk of these are re-imported into the nucleus in early G1, after a sealed NE has been formed31,105 (FIG. 5). The reassembly of B-type lamins in mammalian cells has recently been shown to be under the control of protein phosphatase 1 (PP1)106. During mitosis, PP1 is associated with chromatin, but it is targeted to sites of nuclear membrane formation by interaction with AKAP149, an ER-membrane protein. PP1 can then dephosphorylate B-type lamins, which allows them to associate to form the nuclear lamina.

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Although our understanding of NE dynamics during the cell cycle is still incomplete, important themes have been outlined. The finding that the ER functions as a mitotic reservoir for NE-membrane components has forced us to reconsider the mechanisms by which the NE is dispersed during prometaphase, and later how an NE-specific membrane domain is re-established at the end of mitosis. Regarding NEBD, it is clear that mechanical processes that link the mitotic spindle to the stable NE protein network are instrumental. However, the role of NPCs still raises many questions. For instance, do vacated NPCs form the epicentre for microtubuleinduced nuclear membrane tearing? Do NPCs contain a dynein–dynactin binding site that could permit the transmission of tensile forces to the INM? If so, how is this binding regulated? For the better-understood process of NE assembly, interactions of chromatin and INM proteins seem to be the key determinants of nuclear membrane re-formation and drive the segregation of the INM domain from the ONM and bulk ER by selective retention of membrane proteins. Likewise, chromatin-associated nucleoporins might define assembly sites for NPCs by binding and retaining POM121 early during NE assembly. More chromatin proteins than identified so far will probably be involved in the complex orchestration of rebuilding nuclear architecture. The lamins, although essential for the maintenance of nuclear architecture, turn out to be dispensable with respect to the formation of a minimal NE that contains ONM and INM domains and NPCs. There are still gaps in our understanding of the essential mechanisms behind NE assembly. It is not clear what machinery underlies NPC reassembly, especially with respect to fusion of the INM and ONM to create aqueous channels between the nucleus and cytoplasm. Similarly, the role of Ran in the formation of the early chromatin-associated membrane network and the function of p97–Ufd1–Npl4 in the sealing of the NE remain largely obscure. From the pace of recent progress, the answers to these questions could be forthcoming in the not-too-distant future.

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Acknowledgements The authors would like to thank P. Lénárt for preparing the illustrations and K. Ribbeck for preparing FIG. 2.

Online links DATABASES The following terms in this article are linked online to: Interpro: http://www.ebi.ac.uk/interpro/ LEM domain LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink AKAP149 | MAN1 | RanBP2 Saccharomyces Genome Database: http://genomewww.stanford.edu/Saccharomyces/ CDC48 | Npl4 | Ufd1 Swiss-Prot: http://www.expasy.ch/ cdc2 | emerin | GFP | gp210 | Nup98 | Nup133 | Nup153 | p34cdc2 | POM121 | Ran Access to this interactive links box is free online.

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