A microtubule-independent role for centrosomes and aurora a in nuclear envelope breakdown

Aurora A kinase localizes to centrosomes and is required for centrosome maturation and spindle assembly. Here we describe a microtubule-independent role for Aurora A and centrosomes in nuclear envelope breakdown (NEBD) during the first mitotic division of the C. elegans embryo. Aurora A depletion does not alter the onset or kinetics of chromosome condensation, but dramatically lengthens the interval between the completion of condensation and NEBD. Inhibiting centrosome assembly by other means also lengthens this interval, albeit to a lesser extent than Aurora A depletion. By contrast, centrosomally nucleated microtubules and the nuclear envelope-associated motor dynein are not required for timely NEBD. These results indicate that mitotic centrosomes generate a diffusible factor, which we propose is activated Aurora A, that promotes NEBD. A positive feedback loop, in which an Aurora A-dependent increase in centrosome size promotes Aurora A activation, may temporally couple centrosome maturation to NEBD during mitotic entry.     

Nuclear envelope disassembly and nuclear lamina depolymerization during germinal vesicle breakdown in starfish

During germinal vesicle breakdown (GVBD) in starfish, the nuclear envelope disassembles before the nuclear lamina completely depolymerizes, judging from correlative ultrastructural, immunolabeling, and light microscopic analyses. At 13 degrees C, prophase-arrested oocytes of Pisaster ochraceus begin GVBD and rapidly undergo nuclear envelope disassembly about 50 min after addition of the maturation-inducing hormone 1-methyladenine (1-MA). The nuclear lamina of these oocytes, however, remains present for 10-20 min following the vesiculation of the nuclear envelope. Completion of GVBD, as evidenced by a blending of the nuclear contents with the surrounding cytoplasm, occurs within about 15 min after the nuclear lamina has fully depolymerized. Immunofluorescence studies also indicate that a marked increase in the phosphorylations of nuclear proteins precedes the structural reorganizations of the nuclear envelope and nuclear lamina during GVBD.     

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.     

Virtual breakdown of the nuclear envelope in fission yeast meiosis

Asymmetric localization of Ran regulators (RanGAP1 and RanGEF/RCC1) produces a gradient of RanGTP across the nuclear envelope. In higher eukaryotes, the nuclear envelope breaks down as the cell enters mitosis (designated "open" mitosis). This nuclear envelope breakdown (NEBD) leads to collapse of the RanGTP gradient and the diffusion of nuclear and cytoplasmic macromolecules in the cell, resulting in irreversible progression of the cell cycle. On the other hand, in many fungi, chromosome segregation takes place without NEBD (designated "closed" mitosis). Here we report that in the fission yeast Schizosaccharomyces pombe, despite the nuclear envelope and the nuclear pore complex remaining intact throughout both the meiotic and mitotic cell cycles, nuclear proteins diffuse into the cytoplasm transiently for a few minutes at the onset of anaphase of meiosis II. We also found that nuclear protein diffusion into the cytoplasm occurred coincidently with nuclear localization of Rna1, an S. pombe RanGAP1 homolog that is usually localized in the cytoplasm. These results suggest that nuclear localization of RanGAP1 and depression of RanGTP activity in the nucleus may be mechanistically tied to meiosis-specific diffusion of nuclear proteins into the cytoplasm. This nucleocytoplasmic shuffling of RanGAP1 and nuclear proteins represents virtual breakdown of the nuclear envelope.     

Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

Till disassembly do us part: a happy marriage of nuclear envelope and chromatin

A characteristic feature of eukaryotic cells is the presence of nuclear envelope (NE) which separates genomic DNA from cytoplasm. NE is composed of inner nuclear membrane (INM), which interacts with chromatin, and outer nuclear membrane, which is connected to endoplasmic reticulum. Nuclear pore complexes are inserted into NE to form transport channels between nucleus and cytoplasm. In metazoan cells, an intermediate filament-based meshwork called as nuclear lamina exists between INM and chromatin. Sophisticated collaboration of these molecular machineries is necessary for the structure and functions of NE. Recent research advances have revealed that NE dynamically communicates with chromatin and cytoskeleton to control multiple nuclear functions. In this mini review, I briefly summarize the basic concepts and current topics of functional relationships between NE and chromatin.     

Relation between nuclear envelope and nuclear lamina in nuclear assemblyin vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitution in vitro. The experimental results showed that lamin was involved in the nuclear assembly in vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear lamina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Inhibition of cAMP-dependent protein kinase plays a key role in the induction of mitosis and nuclear envelope breakdown in mammalian cells.

Inhibiting cAMP-dependent protein kinase (A-kinase) in mammalian fibroblasts through microinjection of a modified specific inhibitor peptide, PKi(m) or the purified inhibitor protein, PKI, resulted in rapid and pronounced chromatin condensation at all phases of the cell cycle. Together with these changes in chromatin, a marked reorganization of microtubule network occurred, accompanied in G2 cells by extensive alterations in cell shape which have many similarities to the premitotic phenotype previously observed after activation of p34cdc2 kinase, including the lack of spindle formation and the persistence of a nuclear envelope. In order to examine whether A-kinase inhibition and p34cdc2 kinase form part of the same or different inductive pathways, PKI and p34cdc2 kinase were injected together. Co-injection of both components resulted in nuclear envelope disassembly, an event not observed with injection of either component alone. This result implies that p34cdc2 and A-kinase inhibition have complementary and additive effects on the process of nuclear envelope breakdown in living fibroblasts, a conclusion further supported by our observation of a pronounced dephosphorylation of lamins A and C in cells after injection of PKi(m). Taken together, these data suggest that down-regulation of A-kinase is a distinct and essential event in the induction of mammalian cell mitosis which co-operates with the p34cdc2 pathway.     

Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown

The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea. During nuclear envelope breakdown the lamin is released as a mixture of oligomers in which disulfide-stabilized dimers are associated noncovalently with lamin monomers. Concurrent with solubilization, both dimers and monomers are phosphorylated to a similar extent, indicating that the interactions which maintain these complexes are not destabilized by lamin phosphorylation. Our results suggest the existence of two types of interactions between the lamin molecules in the polymer, which react differently to phosphorylation during nuclear envelope breakdown.     

Meiotic breakdown of nuclear envelope in oocytes of Spisula solidissima involves phosphorylation and release of nuclear lamin

During meiotic nuclear envelope breakdown (NEBD) in maturing oocytes of the surf clam, Spisula solidissima, the 67-kDa lamin is extensively phosphorylated, concurrently with its solubilization. This is accompanied by a reduction of the nuclear diameter. Quercetin, a protein kinase inhibitor, does not affect lamin phosphorylation and release, nor NEBD per se, but specifically inhibits the early phosphorylation of a set of proteins, on which NEBD seems to depend. Our results suggest that meiotic NEBD in Spisula oocytes may be controlled by a mechanism which involves lamin phosphorylation, similar to that which is thought to operate in mitosis.     

A radiation-induced inhibitor of chromosome condensation and nuclear envelope breakdown in HeLa cells.

An in vitro microscopic assay for mitosis-inducing activity in mitotic HeLa cells was developed and used to demonstrate that cells irradiated and arrested in G2 phase of the cell cycle contain an inhibitor of mitosis. This assay system has a number of advantages over other assays including the use of autologous components (HeLa nuclei and mitotic cell extracts) in contrast to the microinjection method with Xenopus oocytes and without the requirements for microinjection expertise and Xenopus oocytes. The radiation-inducible inhibitor was detected at the lowest radiation dose tested (2 Gy) with maximal activity achieved within 30 min after radiation. Inhibitor activity decayed with time after radiation (2 Gy) with no activity detected at 6 h even though the cells remained in G2 phase, suggesting that either synthesis or activation of additional components is necessary for recovery from G2 arrest. The inhibitor activity was not detected in irradiated cells treated with caffeine to induce premature recovery from G2 arrest.     

The use of field emission in-lens scanning electron microscopy to study the steps of assembly of the nuclear envelope in vitro.

At mitosis the nuclear envelope (NE) is disassembled to allow chromosome separation. In telophase it is reassembled as the chromosomes decondense. Cell-free extracts of Xenopus eggs have been used extensively to study assembly of the NE and the nuclear pore complexes (NPCs), providing several models for the steps involved. The NE is a surface structure which in cell-free extracts is easily exposed. It is appropriate, therefore, to use a surface imaging technique to study NE dynamics. Field emission in-lens scanning electron microscopy (FEISEM) provides the opportunity to image surfaces, directly, and to visualise details of structures such as the NPC. Here we show the feasibility and value of FEISEM to study the steps of NE formation. Nuclei have been assembled in vitro and fixed at different time points during assembly, followed by conductive staining, platinum coating, and visualisation by FEISEM. Changes on the nuclear surface with time are shown. Details of the surface of chromatin and the cytoplasmic face of NPC structure are demonstrated without the need to isolate the structures from the nucleus.     

Lis1 and Ndel1 influence the timing of nuclear envelope breakdown in neural stem cells

Lis1 and Ndel1 are essential for animal development. They interact directly with one another and with cytoplasmic dynein. The developing brain is especially sensitive to reduced Lis1 or Ndel1 levels, as both proteins influence spindle orientation, neural cell fate decisions, and neuronal migration. We report here that Lis1 and Ndel1 reduction in a mitotic cell line impairs prophase nuclear envelope (NE) invagination (PNEI). This dynein-dependent process facilitates NE breakdown (NEBD) and occurs before the establishment of the bipolar spindle. Ndel1 phosphorylation is important for this function, regulating binding to both Lis1 and dynein. Prophase cells in the ventricular zone (VZ) of embryonic day 13.5 Lis1^+/− mouse brains show reduced PNEI, and the ratio of prophase to prometaphase cells is increased, suggesting an NEBD delay. Moreover, prophase cells in the VZ contain elevated levels of Ndel1 phosphorylated at a key cdk5 site. Our data suggest that a delay in NEBD in the VZ could contribute to developmental defects associated with Lis1–Ndel1 disruption.     

The COPI complex functions in nuclear envelope breakdown and is recruited by the nucleoporin Nup153

Nuclear envelope breakdown is a critical step in the cell cycle of higher eukaryotes. Although integral membrane proteins associated with the nuclear membrane have been observed to disperse into the endoplasmic reticulum at mitosis, the mechanisms involved in this reorganization remain to be fully elucidated. Here, using Xenopus extracts, we report a role for the COPI coatomer complex in nuclear envelope breakdown, implicating vesiculation as an important step. We have found that a nuclear pore protein, Nup153, plays a critical role in directing COPI to the nuclear membrane at mitosis and that this event provides feedback to other aspects of nuclear disassembly. These results provide insight into how key steps in nuclear division are orchestrated.     

The polo-like kinase PLK-1 is required for nuclear envelope breakdown and the completion of meiosis in Caenorhabditis elegans

The Polo-like kinases are key regulatory molecules required during the cell cycle for the successful completion of mitosis. We have cloned a C. elegans homolog of the Drosophila melanogaster polo gene (designated plk-1 for C. elegans polo-like kinase-1) and present the subcellular localization of the PLK-1 protein during the meiotic and mitotic cell cycles in C. elegans oocytes and embryos, respectively. Disruption of PLK-1 expression by RNA-mediated interference (RNAi) disrupts normal oocyte and embryonic development. Inspection of oocytes revealed a defect in nuclear envelope breakdown (NEBD) before ovulation. This defect in NEBD was also observed in oocytes that were depleted of the cyclin-dependent kinase NCC-1 (C. elegans homolog of Cdc2). The plk-1 RNAi oocytes were fertilized; however the resulting embryos were unable to separate their meiotic chromosomes or form and extrude polar bodies. These defects led to embryonic arrest as single cells. genesis 26:26-41, 2000. Published 2000 Wiley-Liss, Inc.     

Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.     

Microtubule dynamics at the G2/M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implications for spindle morphogenesis

We recently developed a direct fluorescence ratio assay (Zhai, Y., and G.G. Borisy. 1994. J. Cell Sci. 107:881-890) to quantify microtubule (MT) polymer in order to determine if net MT depolymerization occurred upon anaphase onset as the spindle was disassembled. Our results showed no net decrease in polymer, indicating that the disassembly of kinetochore MTs was balanced by assembly of midbody and astral MTs. Thus, the mitosis-interphase transition occurs by a redistribution of tubulin among different classes of MTs at essentially constant polymer level. We now examine the reverse process, the interphase-mitosis transition. Specifically, we quantitated both the level of MT polymer and the dynamics of MTs during the G2/M transition using the fluorescence ratio assay and a fluorescence photoactivation approach, respectively. Prophase cells before nuclear envelope breakdown (NEB) had high levels of MT polymer (62%) similar to that previously reported for random interphase populations (68%). However, prophase cells just after NEB had significantly reduced levels (23%) which recovered as MT attachments to chromosomes were made (prometaphase, 47%; metaphase, 56%). The abrupt reorganization of MTs at NEB was corroborated by anti- tubulin immunofluorescence staining using a variety of fixation protocols. Sensitivity to nocodazole also increased at NEB. Photoactivation analyses of MT dynamics showed a similar abrupt change at NEB, basal rates of MT turnover (pre-NEB) increased post-NEB and then became slower later in mitosis. Our results indicate that the interphase-mitosis (G2/M) transition of the MT array does not occur by a simple redistribution of tubulin at constant polymer level as the mitosis-interphase (M/G1) transition. Rather, an abrupt decrease in MT polymer level and increase in MT dynamics occurs tightly correlated with NEB. A subsequent increase in MT polymer level and decrease in MT dynamics occurs correlated with chromosome attachment. These results carry implications for understanding spindle morphogenesis. They indicate that changes in MT dynamics may cause the steady-state MT polymer level in mitotic cells to be lower than in interphase. We propose that tension exerted on the kMTs may lead to their lengthening and thereby lead to an increase in the MT polymer level as chromosomes attach to the spindle.     

In vitro assay for the quantitation of apoptosis-induced alterations of nuclear envelope permeability

This protocol describes how to perform comparative measurements of the permeability of the nuclear envelope in adherent cells. The plasma membrane is permeabilized at low digitonin concentrations, leaving the nuclear membrane intact. These semi-permeabilized cells are incubated with cytosolic extracts prepared in advance and with a fluorescent reporter molecule whose molecular weight exceeds the size-exclusion limit of the nuclear envelope. Images are taken with a confocal microscope and subsequently analyzed using a custom-made software program that recognizes the nuclei automatically and calculates the mean nuclear fluorescence signal. Here, we measure the increase in nuclear permeability triggered by cytosolic extracts from cells dying by apoptosis. This method can be employed for the study of processes that affect the nucleocytoplasmic distribution of fluorescent molecules in cell populations. The large size of the samples means that subtle fluctuations in nuclear fluorescence can be detected with a high confidence level. Isolation of cell extracts takes 5-6 h, and the preparation and imaging of 15 or so specimens takes 4-5 h.