Microtubule dynamics alter the interphase nucleus

Microtubules are known to drive chromosome movements and to induce nuclear envelope breakdown during mitosis and meiosis. Here we show that microtubules can enforce nuclear envelope folding and alter the levels of nuclear envelope-associated heterochromatin during interphase, when the nuclear envelope is intact. Microtubule reassembly, after chemically induced depolymerization led to folding of the nuclear envelope and to a transient accumulation of condensed chromatin at the site nearest the microtubule organizing center (MTOC). This microtubule-dependent chromatin accumulation next to the MTOC is dependent on the composition of the nuclear lamina and the activity of the dynein motor protein. We suggest that forces originating from simultaneous polymerization of microtubule fibers deform the nuclear membrane and the underlying lamina. Whereas dynein motor complexes localized to the nuclear envelope that slide along the microtubules transfer forces and/or signals into the nucleus to induce chromatin reorganization and accumulation at the nuclear membrane folds. Thus, our study identified a molecular mechanism by which mechanical forces generated in the cytoplasm reshape the nuclear envelope, alter the intranuclear organization of chromatin, and affect the architecture of the interphase nucleus.     

The plant nuclear envelope

This review summarizes our present knowledge about the composition and function of the plant nuclear envelope. Compared with animals or yeast, our molecular knowledge of the nuclear envelope in higher plants is in its infancy. However, there are fundamental differences between plants and animals in the structure and function of the nuclear envelope. This review will compare and contrast these differences for nuclear pore complexes, nuclear transport, inner nuclear envelope proteins and the role of the nuclear envelope during mitosis. In some cases, seemingly 'novel' aspects of plant nuclear envelope function may provide new insight into the animal cell nucleus.     

The nuclear envelope: emerging roles in development and disease

The chromosomes of eukaryotic cells are separated from the cytoplasm by the nuclear envelope. The nuclear envelope includes two riveted membranes, plus embedded pore complexes that mediate nuclear import and export. In this sense, the nuclear envelope is truly a border zone. However, the envelope also links directly to chromosomes, and anchors two major infrastructures--the nuclear lamina and Tpr filaments--to the nuclear perimeter. Proteins of the nuclear envelope mediate a variety of fundamental activities, including DNA replication, gene expression and silencing, chromatin organization, cell division, apoptosis, sperm nuclear remodeling, the behavior of pronuclei, cell fate determination, nuclear migration and cell polarity. Furthermore, mutations in nuclear lamins and lamin-binding proteins cause tissue-specific inherited diseases. This special issue of Cell and Molecular Life Sciences is devoted to recent major advances in the characterization of nuclear envelope proteins and their roles. We offer here an overview of the topics covered in this issue of CMLS, and also discuss the emerging recognition that the nuclear envelope is an organelle critical for a wide range of genetic and developmental activity in multicellular organisms.     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.     

Dissection of the molecular mechanisms that control the nuclear accumulation of transport factors importin-α and CAS in stressed cells

The physiological state of eukaryotic cells controls nuclear trafficking of numerous cargos. For example, stress results in the inhibition of classical protein import, which is characterized by the redistribution of several transport factors. As such, importin-alpha and cellular apoptosis susceptibility protein (CAS) accumulate in nuclei of heat-shocked cells; however, the mechanisms underlying this relocation are not fully understood. We now show that heat upregulates the initial docking of importin-alpha at the nuclear envelope and stimulates the translocation of CAS into the nuclear interior. Moreover, heat exposure compromises the exit of importin-alpha from nuclei and drastically increases its retention in the nucleoplasm, whereas CAS nuclear exit and retention are less affected. Taken together, our results support the idea that heat shock regulates importin-alpha and CAS nuclear accumulation at several levels. The combination of different stress-induced changes leads to the nuclear concentration of both transport factors in heat-stressed cells.     

Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium

The Dictyostelium centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates, yielding two spindle poles. CP148 is a novel coiled coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle-dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage.     

Programmed cell clearance

Apoptosis, a physiological process of self-annihilation, is essential during development and for the maintenance of tissue homeostasis. Considerable efforts have been made in recent years to elucidate the molecular mechanisms that govern this mode of cellular demise; however, the subsequent recognition and removal of apoptotic corpses by neighboring phagocytes has received less attention. Nevertheless, macrophage engulfment of apoptotic cells is known to be important in the remodeling of tissues, and contributes to the resolution of inflammation through the removal of effete cells prior to the release of noxious cellular constituents. Moreover, apoptotic cells are a potential source of self-antigens, and clearance of cell corpses is thought to preclude the induction of autoimmune responses. The view is thus emerging that tissue homeostasis is dependent not only on the balance between mitosis and apoptosis, but also on the rate of apoptosis versus that of cell clearance. This review aims to discuss the mechanisms and consequences of macrophage recognition and disposal of apoptotic cells, a process which will be referred to as programmed cell clearance.Received 16 April 2003; received after revision 22 May 2003; accepted 26 May 2003     

Action and interactions at microtubule ends

Microtubule dynamic instability is fundamentally important to the way cells respond to their environment and segregate their genetic material. A disparate class of proteins defined by their localisation to growing microtubule plus ends ('+TIPS') play a key role in controlling microtubule dynamics and organisation. They directly impact upon the behaviour of the microtubule tip and link this structure to interfaces that include kinetochores and the cortex of the cell. Surprisingly, some +TIPs also have important functions at the microtubule minus end. These properties contribute to the important roles played by +TIPs in processes such as mitosis and cell migration. This review examines how recent advances have impacted our understanding of +TIP function in mammalian cells, with emphasis on the emergence of the EB1 family as a core component of +TIP activities. An overview of the use of +TIP imaging as a tool for the cell biologist is also presented.     

A stereoscan analysis of cell surface characteristics during the interkinetic nuclear migration in normal and colchicine-treated developing chick retina

Summary The cell surface and shape changes taking place during the interkinetic nuclear migration are reported in normal neuroepithelial cells of chick retina, and after metaphase-arrest induced by colchicine. Persistence of the apical junctions and loss of basal connections during the preparation for mitosis have been demonstrated in these cells.     

Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

Nuclear envelope complexity is expanding with respect to identification of protein components. Here we test the validity of proteomics results that identified 67 novel predicted nuclear envelope transmembrane proteins (NETs) from liver by directly comparing 30 as tagged fusions using targeting assays. This confirmed 21 as NETs, but 4 only targeted in certain cell types, underscoring the complexity of interactions that tether NETs to the nuclear envelope. Four NETs accumulated at the nuclear rim in normal fibroblasts but not in fibroblasts lacking lamin A, suggesting involvement of lamin A in tethering them in the nucleus. However, intriguingly, for the NETs tested alternative mechanisms for nuclear envelope retention could be found in Jurkat cells that normally lack lamin A. This study expands by a factor of three the number of liver NETs analyzed, bringing the total confirmed to 31, and shows that several have multiple mechanisms for nuclear envelope retention.     

Telomeres and meiosis in health and disease

Telomeres are important segments of chromosomes that protect chromosome ends from nucleolytic degradation and fusion. At meiosis telomeres display an unprecedented behavior which involves their attachment and motility along the nuclear envelope. The movements become restricted to a limited nuclear sector during the so-called bouquet stage, which is widely conserved among species. Recent observations suggest that telomere clustering involves actin and/or microtubules, and is altered in the presence of impaired recombinogenic and chromosome related functions. This review aims to provide an overview of what is currently known about meiotic telomere attachment, dynamics and regulation in synaptic meiosis.     

Telomeres and meiosis in health and disease

Telomeres are important segments of chromosomes that protect chromosome ends from nucleolytic degradation and fusion. At meiosis telomeres display an unprecedented behavior which involves their attachment and motility along the nuclear envelope. The movements become restricted to a limited nuclear sector during the so-called bouquet stage, which is widely conserved among species. Recent observations suggest that telomere clustering involves actin and/or microtubules, and is altered in the presence of impaired recombinogenic and chromosome related functions. This review aims to provide an overview of what is currently known about meiotic telomere attachment, dynamics and regulation in synaptic meiosis.     

Inhibitors of phosphatidylinositol 3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells

Phosphatidylinositol 3-kinase (PI3-kinase) activity has been implicated in regulating cell cycle progression at distinct points in the cell cycle by preventing cell cycle arrest or apoptosis. In this study, the role of PI3-kinase activity during the entire G1 phase of the ongoing cell cycle was studied in Chinese hamster ovary (CHO) cells synchronized by mitotic shake-off. We show that inhibition of PI3-kinase activity during and 2 h after mitosis inhibited cell cycle progression into S phase. In the presence of the PI3-kinase inhibitor wortmannin or LY294002, cells were arrested during early G1 phase, leading to the expression of the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PI3-kinase activity is required for progression through the M/G1 phase. In the absence of PI3-kinase activity, cells are induced for apoptosis in this particular phase of the cell cycle. Received 7 September 2005; received after revision 26 October 2005; accepted 11 November 2005     

A kinesin-mediated mechanism that couples centrosomes to nuclei

The M-type kinesin isoform, Kif9, has recently been implicated in maintaining a physical connection between the centrosome and nucleus in Dictyostelium discoideum. However, the mechanism by which Kif9 functions to link these two organelles remains obscure. Here we demonstrate that the Kif9 protein is localized to the nuclear envelope and is concentrated in the region underlying the centrosome point of attachment. Nuclear anchorage appears mediated through a specialized transmembrane domain located in the carboxyl terminus. Kif9 interacts with microtubules in in vitro binding assays and effects an endwise depolymerization of the polymer. These results suggest a model whereby Kif9 is anchored to the nucleus and generates a pulling force that reels the centrosome up against the nucleus. This is a novel activity for a kinesin motor, one important for progression of cells into mitosis and to ensure centrosome-nuclear parity in a multinuclear environment.     

VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3–Thr3ph–survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.     

Structure,function and evolution of haspin and haspinrelated proteins,a distinctive group of eukaryotic protein kinases

The haspins constitute a newly defined protein family containing a distinctive C-terminal eukaryotic protein kinase domain and divergent N termini. Haspin homologues are found in animals, plants and fungi, suggesting an origin early in eukaryotic evolution. Most species have a single haspin homologue. However, Saccharomyces cerevisiae has two such genes, while Caenorhabditis elegans has at least three haspin homologues and approximately 16 haspin-related genes. Mammalian haspin genes have features of retrogenes and are strongly expressed in male germ cells and at lower levels in some somatic tissues. They encode nuclear proteins with serine/threonine kinase activity. Murine haspin is reported to inhibit cell cycle progression in cell lines. One of the S. cerevisiae homologues, ALK1, is a member of the CLB2 gene cluster that peaks in expression at M phase and thus may function in mitosis. Therefore, the haspins are an intriguing group of kinases likely to have important roles during or following both meiosis and mitosis.     

Inhibition of protein deacetylation by trichostatin A impairs microtubule-kinetochore attachment

Inhibition of protein deacetylation arrests cells in mitosis, but the mechanism is unknown. To understand why inhibiting protein deacetylation causes cell cycle arrest, we treated HeLa cells beyond G1/S transition with trichostatin A (TSA), a potent protein deacetylase inhibitor, and found that the cells arrested at prometaphase with ectopic spindles and unaligned chromosomes. The hyper-acetylated cells encountered a serious microtubule (MT)-kinetochore attachment problem, although the kinetochores are intact at ultrastructural level. By immunofluorescence staining of kinetochore proteins, we found that the pericentromeric H3K9Me3-HP1 pathway was disrupted and that the CENP-A-dependent outer plate protein dynamics of kinetochores was greatly diminished by the drug treatment. The treatment also caused the loss of chromosome passenger complex (CPC), the proposed error checking system, from centromere and impaired the microtubule dynamics of the cells. Overall, we propose that deacetylation inhibition impairs MT-kinetochore attachment through disrupting the centromere function and altering the kinetochore composition and MT dynamics. Received 30 April 2008; received after revision 28 July 2008; accepted 14 August 2008     

Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplasts

Detection of stereotypic hallmarks of apoptosis during cell death induced by menadione, including DNA laddering and the formation of apoptotic bodies, is reported. Comet assay and the TdT-mediated dUTP nick end labelling (TUNEL) procedure were also performed to detect DNA fragmentation. Inhibition of DNA fragmentation by Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and phenylmethylsulfosyl (PMSF) implicated the involvement of caspase-like proteases in menadione-induced apoptosis in plants. We further studied the cleavage of lamin-like proteins during apoptosis in menadione-treated tobacco protoplasts. In animals, it has been reported that the solubilization of nuclear lamina and lamin degradation occurs during apoptotic cell death. However, little is known about the fate of lamins in apoptotic plant cells. Our study provided evidence that lamin-like proteins degraded into 35-kDa fragments in tobacco protoplasts induced by menadione, and this preceded DNA fragmentation. The results thus indicated that proteolytic cleavage of nuclear lamins was also conserved in programmed cell death in plants. Received 16 November 1998; received after revision 21 December 1998; accepted 23 December 1998