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Dynamics of Nuclear Envelope Proteins During the Cell Cycle in Mammalian Cells

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Breakdown and reformation of the nuclear envelope (NE) during cell division is one of the most dramatic structural and functional changes in higher eukaryotic cells. NE breakdown (NEBD) marks a highly regulated switch in chromosome confinement by membranes in interphase to microtubules in M-phase. The boundary of interphase nuclei has a rigid and highly interconnected architecture made up of a concentric double membrane with embedded nuclear pores, underlying intermediate filaments and the connected chromosome territories. Upon entering mitosis, cells completely and rapidly dismantle the connections between these structures to allow chromosomes to condense and be captured by the mitotic spindle which then accurately partitions them to daughter cells. Once segregation is accomplished, the complex interphase architecture is quickly re-established to enable essential functions such as transcription and replication to start anew. Several excellent recent reviews have touched upon this subject from several angles.1-6 In this Chapter, I intend to present a global picture of the dynamics of nuclear envelope proteins during mitosis in mammalian cells and also touch upon other cellular structures important for nuclear envelope remodeling including chromosomes and the mitotic spindle.

Why Should Nuclear Envelope Proteins Be Dynamic?

The NE forms a selective boundary around the chromosomes and acts as a peripheral scaffold to spatially organize chromatin. As a consequence, most NE proteins have structural functions in organizing the interphase nuclear architecture. For structural proteins the intuitive assumption is that their behavior is rather static. However, both in non-dividing and dividing cells there are aspects of NE function that require dynamic exchange of its proteins. Before we review these, it is useful to remind ourselves that the NE has a unique topology. Its two membranes, inner nuclear membrane (INM) and outer nuclear membrane (ONM) are connected at several thousand nuclear pores via a short stretch of lipid bilayer sometimes referred to as the pore membrane (POM) (Fig. 1). The outer membrane is continuous with the endoplasmic reticulum (ER) and is indistinguishable from the ER in terms of its protein composition including attached and translating ribosomes. Viewed from the cytoplasm, the NE is simply a specialized subcompartment of the ER, a large spherical ER cisterna studded by nuclear pores and wrapped around lamins and chromosomes (Fig. 1).

Figure 1. Schematic view of the organization of the interphase nuclear envelope.

Figure 1

Schematic view of the organization of the interphase nuclear envelope. (Left) Nuclear membranes can be seen clearly as a subcompartment of the ER studded by nuclear pores and closely apposed to the nuclear lamina and peripheral chromatin. Also shown are (more...)

What then are the situations in which NE proteins have to be dynamic? The first need arises when cells replicate their set of chromosomes which causes nuclear volume and NE surface to grow significantly. This expansion requires the targeting of proteins to the NE to equip it with new molecules. A good example for this is that the number of nuclear pores doubles during this time.7 Secondly, nuclear architecture needs to be remodeled in response to external stimuli. It is becoming increasingly clear that chromosome attachment to the nuclear envelope can influence replication timing and transcription activity.8-10 When cells activate peripherally located genes or replicate them, these attachments must be remodeled in a dynamic fashion. NE protein dynamics become essential when a cell divides. The stable structure of the NE poses a formidable barrier to mitosis in metazoan cells which have exclusively cytoplasmic microtubules. These cells undergo an open mitosis, disassembling their NE at the transition to M-phase so that the mitotic spindle can access and attach to the chromosomes. Conversely, after sister chromatids have been successfully separated, new NEs have to be reformed quickly to allow a new cycle of metabolic activity.

What is the Nuclear Envelope Made of?

Over the recent years we have obtained an almost comprehensive list of the proteins present in the NE in vertebrates especially with the advances made by recent proteomics studies.11,12 Based both on the identified proteins and on morphological considerations it makes sense to subdivide the NE into four main structures (Fig. 1), each of which are reviewed in more detail in other Chapters of this volume. The first of these, the nuclear lamina consists of lamins, proteins of the intermediate filament family that are divided into two classes, namely B type (ubiquitous) and A/C type lamins (found only in differentiated cells).13,14 These rod-shaped proteins form a peripheral branched polymer of 10-nm filaments which provides structural support to the NE.15 The second NE structure is the inner nuclear membrane (INM). It contains a unique set of membrane proteins and protein families which reside only at low levels in the ER and the secretory pathway. Most of these more than 10 proteins function as adaptors linking the INM to the lamina and/or chromosomes2 and some authors have now extended the definition of the lamina to encompass also the lamina associated proteins.16,17 The third structure is the nuclear pore complex (NPC), a 125-MDa large protein assembly that forms an aqueous channel through the NE, thereby joining the inner and outer NM. Mammalian cells contain one to several thousand of these channels per nucleus. Each NPC is made of nucleoporins (Nups), a class of more than 20 soluble and only two integral membrane proteins. The NPC mediates all nucleocytoplasmic traffic18 but may also be involved in nuclear organization in general.19 Some nucleoporins interact both with the lamina and chromosomes19,20 and direct connections between the lamina and the nuclear face of the NPC can be visualized by electron microscopy.21 The last structure of the NE, which is classically not counted among NE components is the peripheral chromatin which contains several proteins that interact with the lamina and/or the INM. In interphase these four units of NE architecture are connected by a multitude of protein-protein interactions and the NE appears as a complex, highly cross-linked structural protein network (Fig. 1).16

Studying Nuclear Envelope Protein Dynamics

True insight into NE protein dynamics has mostly come from studying these proteins in their natural environment in living cells. In mammalian cells this has been achieved through the analysis of fluorescently labeled derivatives of NE proteins. Fusion to green fluorescent protein (GFP)22 and subsequent stable or transient expression has been the method of choice in many cases, especially for the many transmembrane proteins, for which recombinant expression, labeling with chemical fluorophores and reintroduction into live cells is not feasible. Once the NE protein of interest has been labeled successfully (and without impairing its function!) several techniques can be used to characterize its dynamics. In this Chapter, I will describe results mostly from two approaches. The first is time-lapse fluorescence imaging. Here a fluorescence microscope, either confocal or wide field, is used to take images of the protein distribution in live cells and document changes of localization over time. Time-lapse imaging, if performed quantitatively, can document the fluxes of a given protein within the cell with high spatial resolution and even in three dimensions.23 The second method is fluorescence recovery after photobleaching or FRAP.24 In FRAP a portion of the fluorescently labeled protein is bleached irreversibly with a high intensity laser beam. After the bleach, the exchange of the bleached molecules with the surrounding unbleached molecules is then measured by monitoring the recovery of fluorescence in the bleached area. If the bleached molecules do not exchange during the time of the experiment, fluorescence does not recover and the patterns bleached by the laser can be used to mark regular geometries inside cells. This approach is referred to as pattern bleaching and has been very useful to characterize surface dynamics of the NE as we will see below.

Dynamics in Interphase

INM Proteins Are Targeted by Selective Retention

INM proteins are defined by their specific localization to the nucleoplasmic face of the nuclear membranes. Since the NE is an ER subcompartment, it is interesting to ask how these proteins are confined to just the INM and largely excluded from the ER. Initial experiments focused on identifying “sorting signals” in INM proteins, analogous to the short consensus sequences that govern localization of membrane proteins in the secretory pathway.25,26 However, the sequences identified turned out to be binding motifs to nuclear proteins rather than classical signals for transport adaptors. We now know that most INM proteins contain sequence motifs in their nucleoplasmic domains that mediate interactions to lamins, chromatin or other INM proteins in an often redundant fashion. The ability of INM proteins to bind to nuclear partners turns out to be sufficient to account for their specific localization by a mechanism based on selective retention (Fig. 2A). INM proteins start their life in the ER where they are inserted into the membrane. In the ER, their binding domains are exposed to the cytoplasm and do not encounter nuclear proteins. As a result, INM proteins can freely diffuse within the ER and also have access to the INM through the membrane connection between ONM and INM at the periphery of each NPC (Figs. 1 and 2A). Importantly, this access is driven by diffusion and is thus independent of signals and not directional. The only restriction to diffusion through the POM appears to be the size of the cytoplasmic domain; it can inhibit localization when it becomes too bulky to pass through the peripheral channels of the NPC.27 Once an INM protein has reached the inner face of the NE, its now nucleoplasmic binding domain encounters nuclear interaction partners to which it attaches, preventing its diffusion back into the ER. This selective retention of INM proteins but not of general ER proteins in the INM elegantly explains their retention and concentration in the INM ER-subdomain (Fig. 2A). Selective retention makes two clear predictions for the dynamics of integral INM proteins: (i) INM proteins can be targeted in interphase (as opposed to just after mitosis) and (ii) the mobility of these proteins should be reduced upon localization to the NE. Indeed, interphase targeting was demonstrated by following the localization of newly synthesized GFP-tagged lamin B receptor (LBR) after microinjection of an expression plasmid in interphase cells. Initially fluorescence was equally distributed between ER and NE, but after a few hours it was five times more concentrated in the NE.28 The reduced mobility in the INM has been confirmed by FRAP of three INM proteins, GFP-tagged emerin, LBR and MAN1 28-30. The fact that localization of emerin to the INM depends in part upon lamin A provides further evidence for this mechanism.31,32 We will revisit selective retention again when discussing nuclear membrane dynamics in mitosis where switching on and off the retaining interactions is responsible for loss and reestablishment of the INM domain of the ER (Fig. 2B,C).

Figure 2. Selective retention in interphase and mitosis.

Figure 2

Selective retention in interphase and mitosis. Schematic illustrating how INM proteins can be localized to the ER and INM-subdomain in interphase and mitosis. ER/nuclear membranes contain a typical chromatin binding INM protein (dots) and are in close (more...)

The Interphase Lamina: A Stable but Elastic Polymer

Several recent studies have examined the properties of GFP tagged A and B type lamins.3335 Time lapse sequences on interphase cells demonstrate that the lamina can undergo dynamic deformations, such as folds and indentations that typically occur during cellular movements or nuclear rotations (Fig. 3A). To assay how stable fluorescent lamins were incorporated into the lamin polymer, FRAP was used to determine if bleached lamin molecules could be replaced by new fluorescent lamins. Both for A and B type lamins, recovery was found to be extremely slow and complete recovery could not be observed in experiments ranging from 10 minutes 35 to more than 40 hours.34 This indicated a very low dissociation rate of lamins from the polymer in interphase. On the other hand overexpressed lamins can be incorporated into the lamina of interphase cells in less than 20 h probably reflecting the capability of excess lamin monomers to be absorbed into the lamina in addition to, but not replacing the already polymerized filaments. The elasticity of the lamina was directly addressed by taking advantage of the very slow recovery of GFP-tagged B type lamins in pattern bleaching experiments. Here bleaching by a laser beam is used to create geometrical patterns such as stripes and grids on the surface of the smooth peripheral lamina surface, which can then be tracked during cellular movements (Fig. 3B). These experiments clearly demonstrated that the lamina behaves as a two dimensional polymer that can undergo elastic deformations during cellular movement but relaxes back into the original geometrical arrangement when movement ceases.34,36 The stable and elastic properties of the lamin polymer have confirmed in vivo what could be predicted from its ultrastructural mesh-like appearance15 and its resistance to biochemical extractions since the 70's.37

Figure 3. Dynamic properties of the peripheral lamin polymer.

Figure 3

Dynamic properties of the peripheral lamin polymer. (A) 3D confocal time-lapse sequence of a PtK2 cell expressing GFP-lamin B1 in interphase. DIC and fluorescence images are overlaid. Insets show a top projection of GFP fluorescence only. Note nuclear (more...)

NPCs Form Networks and Have a Stable Core

So far only three studies have started to characterize the dynamics of NPCs in intact mammalian cells.34,38,39 The NPC is a remarkable protein complex in many ways. It is very large (125 MDa), consists of more than 30 different proteins in vertebrates each of which occurs in probably 8-24 copies, reflecting the eightfold rotational symmetry of the complex.12 The core of the NPC forms a flat hollow cylinder with dimensions of ~120 nm in width and ~40 nm in length, and an inner channel diameter of ~40 nm whose walls are embedded in the POM (Fig. 1). This cylinder surrounds the so-called central plug, proteinaceous material located in the middle of the aqueous channel. From the rims of the cylinder emanate eight cytoplasmic and nuclear filaments the latter being joined by a distal ring to form the nuclear basket (Fig. 1). Using five GFP-tagged nucleoporins Nup98, Nup 153, POM121, and Nup107/Nup 133, these studies again employed time-lapse fluorescence microscopy and FRAP to assay the dynamics of nucleoporins in interphase. The core of the NPC represented by the transmembrane protein POM121 and Nup107/Nup133 was found to form an extremely stable complex that did not exchange any of the three Nups over many hours in interphase. Strikingly, Nup153 and Nup98 which are both localized to the nuclear face of the NPC were found to associate only transiently with the NPC.34,39

Using markers of the NPC core, the mobility of the whole NPC itself in the plane of the NE was also examined. The notion that NPCs might be mobile was prompted by earlier studies in yeast, which reported movement of NPC across the surface of nuclei after karyogamy of haploid cells.40,41 In contrast to yeast, mammalian NPCs were found to be completely immobile in the surface of the NE unless it was deformed by folds and indentations. Under those circumstances NPC movements correlated precisely with those of the underlying lamina.34 These in vivo experiments support ultrastructural data that proposed a direct link between the NPC and the lamina meshwork.21

Chromosomes Do Not Move Much in Interphase

Our insight into the dynamics of the chromatin class of NE envelope proteins is unfortunately very limited at the moment. Only for one of them, the DNA crosslinker barrier to autointegration factor (BAF), do we have any data from living cells, which mostly addresses the localization of a GFP fusion during nuclear assembly.42 No FRAP data on the lifetime of chromatin-NE interactions are available at the moment, although for BAF and heterochromatin protein 1 it appears as if they might be more dynamic than those reported for the INM, NPC and lamina (J.E., unpublished observations). However, we know more about the dynamics of chromosomes themselves in interphase mammalian nuclei from several approaches. In one approach developed by Daniele Zink and coworkers, chromatin domains are labeled with pulses of microinjected fluorescent nucleotides during replication and can then be traced over several cell cycles.43-45 A second approach pioneered by Andrew Belmont and coworkers employs a system of multimeric repeats of lac operators integrated into the genome of cell lines. These arrays can then be labeled by expression of lac repressor-GFP fusion proteins.46 Using global DNA labeling with intercalating dyes, FRAP has also been used to address chromatin dynamics 47. The consensus from all of these studies is that chromatin typically does not undergo long range movement over several hours in interphase but is restricted to local constrained motion. However this rather static picture can change if transcription is activated, which can lead to decondensation and movement to the interior of the affected locus.48,49 Another phase of repositioning seems to be replication of a locus, which again can be associated with movement towards the interior.50 In summary, we can assume that the position of peripheral chromatin is rather static during interphase, consistent with the exceptional stability of the NE protein network. It will be important to find out in the future how long chromatin-NE adaptors stay bound to chromosomes and if these interactions are specifically regulated during transcription activation or replication of peripheral chromatin.

Overall the interphase dynamics of all NE proteins studied so far have reinforced the view of a protein network that is very stable, made up of long lived interactions that serve to maintain the structure of the interphase nucleus.

Dynamics in Mitosis

The interphase NE which so efficiently separates nuclear from cytoplasmic processes complicates life of metazoan cells when it is time to divide. To successfully complete mitosis, the microtubules of the spindle apparatus which are exclusively cytoplasmic must come in contact with chromosomes which are shielded by the NE protein network. To achieve this, mammalian cells break down their NE completely in prometaphase and undergo an “open” mitosis, releasing chromosomes into the cytoplasm to accomplish segregation. The process of NE breakdown (NEBD) and reformation involves the disassembly and dispersal of all four structural units of the NE. Once mitosis is completed, the dispersed NE proteins are then used again to assemble new nuclei in the next cell generation. As expected from the complex interphase architecture, NE breakdown and assembly are complicated processes that require the coordinated action of many cellular activities such as mitotic phosphorylation/ dephosphorylation, nucleocytoplasmic transport, membrane fusion as well as the action of microtubule motor-proteins. Currently, a consensus model of NE dynamics is emerging that can explain all the changes in NE structure and dynamics that have been documented during cell division.

INM Proteins: Switching Retention Off and Back On

The Old Model: Mitotic Phosphorylation of NE Proteins and Vesiculation of Nuclear Membranes

Many biochemical studies have shown that NE proteins are subject to phosphorylation in M-phase by MPF, the complex of cyclin B and p34cdc2 in mammalian cells. Phosphorylation depolymerizes and disperses lamins34,51–53 and some nucleoporins.54,55 Several INM proteins have also been shown to be targeted by cdc2 (ref. 2) but the consequence of their modification is much less clear. We currently assume that it abolishes their ability to interact with lamins and/or chromatin, which would allow the INM to detach from chromosomes. The fate of nuclear membrane proteins during M-phase has been an issue of some contention in the recent literature.1 Nevertheless, most textbooks present a seemingly simple model according to which the NE vesiculates after the lamin polymer has been depolymerized through mitotic phosphorylation.56 It is useful to take a brief look at how we arrived at this model. In the early '80s nuclear assembly and breakdown was reconstituted in amphibian oocyte extracts57,58 a system that subsequently lead to a wealth of biochemical data from many other laboratories. Since the procedure of this assay results in fragmented membrane homogenates, such “vesicles” were assumed to be the natural starting material to assemble new nuclear membranes. Additional support for mitotic NE vesicles came from a contemporary EM study showing ER vesiculation in dividing rat thyroid cells.59 Based on these two lines of evidence, NE vesiculation was quickly accepted as the mechanism that would do in cells what homogenization did in nuclear reconstitution assays: produce precursor membrane fragments for nuclear assembly. Another attractive feature postulated by this model was that many small precursor membrane fragments can be partitioned efficiently by a stochastic mechanism such as diffusion between the two daughter cells.

The Modern (and Traditional!) View: ER Absorption by Switching Off Retention

However, if one steps back even further in time and looks at the pioneering electron microscopic work done on mitotic plant and animal cells in the '60s60-62 it is clear that assays in extracts are not ideally suited to evaluate the dynamic morphological changes nuclear membranes undergo in mitosis. The first EM observations of mitotic cells already documented that mitotic nuclear membranes became indistinguishable from tubules and cisternae of the ER when cells entered M-phase and that nuclear membranes assembled after mitosis seemed to derive from the ER. This view has been confirmed strongly in recent studies in intact mammalian cells that revisited the fate of the NE in mitosis and demonstrated that the ER serves as the reservoir for nuclear membrane proteins in M-phase.28,34,36,63 That the ER network, rather than membrane vesicles, is the precursor for NE assembly is also suggested by recent dynamic in vitro studies on NE assembly, which show that also in Xenopus egg extracts, network formation from vesicles is an intermediate step prior to NE assembly.64-66

How then do INM proteins move back into the ER in prometaphase and how is the INM subdomain of the ER reestablished? If we remind ourselves how INM proteins are targeted in interphase, and take into account the disruptive force of mitotic phosphorylation on protein-protein interactions the answer becomes immediately clear. In interphase nuclear membrane proteins diffuse between the ER and the INM but are trapped in the latter by selective binding interactions when they meet lamins and chromatin (Fig. 2A). When these interactions are switched off by mitotic phosphorylation in prophase, INM proteins will equilibrate with the ER, since they are no longer retained and set free to diffuse back into the ER (Fig. 2B). Simple diffusion can equilibrate the INM pool with the ER efficiently and rapidly through many connections between INM and ONM and the continuity between ONM and ER. Exactly such an equilibration process from nuclear rim to the ER network can be observed in vivo for several INM proteins at different times in prophase36 (J. Beaudouin and J.E., unpublished observations) leading to a uniform dispersed distribution of INM proteins in the intact mitotic ER.28 The reverse mechanism, i.e., switching the retaining binding interactions back on by dephosphorylation at the end of mitosis, elegantly explains how the INM subdomain can be reformed. Degradation of cyclin B after metaphase inactivates MPF kinase and allows dephosphorylation to reactivate the interactions between INM proteins and their chromatin binding partners. In anaphase, when more and more attachment sites for membranes are becoming available through the combined effect of dephosphorylation and chromosome decondensation, NE assembly can proceed by coating of the chromosome surface with ER cisternae. The cisternae contain INM proteins which bind to chromatin as soon as they are in close proximity (Fig. 2C). Thus, nuclear membrane proteins are immediately concentrated at the membrane chromatin interface, again by diffusion from the ER and selective retention on chromatin, which drives an increases in the membrane surface around the chromosome template. Precisely this process can be observed in living cells by following GFP-labeled INM and ER proteins (Fig. 4).28,42,67 However, even with the ER network as a precursor for nuclear membranes, membrane fusion will be necessary to enclose the chromosomes by a sealed NE.68 Recent studies have begun to shed light on the molecular machinery in NE fusion processes65,66 and it will be very interesting to investigate the dynamics of this process in intact cells.

Figure 4. Reestablishment of the INM subdomain from the ER.

Figure 4

Reestablishment of the INM subdomain from the ER. 2D confocal time-lapse sequences of a NRK cells expressing the ER membrane protein SRb-CFP (A) and the INM protein LBR-GFP (B). Note how ER cisternae and tubules surround the chromatin area in anaphase (more...)

Lamina: Tearing of a Polymer, Dispersion and Re-Import of Monomers

The same pioneering ultrastructural studies that reported the merging of NE and ER in mitosis, also noted that centrosomes were closely associated with the NE and often buried in an invagination in prophase.60,61 More recent biochemical and genetic studies of microtubule motors have shown that cytoplasmic dynein is required to attach centrosomes to the nucleus in C. elegans and Drosophila.69-71 In addition, dynein localizes to the NE of mammalian cells in prophase.72,73 Although the molecular basis of the dynein-NE interaction is still unclear, we have gained some insight into its functions such as centrosome separation and nuclear movement.74 Two recent studies have now also linked NEBD to the action of dynein and the mitotic spindle. Using quantitative live cell imaging and electron microscopy, these studies showed that spindle microtubules facilitate NEBD by literally tearing the lamin polymer open. This is apparently accomplished by immobilizing dynein on the outer surface of the nuclear envelope, which is then drawn towards the centrosomes of the forming mitotic spindle by dynein's minus end directed motion. Pulling on the nucleus by the mitotic spindle results in massive distortion of nuclear shape, which could be documented by pattern photobleaching of the nuclear lamina (Fig. 3B). Most prominently deep invaginations are formed close to the centrosomes while the lamina is stretched further away from the asters.36,73 The NE remained intact during these deformations until holes appeared in the lamina at the sites of maximum stretching, suggesting a tearing mechanism. The opening of this physical discontinuity in the NE allows even large cytoplasmic molecules to freely enter the nucleus. This then triggers the gradual disassembly of the lamina, a process that is only completed in metaphase, when even the lamina fragments that have been drawn to the centrosomes by dynein are completely solubilized. These observations nicely demonstrate that formation of the mitotic spindle and NEBD are two mitotic processes which are highly coordinated. By doing this the mammalian cells could have evolved an additional mechanism to control the transition of chromosome organization by nuclear membranes to microtubules.

Although it is clear that the lamina plays an essential role in maintaining nuclear integrity and shape in somatic cells and is probably a key structure resisting transition into mitosis, it seems to play only a minor role in the early stages of nuclear assembly. According to most studies, the majority of both A and B type lamins are re-imported into post-mitotic nuclei that have already assembled a fully sealed nuclear membrane containing functional nuclear pores,34,35,75 although some studies have suggested an earlier association.33 Interestingly, recent work has shown that the assembly of B type lamins is regulated by protein phosphatase 1. This protein binds to the integral membrane protein A-kinase anchoring protein (AKAP)149 and then dephosphorylates lamins at sites of contact between ER and chromosomes.76 Without the interactions of PP1 and AKAP149 lamins do not assemble, but cells still complete mitosis. Thus it appears that the assembly of a functional nuclear lamina is secondary to the assembly of nuclear membranes and dispensable for nuclear assembly.

Pore Complex Disassembly and Assembly: Many Open Questions

The nuclear pore is a topologically unique structure. It forms an aqueous channel that spans and connects a double membrane. We know very little about the mechanism of disassembling or reassembling the NPC, apart from the fact that some nucleoporins undergo mitotic phosphorylation.54,55 It is completely unclear how or in which order this large complex is disassembled. In mammalian cells we only know that dispersal of a core nucleoporin such as POM121 only starts after the NE is permeabilized by tearing of the lamina.36 However there is evidence from very different cell systems such as starfish oocytes and Drosophila embryos that point to a key role for NPC disassembly in triggering nuclear permeabilization77,78 and it will be very interesting to investigate this process in more detail in mammalian cells. Once disassembly is accomplished, the NPC is not broken down to individual polypeptides but rather into Nup subcomplexes that are stable in mitosis and probably form the building blocks from which the NPC can be assembled anew after mitosis.38,79 While the majority of nucleoporins show a dispersed cytoplasmic distribution in mitotic cells, the transmembrane nucleoporins are absorbed by the ER similar to INM proteins.34,63 Some nucleoporin (subcomplexes) however show striking localizations in mitosis. The Nup133/107 complex binds to kinetochores from prophase to anaphase38 while Nup358 (RanBP2) can be seen to localize to the spindle apparatus in mitotic cells.80 So far however, the mitotic functionif anyof these nucleoporins is unclear. The reassembly of the NPC after mitosis is also mysterious. Two principally different ways of NPC assembly can be envisioned and available data are supporting aspects of both mechanisms. In the first mechanism the soluble core structure of the NPC would be assembled on the surface of chromosomes and then connect to ER cisternae that attach to chromosomes and the side of the core NPC. This model does not require a fusion event between the INM and ONM and is supported by the very early appearance of some nucleoporins on the chromosome surface during anaphase.34,38,81 Alternatively, NPCs could be inserted into large intact double membranes by a specific intralumenal fusion event. This model is supported by studies on artificial nuclei in the presence of inhibitors as well as in Drosophila embryos where different stages of NPC assembly on the surface of intact membranes can be distinguished by electron microscopy.77,82,84 It will be a main challenge of future work to shed more light on this mechanism and identify key molecules involved in this process.

Chromosomes: A Complex Template for Nuclear Assembly

We understand even less about the mitotic dynamics of peripheral chromatin proteins linked to the NE than we know about the mechanism of NPC disassembly and reassembly. From the limited data available, it appears that chromosomes retain at least some of their NE adaptor proteins in mitosis. In intact cells, only the behavior of a GFP fusion to barrier to autointegration factor (BAF) has been described. The GFP tagged protein appeared soluble in mitosis and assembled on chromatin concomitantly with one of its binding partners the INM protein emerin.42 However this data is in conflict with previous localization of BAF to mitotic chromosomes85 and it remains to be tested if the GFP fusion employed is DNA binding competent, as other GFP-BAF fusions show different behavior (J.E. unpublished observations). Another important group of peripheral chromatin proteins, the heterochromatin protein 1 family has also been localized to chromosomes in mitotic cells using antibodies.86 Similar to BAF, a second study reported a different localization87 and more experiments are required to clarify the picture. It is interesting to note that both the HP1 family as well as the third peripheral chromatin protein lamina associated protein 2α (LAP2α) localize to specific subchromosomal domains such as centromeres and telomeres.88 In anaphase this creates a patchwork like template for nuclear membrane assembly and probably explains the differential localization patterns found for different INM proteins at this time.67,89

Concluding Remarks

Our understanding of NE dynamics during the cell cycle has increased dramatically over the recent years. Although areas such as NPC assembly and the precise role of the heterochromatin proteins remain poorly studied, we have arrived at several important mechanistic conclusions. In mammalian cells it is clear now that the ER functions as the mitotic reservoir for all nuclear membrane proteins tested so far and this has had fundamental implications to interpret nuclear membrane protein dispersal and the reformation of the INM ER-subdomain after mitosis. For the latter it seems clear that the binding interactions between INM proteins and chromatin are the driving force of nuclear reformation and probably important in determining the nuclear architecture of the next cell generation. Most likely we still have to discover many chromatin bound factors involved in this process. At the G2/M transition we have seen that mechanical forces exerted by the mitotic spindle on the stable NE protein network facilitate NEBD and complement the biochemical machinery that disrupts protein-protein interactions by phosphorylation. Functional dynamics of the NE promises to be an exciting subject for future research in the coming years.

Acknowledgments

The author would like to thank Péter Lénárt for preparing Figures 1 and 3, Joël Beaudouin for preparing Figure 2B and Nathalie Daigle for preparing Figure 4.

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