Time Of Nuclear Envelope Breakdown Research Articles

Chromosome dynamics and spindle microtubule establishment in mouse embryos.

Chromosome segregation errors in mammalian embryos are common and jeopardize embryo health. Here, we perform for the first time 4-Dimensional imaging and tracking of chromosomes and centromeres through each preimplantation mitotic cell division in mouse embryos to define the normal dynamics of chromosome segregation. We show that a microtubule (MT)-dependent inward movement of chromosomes occurs at the time of nuclear envelope breakdown (NEBD), particularly in the earliest cell divisions, to position chromosomes prior to spindle assembly. Establishment of a rudimentary metaphase plate occurs immediately after NEBD, and is followed by a progressive alignment and biorientation of mitotic chromosomes. Stable end-on kinetochore-MT attachments form rapidly and attachment errors are uncommon. Altogether our data describe a rapid and efficient spindle assembly pathway that apparently minimizes the need for canonical MT attachment error correction in normally dividing embryos.

NuMA assemblies organize microtubule asters toestablish spindle bipolarity in acentrosomal human cells.

In most animal cells, mitotic spindle formation is mediated by coordination of centrosomal and acentrosomal pathways. At the onset of mitosis, centrosomes promote spindle bipolarization. However, the mechanism through which the acentrosomal pathways facilitate the establishment of spindle bipolarity in early mitosis is not completely understood. In this study, we show the critical roles of nuclear mitotic apparatus protein (NuMA) in the generation of spindle bipolarity in acentrosomal human cells. In acentrosomal human cells, we found that small microtubule asters containing NuMA formed at the time of nuclear envelope breakdown. In addition, these asters were assembled by dynein and the clustering activity of NuMA. Subsequently, NuMA organized the radial array of microtubules, which incorporates Eg5, and thus facilitated spindle bipolarization. Importantly, in cells with centrosomes, we also found that NuMA promoted the initial step of spindle bipolarization in early mitosis. Overall, these data suggest that canonical centrosomal and NuMA-mediated acentrosomal pathways redundantly promote spindle bipolarity in humancells.

Bridging centrioles and PCM in proper space and time.

Throughout biology, specifying cellular events at the correct location and time is necessary for ensuring proper function. The formation of robust microtubule organizing centers (MTOCs) in mitosis is one such event that must be restricted in space to centrosomes to prevent ectopic MTOC formation elsewhere in the cell, a situation that can result in multipolar spindle formation and aneuploidy. The process of reaching maximum centrosome MTOC activity in late G2, known as centrosome maturation, ensures accurate timing of nuclear envelope breakdown and proper chromosome attachment. Although centrosome maturation has been recognized for over a century, the spatial and temporal regulatory mechanisms that direct MTOC activation are poorly understood. Here, we review Sas-4/CPAP, Asterless/Cep152, Spd-2/Cep192, and PLP/Pericentrin, a group of proteins we refer to as ‘bridge’ proteins that reside at the surface of centrioles, perfectly positioned to serve as the gatekeepers of proper centrosome maturation at the perfect place and time.

Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females

The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.

Kinesin-2 Controls the Motility of RAB5 Endosomes and Their Association with the Spindle in Mitosis.

RAB5 is a small GTPase that belongs to the wide family of Rab proteins and localizes on early endosomes. In its active GTP-bound form, RAB5 recruits downstream effectors that, in turn, are responsible for distinct aspects of early endosome function, including their movement along microtubules. We previously reported that, at the onset of mitosis, RAB5positive vesicles cluster around the spindle poles and, during metaphase, move along spindle microtubules. RNAi-mediated depletion of the three RAB5 isoforms delays nuclear envelope breakdown at prophase and severely affects chromosome alignment and segregation. Here we show that depletion of the Kinesin-2 motor complex impairs long-range movement of RAB5 endosomes in interphase cells and prevents localization of these vesicles at the spindle during metaphase. Similarly to the effect caused by RAB5 depletion, functional ablation of Kinesin-2 delays nuclear envelope breakdown resulting in prolonged prophase. Altogether these findings suggest that endosomal transport at the onset of mitosis is required to control timing of nuclear envelope breakdown.

Timing of centrosome separation is important for accurate chromosome segregation

Spindle assembly, establishment of kinetochore attachment, and sister chromatid separation must occur during mitosis in a highly coordinated fashion to ensure accurate chromosome segregation. In most vertebrate cells, the nuclear envelope must break down to allow interaction between microtubules of the mitotic spindle and the kinetochores. It was previously shown that nuclear envelope breakdown (NEB) is not coordinated with centrosome separation and that centrosome separation can be either complete at the time of NEB or can be completed after NEB. In this study, we investigated whether the timing of centrosome separation affects subsequent mitotic events such as establishment of kinetochore attachment or chromosome segregation. We used a combination of experimental and computational approaches to investigate kinetochore attachment and chromosome segregation in cells with complete versus incomplete spindle pole separation at NEB. We found that cells with incomplete spindle pole separation exhibit higher rates of kinetochore misattachments and chromosome missegregation than cells that complete centrosome separation before NEB. Moreover, our mathematical model showed that two spindle poles in close proximity do not "search" the entire cellular space, leading to formation of large numbers of syntelic attachments, which can be an intermediate stage in the formation of merotelic kinetochores.

Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly

Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein-dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.

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.

Cyclin A2 Regulates Nuclear-Envelope Breakdown and the Nuclear Accumulation of Cyclin B1

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.

An increase in [Ca2+]i is sufficient but not necessary for driving mitosis in early mouse embryos

An increase in intracellular Ca2+ concentration ([Ca2+]i) has been shown to drive sea-urchin embryos and some fibroblasts through nuclear-envelope breakdown (NEBD) and the metaphase-to-anaphase transition. Mitotic Ca2+ transients can be pan-cellular global events or localized to the perinuclear region. It is not known whether Ca2+ is a universal regulator of mitosis or whether its role is confined to specific cell types. To test the hypothesis that Ca2+ is a universal regulator of mitosis, we have investigated the role of Ca2+ in mitosis in one-cell mouse embryos. Fertilized embryos generate Ca2+ transients during the first mitotic division. Imposing a Ca2+ transient by photorelease of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] resulted in acceleration of mitosis entry, suggesting that a [Ca2+]i increase is capable of triggering mitosis. Mitotic Ca2+ transients were inhibited using three independent approaches: injection of intracellular Ca2+ buffers; downregulation of Ins(1,4,5)P3 receptors; and removal of extracellular Ca2+. None of the interventions had any effects on the timing of NEBD or cytokinesis. The possibility that NEBD is driven by localized perinuclear Ca2+ transients was examined using two-photon microscopy but no Ca2+-dependent increases in fluorescence were found to precede NEBD. Finally, the second mitotic division took place in the absence of any detectable [Ca2+]i increase. Thus, although an induced [Ca2+]i increase can accelerate mitosis entry, neither cytosolic nor perinuclear [Ca2+] increases appear to be necessary for progression through mitosis in mouse embryos.

Pulling the spindle into shape

Mutants, RNAi, and extracts were unanimous: myosin II is not needed in mitosis before cytokinesis. But now Jody Rosenblatt, Karen McGee (University College London, England), and colleagues report that myosin II does, indeed, function in mitosis to pull apart and position centrosomes. Figure Spindles are defective without cortical myosin pulling on them. Rosenblatt did not set out to contradict the myosin II dogma. She was working on her primary interest of cell extrusion, checking the effect of blocking myosin, when “one day I was staining for tubulin and noticed spindle defects.” One colleague told her, “you really need to focus,” but she continued to see the unexpected phenotype. “Basically it was really crazy but it was happening,” she says. “It took me a long time to convince myself.” That convincing involved getting the same phenotype—defective spindles and displaced or misaligned chromosomes—after any one of four treatments: myosin RNAi, an immobilizing cross-linking of the cortex with extracellular lectins, and applying either one of two different myosin-inhibiting chemicals. Some cells were not affected because their centrosomes had migrated sufficiently far apart along the nuclear envelope by the time of nuclear envelope breakdown. (This lack of an absolute requirement may have obscured myosin effects in earlier experiments.) But up to half needed some later maneuvering by myosin II. Latex beads on the surface of cultured cells moved in the direction of centrosome movement. Rosenblatt suggests that myosin II activity in the cortex near centrosomes may be dampened by microtubules contacting a cortical protein. That would leave active only the myosin-based contraction on the opposite side of the cell. Centrosomes would be pulled apart toward that other side of the cell until microtubule–cortex contacts were equalized. ▪ Reference: Rosenblatt, J., et al. 2004. Cell. 117: 361–372. [PubMed]

Calcium- and Cell Cycle-dependent Association of Annexin 11 with the Nuclear Envelope

Annexin 11 is a widely expressed calcium- and phospholipid-binding protein that resides in the nucleoplasm in many cultured cell lines. This is in contrast to its most extensively characterized in vitro ligand, the small calcium-binding protein S100A6 (calcyclin), which is concentrated in the nuclear envelope. Here we have examined the significance of the association of annexin 11 and S100A6 by asking whether circumstances exist in which the two proteins occupy the same subcellular localization. First, we show that in both A431 and vascular smooth muscle cells, elevation of intracellular Ca2+ leads to translocation of annexin 11 from the nucleus to the nuclear envelope where it co-localizes with S100A6. We also demonstrate, using fusions of annexin 11 with green fluorescent protein, that whereas the C-terminal core domain of annexin 11 is essential for Ca2+ sensitivity, the N-terminal domain is required for targeting to the nuclear envelope. Second, we show that annexin 11 relocalizes to the nuclear envelope as A431 cells transit from early to mid-prophase. In late prophase, at the time of nuclear envelope breakdown, annexin 11 and S100A6 become intensely localized with lamina-associated polypeptide 2 to folds in the nuclear envelope. From metaphase to telophase S100A6 is degraded, but in late telophase annexin 11 associates with the reforming nuclear envelope before resuming a nucleoplasmic location in interphase. These results show that co-localization of annexin 11 and S100A6 at the nuclear envelope may be regulated either by elevation of intracellular Ca2+ or by cell cycle progression and provide the first evidence that these proteins may associate in vivo.

Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells.

Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.

Changes in organization of the endoplasmic reticulum during Xenopus oocyte maturation and activation.

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releasability of Ca(2+) by IP(3). The clusters dispersed during the Ca(2+) wave at activation. Possible relationships of ER structure and Ca(2+) regulation are discussed.

C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis.

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes.

Dependence of timing of mitotic events on the rate of protein synthesis and DNA replication in sea urchin early cleavages.

To understand what processes affect the cell-cycle timing of mitotic events in early cleavage cycles of sea urchin embryos, a study was made on the effects of (a) reducing protein synthesis with emetine and (b) DNA replication with aphidicolin, on the timing of nuclear envelope breakdown, anaphase onset and cytokinesis. When protein synthesis was slightly inhibited by administration of emetine, the delay in the mitotic events increased, with an increase in the delay in accumulation of proteins up to the levels to which cells must synthesize the proteins to execute the cleavage. This indicated that protein synthesis affects the timing of mitotic events. The delay in cleavage cycles caused by a slight inhibition of DNA replication with aphidicolin was in proportion to the concentration of aphidicolin administered, suggesting that DNA replication also affects the timing of mitotic events. Furthermore, it was confirmed that accumulation of the proteins to the levels required for execution of the first cleavage precedes completion of DNA replication as a requirement for execution of the first cleavage. These results imply the existence of process(es) affected by protein synthesis that are included in a feedback control system which prevents the initiation of mitosis until after the completion of DNA replication; it is the characteristic of a cell-cycle control system that has been predicted theoretically.

Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E.

To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.

Gamma Tubulin is Localized to a New Organelle, The Multivesicular Aggregate, In Mammalian Oocytes

Abstract Oocyte meiotic maturation occurs without the aid of centrioles in the mouse; instead the division spindles are organized by a number of microtubule organizing centers (MTOC). Immunofluorescence microscopy using anti-γ tubulin shows that multiple MTOCs migrate to surround the central nucleus (germinal vesicle, GV) during early maturation.. However, at the EM level, recognizable MTOCs have never been seen prior to the nucleation of microtubules (MT) which occurs near the time of nuclear envelope breakdown. Recently, I described multivesicular aggregates (MVAs) as the organelles most likely to represent immature MTOCs prior to MT nucleation4. These are first seen at time zero by EM as two large aggregates in the cortex, with sizes averaging 10um. MVA subsequently fragment into smaller units as they migrate centrally. Materials and Methods: Female ICR mice were injected IP with 100 iu of chorionic gonadotropin (Sigma) to stimulate follicle growth, and ovaries were removed 48 hours later.

Mitotic cyclin distribution during maize cell division: Implications for the sequence diversity and function of cyclins in plants

Cyclin proteins are components of the regulatory system that controls the orderly progression of the events of cell division. Their sub-cellular location, as well as their fluctuating abundance and their affinities for the cyclin-dependent kinases (CDKs) to which they bind, determine their successive roles during the cell cycle. Here we employ species-specific antibodies to monitor changes in quantity and location of four maize cyclins and maize Cdc2-kinase in dividing maize root tip cells. Maize cyclin Ia occurs in the nuclear matrix and is released when the nuclear envelope breaks down. In contrast, cyclin Ib is cytoplasmic until prophase; it associates transiently with the nuclear envelope and preprophase band (PPB) just before these structures break down and then associates with the condensed chromosomes and spindle region before declining at anaphase. Cyclin II and Cdc2 also occur in the PPB. Occurrence of cyclin Ib and Cdc2 at the PPB concurrent with initiation of breakdown is consistent with previous studies in which microinjection of cyclin-dependent protein kinase indicated that removal of the PPB at the time of nuclear-envelope breakdown is catalysed by a CDK. While cyclins Ia and III are predominantly nuclear prior to mitosis, cyclins Ib and II are predominantly cytoplasmic until prophase then become nuclear. The initial cytoplasmic retention of cyclins Ib and II correlates with their possession of a sequence similar to the cytoplasmic-retention signal of animal cyclin B1. Cyclin II binds to all microtubule arrays during the cell cycle, becoming markedly concentrated in the phragmoplast, and cyclin III associates with the spindle and then the phragmoplast. Cdc2 also occurs in the phragmoplast. Persistence of mitotic cyclins and CDK after mitosis into the cytokinetic stage, as seen in maize, is not paralleled in animal cells, where the cytokinetic mid-body is not so labelled, presumably reflecting the key role of the phragmoplast apparatus in plant cell division.

Probing The Mechanisms Underlying Kinetochore Behavior In Vertebate Cells Using Combinations of Advanced Light and 3-D Electron Microscopy

Abstract For daughter cells to receive equal copies of the genome during mitosis, the replicated chromosomes must attach to and move bi-directionally on the mitotic spindle. A chromosome becomes attached to the spindle via a pair specialized structures, known as kinetochores, that are positioned on opposite sides of its primary constriction (one on each of the two chromatids). In addition to being the spindle attachment site, kinetochores are also involved in producing and/or transmitting the forces for chromosome motion. In vertebrates the kinetochore closest to a spindle pole at the time of nuclear envelope breakdown usually is the first to attach to the spindle. As a result of this attachment the now “monooriented” chromosome moves toward the closest pole where its only attached kinetochore initiates oscillatory motions toward and away from that pole until the unattached sister kinetochore acquires microtubules (Mts) from the opposite spindle pole.