Nuclear Envelope Reassembly Research Articles

Orchestrating nuclear envelope disassembly and reassembly during mitosis

Cell division in eukaryotes requires extensive architectural changes of the nuclear envelope (NE) to ensure that segregated DNA is finally enclosed in a single cell nucleus in each daughter cell. Higher eukaryotic cells have evolved 'open' mitosis, the most extreme mechanism to solve the problem of nuclear division, in which the NE is initially completely disassembled and then reassembled in coordination with DNA segregation. Recent progress in the field has now started to uncover mechanistic and molecular details that underlie the changes in NE reorganization during open mitosis. These studies reveal a tight interplay between NE components and the mitotic machinery.

Two Distinct Interacting Classes of Nuclear Envelope–Associated Coiled-Coil Proteins Are Required for the Tissue-Specific Nuclear Envelope Targeting ofArabidopsisRanGAP

Ran GTPase plays essential roles in multiple cellular processes, including nucleocytoplasmic transport, spindle formation, and postmitotic nuclear envelope (NE) reassembly. The cytoplasmic Ran GTPase activating protein RanGAP is critical to establish a functional RanGTP/RanGDP gradient across the NE and is associated with the outer surface of the NE in metazoan and higher plant cells. Arabidopsis thaliana RanGAP association with the root tip NE requires a family of likely plant-specific nucleoporins combining coiled-coil and transmembrane domains (CC-TMD) and WPP domain-interacting proteins (WIPs). We have now identified, by tandem affinity purification coupled with mass spectrometry, a second family of CC-TMD proteins, structurally similar, yet clearly distinct from the WIP family, that is required for RanGAP NE association in root tip cells. A combination of loss-of-function mutant analysis and protein interaction data indicates that at least one member of each NE-associated CC-TMD protein family is required for RanGAP targeting in root tip cells, while both families are dispensable in other plant tissues. This suggests an unanticipated complexity of RanGAP NE targeting in higher plant cells, contrasting both the single nucleoporin anchor in metazoans and the lack of targeting in fungi and proposes an early evolutionary divergence of the underlying plant and animal mechanisms.

Histone Acetyltransferase hALP and Nuclear Membrane Protein hsSUN1 Function in De-condensation of Mitotic Chromosomes

Replicated mammalian chromosomes condense to segregate during anaphase, and they de-condense at the conclusion of mitosis. Currently, it is not understood what the factors and events are that specify de-condensation. Here, we demonstrate that chromosome de-condensation needs the function of an inner nuclear membrane (INM) protein hsSUN1 and a membrane-associated histone acetyltransferase (HAT), hALP. We propose that nascently reforming nuclear envelope employs hsSUN1 and hALP to acetylate histones for de-compacting DNA at the end of mitosis.

Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth

Reticulon 4a (Rtn4a) is a membrane protein that shapes tubules of the endoplasmic reticulum (ER). The ER is attached to the nuclear envelope (NE) during interphase and has a role in post mitotic/meiotic NE reassembly. We speculated that Rtn4a has a role in NE dynamics. Using immuno-electron microscopy we found that Rtn4a is located at junctions between membranes in the cytoplasm, and between cytoplasmic membranes and the outer nuclear membrane in growing Xenopus oocyte nuclei. We found that during NE assembly in Xenopus egg extracts, Rtn4a localises to the edges of membranes that are flattening onto the chromatin. These results demonstrate that Rtn4a locates to regions of high membrane curvature in the ER and the assembling NE. Previously it was shown that incubation of egg extracts with antibodies against Rtn4a caused ER to form into large vesicles instead of tubules. To test whether Rtn4a contributes to NE assembly, we added the same Rtn4a antibody to nuclear assembly reactions. Chromatin was enclosed by membranes containing nuclear pore complexes, but nuclei did not grow. Instead large sacs of ER membranes attached to, but did not integrate into the NE. It is possible therefore that Rtn4a may have a role in NE assembly.

Protein and lipid signaling in membrane fusion: nuclear envelope assembly

AbstractMembrane fusion is important in many cell processes including membrane trafficking, mitotic reconstitution of organelles, viral infection and fertilization. Several fusion events occur just prior and subsequent to fertilization in the sea urchin, including the sperm acrosomal reaction, fusion of sperm and egg plasma membranes, exocytosis of cortical granules, reassembly of the sperm nuclear envelope and fusion of the male and female pronuclear envelopes leading to the zygote nucleus of the one‐cell embryo. The study of male pronuclear membrane dynamics with cell‐free extracts of fertilized sea urchin eggs has revealed several novel features, in particular a structural role arising from altering phospholipids prior to nuclear membrane formation. Fusion of chromatin‐bound membrane vesicles in vitro can be triggered by either GTP hydrolysis or exogenous phosphatidyl inositol phospholipase C (PI‐PLC). Recent data strongly implicate a role for diacylglycerol in nuclear envelope formation as a structural destabilizing lipid in membrane fusion. Moreover, the endogenous enzyme, PI‐PLCγ, is >100‐fold enriched in a nuclear envelope precursor vesicle population (MV1) that is required for nuclear envelope assembly. NMR and mass spectrometry analyses show that MV1 contains high levels of phosphoinositides, including the substrate of PLCγ, compared to the other nuclear envelope precursor membranes. MV1 exists in eggs as vesicles in the cortex distinct from the endoplasmic reticulum which contributes most of the nuclear envelope membrane. PLCγ is activated by a tyrosine kinase in response to GTP hydrolysis at an early stage of nuclear envelope formation suggesting a role in initiation of fusion and revealing aspects of a signaling mechanism leading to fusion. The binding of MV1 to two poles of the sperm nucleus offers spatial as well as temporal control of the initiation phase.

Electrostatic considerations in nuclear envelope breakdown and reassembly

I have previously proposed a possible role for electrostatic interactions in the dynamics of chromosome motions during prometaphase, metaphase, and anaphase in primitive eukaryotic cells. The present work focuses on a biophysical mechanism for breakdown and reassembly of the nuclear envelope based on electrostatics. Intracellular pH rises to a peak at mitosis, and decreases through cytokinesis. This result, in conjunction with an observed 50% elevation in the whole cell content of sialic acid preceding mitosis, could set the stage for a precisely timed increase in net negative nuclear envelope charge resulting from an observed release of Ca 2 + from nuclear envelope stores just prior to nuclear envelope breakdown. The resulting increased electrostatic stress in these membranes could provide sufficient electrostatic energy for their breakdown. At late anaphase and early telophase, a decrease in the whole cell content of sialic acid is accompanied by a decline in intracellular pH. These changes would decrease the mutual electrostatic repulsion of membrane fragments, allowing membrane reassembly to occur for the nuclear envelopes of nascent daughter cells.

Lamins A and C are differentially dysfunctional in autosomal dominant Emery-Dreifuss muscular dystrophy

Mutations in the LMNA gene, which encodes nuclear lamins A and C by alternative splicing, can give rise to Emery–Dreifuss muscular dystrophy. The mechanism by which lamins A and C separately contribute to this molecular phenotype is unknown. To address this question we examined ten LMNA mutations exogenously expressed as lamins A and C in COS-7 cells. Eight of the mutations when expressed in lamin A, exhibited a range of nuclear mislocalisation patterns. However, two mutations (T150P and delQ355) almost completely relocated exogenous lamin A from the nuclear envelope to the cytoplasm, disrupted nuclear envelope reassembly following cell division and altered the protein composition of the mid-body. In contrast, exogenously expressed DsRed2-tagged mutant lamin C constructs were only inserted into the nuclear lamina if co-expressed with any EGFP-tagged lamin A construct, except with one carrying the T150P mutation. The T150P, R527P and L530P mutations reduced the ability of lamin A, but not lamin C from binding to emerin. These data identify specific functional roles for the emerin–lamin C- and emerin–lamin A-containing protein complexes and is the first report to suggest that the A-type lamin mutations may be differentially dysfunctional for the same LMNA mutation.

Crm1 is a mitotic effector of Ran-GTP in somatic cells

The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.

Molecular aspects of diagnostic nucleolar and nuclear envelope changes in prostate cancer.

Prostate cancer is still diagnosed by pathologists based on subjective assessment of altered cell and tissue structure. The cellular-level structural changes diagnostic of some forms of cancer are known to be induced by cancer genes, but the relation between specific cellular-level structural features and cancer genes has not been explored in the prostate. Two important cell structural changes in prostate cancer-nucleolar enlargement and nuclear envelope (NE) irregularity-are discussed from the perspective that they should also relate to the function of the genes active in prostate cancer. Enlargement of the nucleolus is the key diagnostic feature of high-grade prostatic intraepithelial neoplasia (PIN), an early stage that appears to be the precursor to the majority of invasive prostate cancers. Nucleolar enlargement classically is associated with increased ribosome production, and production of new ribosomes appears essential for cell-cycle progression. Several cancer genes implicated in PIN are known (in other cell types) to augment ribosome production, including c-Myc, p27, retinoblastoma, p53, and growth factors that impact on ERK signaling. However, critical review of the available information suggests that increased ribosome production per se may be insufficient to explain nucleolar enlargement in PIN, and other newer functions of nucleoli may therefore need to be invoked. NE irregularity develops later in the clonal evolution of some prostate cancers, and it has adverse prognostic significance. Nuclear irregularity has recently been shown to develop dynamically during interphase following oncogene expression, without a requirement for post-mitotic NE reassembly. NE irregularity characteristic of some aggressive prostate cancers could reflect cytoskeletal forces exerted on the NE during active cell locomotion. NE irregularity could also promote chromosomal instability because it leads to chromosomal asymmetry in metaphase. Finally, NE irregularity could impact replication competence, transcriptional programming and nuclear pore function.

The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin inXenopus egg extracts

Nuclear envelope is a dynamic structure in the cell cycle. At the beginning of mitosis, nuclear envelope breaks down and its components disperse into the cytoplasm. At the end of mitosis, nuclear envelope reassembles using the dispersed components. Searching for the mechanisms of the nuclear disassembly and reassembly has for a long time been one of the key projects for cell biologists. In this report we show that microtubules take a role in the nuclear envelope assembly around the sperm chromatin inXenopus egg extracts. Microtubule cytoskeleton has been demonstrated to take roles in the transport of intracellular membranes such as Golgi and ER vesicles. We found that the nuclear envelope assembly needs functional microtubules. At the beginning of the nuclear assembly, microtubules nucleated to form a microtubule aster around the centrosome at the base of the sperm head. Using the microtubule drug colchicine to disrupt the microtubule nucleation, nuclear envelope reassembly was seriously inhibited. If the microtubules were stabilized by taxol, another microtubule drug, the nuclear envelope reassembly was also interfered, although a significantly large aster formed around the chromatin. Based on these observations, we propose that microtubules play an important role in the nuclear envelope reassembly maybe by transporting the nuclear envelope precursors to the chromatin surfaces.

An interaction that gets things started

The nuclear envelope clearly helps regulate eukaryotic DNA replication, but many of the molecular pieces of this process are still unknown. On page 177, Martins et al. describe a novel, direct interaction between the nuclear envelope and the genome, mediated by the inner nuclear membrane protein LAP2b and the nuclear matrix–associated protein HA95, that is required for the initiation of replication. Figure Disrupting the chromatin–nuclear envelope link (bottom) prevents DNA replication initiation. Having cloned HA95 two years ago, the authors now find that it interacts with LAP2b through two distinct domains. Abolishing the HA95–LAP2b interaction leads to the proteasome-mediated breakdown of prereplication complex component Cdc6, and a block in replication initiation. The HA95–LAP2b interaction is not required for DNA replication elongation or nuclear envelope reassembly after mitosis.The results add to the growing list of functions for HA95, which is also required for normal nuclear envelope breakdown and chromatin condensation, and is believed to facilitate the export of unspliced viral RNA from the nucleus. In replication initiation, HA95 probably works by protecting the prereplication complex from degradation. Martins et al. propose that the global distribution of HA95 in the nucleus and its ability to bind multiple ligands may explain why it appears in several critical regulatory pathways. ▪

Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4.

The multiple functions of the p97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1-Npl4, both bind ubiquitin conjugates directly and so link p97 to ubiquitylated substrates. In the case of Ufd1-Npl4, which is involved in endoplasmic reticulum (ER)-associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1-Npl4, it binds ubiquitin only when complexed with p97, and binds mono- rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of p97-mediated reactions.

Binding of Heterochromatin Protein 1 to the Nuclear Envelope Is Regulated by a Soluble Form of Tubulin

We have previously shown that the mouse heterochromatin protein 1 homologue M31 interacts dynamically with the nuclear envelope. Using quantitative in vitro assays, we now demonstrate that this interaction is potently inhibited by soluble factors present in mitotic and interphase cytosol. As indicated by depletion and order-of-addition experiments, the inhibitory activity co-isolates with a 55-kDa protein, which binds avidly to the nuclear envelope and presumably blocks M31-binding sites. Purification of this protein and microsequencing of tryptic peptides identify it as alpha2/6:beta2-tubulin. Consistent with this observation, bona fide tubulin, isolated from rat brain and maintained in a nonpolymerized state, abolishes binding of M31 to the nuclear envelope and aborts M31-mediated nuclear envelope reassembly in an in vitro system. These observations provide a new example of "moonlighting," a process whereby multimeric proteins switch function when their aggregation state or localization is altered.

Dynamic associations of heterochromatin protein 1 with the nuclear envelope.

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1alpha, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to purified nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His(6)-tagged peptide representing this region inhibited recruitment of LAP2beta and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.

Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types.

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

Rules to remodel by: what drives nuclear envelope disassembly and reassembly during mitosis?

In higher eukaryotic cells the nuclear envelope is reversibly disassembled during mitosis. Under in vivo conditions this process occurs in a sequential, stepwise fashion and involves a variety of structural intermediates. Here we discuss the topological features of these intermediates and their transient interactions with chromatin and the cytoskeleton. As it becomes apparent, nuclear envelope disassembly and reassembly are regulated at multiple levels by modulating the affinity of protein-protein interactions, limiting the availability of structural subunits in different areas of the mitotic cytoplasm, and redirecting mechanical forces exerted by the microtubules.

Nuclear envelope and nuclear pore assembly: analysis of assembly intermediates by electron microscopy

At mitosis, the nucleus of higher eukaryotic cells disassembles into components which subsequently reform functional nuclear envelopes in the two daughter cells. The molecular mechanisms underlying this remarkable morphological reorganization are the focus of active investigation. Recent electron microscopy techniques have provided intriguing glimpses of intermediate structures in both nuclear envelope and nuclear pore complex reassembly.

The role of keratin filaments during nuclear envelope reassembly in Xenopus egg extracts

We report here a new structure, named `strings-of-pearls', which are seen to form in Xenopus egg extracts after incubation, as 200 nm membrane vesicles attach to 10 nm filaments. These membrane vesicles fuse together along the filaments to form annulate lamellae (AL) or attach to demembranated sperm chromatin to initiate assembly of a nuclear envelope. Immunoassay with anti-keratin antibodies AE3 showed that the filaments were mainly composed of a 56 kDa keratin-like protein. Addition of AE3 to the extracts resulted in inhibition of AL formation and defective assembly of NE. These results suggest a function of keratins in the assembly of nuclear envelopes during Xenopus development.

Chapter 16 Cell-Free Nuclear Reassembly in Mammalian Mitotic Homogenates

The use of cell-free experimental systems in which the topology of cellular organelles is preserved provides a powerful tool in the study of cellular processes. This chapter describes the mammalian nuclear reassembly system that utilizes tissue culture cells synchronized in prometaphase. In the simplest procedure, nuclear envelope reassembly occurs about endogenous chromosomes and chromosome clumps present in a homogenate of these synchronized cells. However, assembly will also take place around exogenous purified chromosomes in which a postchromosomal supernatant of the mitotic homogenate provides all necessary nuclear envelope components. The initiation of the nuclear envelope assembly process in mammalian mitotic cell homogenates is found to be strongly inhibited by adenosine triphosphate (ATP) and ATP-γ-S. This indicates that the dephosphorylation of a key regulatory molecule, for example, cyclin-dependent kinase, is required to get the process started. ATP levels do not vary appreciably during the cell cycle; the initiation of nuclear envelope assembly in the mitotic homogenate is likely to represent a nonphysiological occurrence. However, the subsequent steps in the assembly process appear to closely parallel events observed in vivo and certainly give rise to ultrastructurally normal nuclear envelopes surrounding decondensed chromatin.

Chapter 17 Analysis of Nuclear Envelope Assembly Using Extracts of Xenopus Eggs

Cell division in the majority of eukaryotes occurs by an open mitosis, which includes, among other processes, the disassembly of the nuclear envelope (NE) as cells prepare to divide, and its reassembly as cells complete karyokinesis. The NEs that reform late in mitosis in each of the daughter cells are assembled from precursors that are released into the cytoplasm when the NE of the dividing cell disassembled. Because new protein synthesis is not required for NE reassembly, it is likely that disassembly and reassembly are controlled by reversible posttranslational modifications, such as protein phosphorylation. The best understood changes in NE components during mitosis are those of the lamins, the major components of the nuclear lamina. The lamins become highly phosphorylated during mitosis, and the filaments that comprise the nuclear lamina depolymerize into lamin dimers. The protein kinase activity of cyclin B/p34 cdc2 complexes is thought to play a major role in lamin phosphorylation.