Silencing of Nuclear Mitotic Apparatus protein (NuMA) accelerates the apoptotic disintegration of the nucleus

One main feature of apoptosis is the sequential degradation of the nuclear structure, including the fragmentation of chromatin and caspase-mediated cleavage of various nuclear proteins. Among these proteins is the Nuclear Mitotic Apparatus protein (NuMA) which plays a specific role in the organization of the mitotic spindle. The exact function of NuMA in the interphase nucleus is unknown, but a number of reports have suggested that it may play a role in chromatin organization and/or gene expression. Here we show that upon cleavage in apoptotic cells, the N-terminal cleavage fragment of NuMA is solubilized while the C-terminal fragment remains associated with the condensed chromatin. Using pancaspase inhibitor z-VAD-fmk and caspase-3 deficient MCF-7 cells, we further show that the solubilization is dependent on caspase-mediated cleavage of NuMA. Finally, the silencing of NuMA by RNAi accelerated nuclear breakdown in apoptotic MCF-7 cells. These results suggest that NuMA may provide structural support in the interphase nucleus by contributing to the organization of chromatin.     

NuMA interaction with chromatin is vital for proper chromosome decondensation at the mitotic exit

NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding–deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindle-independent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA.     

Caspase-3 is required in the apoptotic disintegration of the nuclear matrix

Apoptotic breakdown of cellular structures is largely mediated by caspases. One target of degradation is a proteinaceous framework of the nucleus termed the nuclear matrix. We compared the apoptotic changes of the nuclear matrix in staurosporine-treated caspase-3-deficient MCF-7 cells transfected with intact CASP-3 gene (MCF-7c3) or an empty vector (MCF-7v) as a control. Nuclear Mitotic Apparatus protein (NuMA), lamin A/C and lamin B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. In both cell lines, staurosporine induced rapid cytoplasmic shrinkage and partial chromatin condensation. MCF-7c3 cells formed apoptotic bodies, whereas MCF-7v cells did not. NuMA and lamins were actively cleaved in MCF-7c3 cells following caspase-3 activation, but only minimal or no cleavage was detected in MCF-7v cells. Interestingly, lamin B but not lamin A/C was relocated into cytoplasmic granules in apoptotic MCF-7v cells. Pancaspase inhibitor, z-VAD-fmk, prevented the apoptotic changes, while caspase-3 inhibitor, z-DEVD-fmk, induced lamin B granules in both cell lines. These results show that caspase-3 is involved in the cleavage of NuMA and lamins either directly or by activating other proteases. This may be essential for disintegration of the nuclear structure during apoptosis.     

Akt phosphorylates acinus and inhibits its proteolytic cleavage, preventing chromatin condensation

Akt promotes cell survival by phosphorylating and inhibiting components of the intrinsic cell death machinery. Akt translocates into the nucleus upon exposure of cells to survival factors, but little is known about its functions in the nucleus. Here, we show that acinus, a nuclear factor required for apoptotic chromatin condensation, is a direct target of Akt. We demonstrate that Akt phosphorylation of acinus on serine 422 and 573 results in its resistance to caspase cleavage in the nucleus and the inhibition of acinus-dependent chromatin condensation. Abolishing acinus phosphorylation by Akt through mutagenesis accelerates its proteolytic degradation and chromatin condensation. Acinus S422, 573D, a mutant mimicking phosphorylation, resists against apoptotic cleavage and prevents chromatin condensation. Knocking down of acinus substantially decreases chromatin condensation, and depletion of Akt provokes the apoptotic cleavage of acinus. Thus, Akt inhibits chromatin condensation during apoptosis by phosphorylating acinus in the nucleus, revealing a specific mechanism by which nuclear Akt promotes cell survival.     

NuMA is required for the proper completion of mitosis

NuMA is a 236-kD intranuclear protein that during mitosis is distributed into each daughter cell by association with the pericentrosomal domain of the spindle apparatus. The NuMA polypeptide consists of globular head and tail domains separated by a discontinuous 1500 amino acid coiled-coil spacer. Expression of human NuMA lacking its globular head domain results in cells that fail to undergo cytokinesis and assemble multiple small nuclei (micronuclei) in the subsequent interphase despite the appropriate localization of the truncated NuMA to both the nucleus and spindle poles. This dominant phenotype is morphologically identical to that of the tsBN2 cell line that carries a temperature-sensitive mutation in the chromatin-binding protein RCC1. At the restrictive temperature, these cells end mitosis without completing cytokinesis followed by micronucleation in the subsequent interphase. We demonstrate that the wild-type NuMA is degraded in the latest mitotic stages in these mutant cells and that NuMA is excluded from the micronuclei that assemble post-mitotically. Elevation of NuMA levels in these mutant cells by forcing the expression of wild-type NuMA is sufficient to restore post-mitotic assembly of a single normal-sized nucleus. Expression of human NuMA lacking its globular tail domain results in NuMA that fails both to target to interphase nuclei and to bind to the mitotic spindle. In the presence of this mutant, cells transit through mitosis normally, but assemble micronuclei in each daughter cell. The sum of these findings demonstrate that NuMA function is required during mitosis for the terminal phases of chromosome separation and/or nuclear reassembly.     

Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

BAPTA blocks DNA fragmentation and chromatin condensation downstream of caspase-3 and DFF activation in HT-induced apoptosis in HL-60 cells

DFF ((DNA Fragmentation Factor) is a heterodimer composed of 40 kDa (DFF40, CAD) and 45 kDa (DFF45, ICAD) subunits. During apoptosis, activated caspase-3 cleaves DFF45 and activates DFF40, a DNase that targets nucleosomal linker region and cleaves chromatin DNA into nucleosomal fragments. We have previously reported that HT induced apoptosis in HL-60 cells, and intracellular Ca²⁺ chelator BAPTA blocked apoptosis-associated DNA fragmentation induced by HT. We report here that HT also induced activation of caspase-3 and cleavage of DFF45. BAPTA prevented neither the caspase-3 activation nor the cleavage of DFF45. Mitochondrial membrane potential was disrupted in BAPTA-AM treated cells. However, BAPTA did prevent DNA fragmentation and chromatin condensation in HT-treated cells. These data suggest a novel role for intracellular calcium in regulating apoptotic nuclease that causes DNA fragmentation and chromatin condensation.     

Overexpression of Helicard,a CARD-containing helicase cleaved during apoptosis,accelerates DNA degradation

Apoptotic cell death is characterized by several morphological nuclear changes, such as chromatin condensation and extensive fragmentation of chromosomal DNA. These alterations are primarily triggered through the activation of caspases, which subsequently cleave nuclear substrates. Caspase-3 induces processing of Acinus, which leads to chromatin condensation. DNA fragmentation is dependent on the DNase CAD, which is released from its inhibitor, ICAD, upon cleavage by caspase-3. DNA degradation is also induced by AIF and endonuclease G, which are both released from mitochondria upon death stimuli but do not require prior processing by caspases for their DNase activity. Here we report the identification of a widely expressed helicase designated Helicard, which contains two N-terminal CARD domains and a C-terminal helicase domain. Upon apoptotic stimuli, Helicard is cleaved by caspases, thereby separating the CARD domains from the helicase domain. While Helicard localizes in the cytoplasm, the helicase-containing fragment is found in the nucleus. Helicard accelerates Fas ligand-mediated DNA degradation, whereas a noncleavable or a helicase-dead Helicard mutant does not, implicating Helicard in the nuclear remodeling occurring during apoptosis.     

NuMA: a protein involved in nuclear structure, spindle assembly, and nuclear re-formation

The abundant coiled-coil protein NuMA is located in the nucleus during interphase, but when the nuclear envelope disassembles in prometaphase it rapidly redistributes to the developing spindle poles. Microinjection of antibodies to NuMA at or before metaphase can block spindle assembly or cause spindle collapse, indicating a role for NuMA in spindle function. NuMA must also play a key role in telophase, as NuMA antibodies or truncations of NuMA cause aberrant nuclear reassembly despite apparently normal chromosome segregation. Consistent with a structural role for NuMA in the nucleus, immunoelectron microscopy reveals NuMA to be a component of nuclear filaments.     

Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation

To study the role of caspase-6 during nuclear disassembly, we generated a chicken DT40 cell line in which both alleles of the caspase-6 gene were disrupted. No obvious morphological differences were observed in the apoptotic process in caspase-6- deficient cells compared with the wild type. However, examination of apoptosis in a cell-free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6-deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild-type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor z-VEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together, these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution.     

Akt phosphorylation of zyxin mediates its interaction with acinus-S and prevents acinus-triggered chromatin condensation

Zyxin, a focal adhesion molecule, contains LIM domains and shuttles between the cytoplasm and the nucleus. Nuclear zyxin promotes cardiomyocyte survival, which is mediated by nuclear-activated Akt. However, the molecular mechanism of how zyxin antagonizes apoptosis remains elusive. Here, we report that zyxin binds to acinus-S, a nuclear speckle protein inducing apoptotic chromatin condensation after cleavage by caspases, and prevents its apoptotic action, which is regulated by Akt. Akt binds and phosphorylates zyxin on serine 142, leading to its association with acinus. Interestingly, 14-3-3gamma, but not zeta isoform selectively, triggers zyxin nuclear translocation, which is Akt phosphorylation dependent. Zyxin is also a substrate of caspases, but Akt phosphorylation is unable to prevent its apoptotic cleavage. Expression of zyxin S142D, a phosphorylation mimetic mutant, diminishes acinus proteolytic cleavage and chromatin condensation; by contrast, wild-type zyxin or unphosphorylated S142A mutant fails. Thus, Akt regulates zyxin/acinus complex formation in the nucleus, contributing to suppression of apoptosis.     

SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF^Slimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF^Slimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF^Slimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.     

Specific attachment of nuclear-mitotic apparatus protein to metaphase chromosomes and mitotic spindle poles: possible function in nuclear reassembly

NuMA protein is the largest, abundant, primate-specific chromosomal protein. The protein was purified from HeLa cells and monospecific monoclonal antibodies were prepared that react exclusively with NuMA protein in immunoblot analysis. These antibodies were used to define the intracellular location and properties of NuMA protein. Using indirect immunofluorescence, NuMA protein was detected only in the nucleus of interphase cells and on the chromosomes in mitotic cells. One class of monoclonal antibody called the 2E4-type antibody, caused NuMA protein (or a complex of proteins including NuMA) to be released from its binding site on metaphase or anaphase chromosomes. The separation of NuMA protein from chromosomes was observed either with the immunofluorescence assay or in electrophoretic analyses of proteins released from isolated metaphase chromosomes after reaction with 2E4 antibody. The immunofluorescence studies also showed that after release of the NuMA protein from chromosomes of metaphase or anaphase cells, the protein bound specifically to the polar region of the mitotic spindle. It was shown that exogenously added NuMA antigen/antibody complex bound only to the mitotic spindle poles of permeabilized primate cells and not to the spindle poles of other mammalian cells, thus demonstrating the specificity of the spindle-pole interaction. The antibody mediated transfer of NuMA from chromosomes to poles was blocked when the chromosomes were treated with cross-linking fixatives. Results suggest that the NuMA protein has specific attachment sites on both metaphase chromosomes and mitotic spindle poles (the site where post-mitotic nuclear assembly occurs). A model is proposed suggesting that a protein having such dual binding sites could function during nuclear reassembly to link mitotic chromosomes into the reforming nucleus.     

HorkaD, a Chromosome Instability-Causing Mutation in Drosophila, Is a Dominant-Negative Allele of lodestar

Correct segregation of chromosomes is particularly challenging during the rapid nuclear divisions of early embryogenesis. This process is disrupted by Horka^D, a dominant-negative mutation in Drosophila melanogaster that causes female sterility due to chromosome tangling and nondisjunction during oogenesis and early embryogenesis. Horka^D also renders chromosomes unstable during spermatogenesis, which leads to the formation of diplo//haplo mosaics, including the gynandromorphs. Complete loss of gene function brings about maternal-effect lethality: embryos of the females without the Horka^D-identified gene perish due to disrupted centrosome function, defective spindle assembly, formation of chromatin bridges, and abnormal chromosome segregation during the cleavage divisions. These defects are indicators of mitotic catastrophe and suggest that the gene product acts during the meiotic and the cleavage divisions, an idea that is supported by the observation that germ-line chimeras exhibit excessive germ-line and cleavage function. The gene affected by the Horka^D mutation is lodestar, a member of the helicase-related genes. The Horka^D mutation results in replacement of Ala777 with Thr, which we suggest causes chromosome instability by increasing the affinity of Lodestar for chromatin.     

A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.     

Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR

Apoptotic DNA degradation could be initiated by the accumulation of single-strand (ss) breaks in vulnerable chromatin regions, such as base unpairing regions (BURs), which might be preferentially targeted for degradation by both proteases and nucleases. We tested this hypothesis in anti-Fas-treated apoptotic Jurkat cells. Several nuclear proteins known for their association with both MARs and the nuclear matrix, that is, PARP, NuMA, lamin B and SATB1, were degraded, but the morphological rearrangement of the BUR-binding SATB1 protein was one of the earliest detected changes. Subsequently, we have identified several genes containing sequences homologous to the 25 bp BUR element of the IgH gene, a known SATB1-binding site, and examined the integrity of genomic DNA in their vicinity. Multiple ss breaks were found in close proximity to these sites relative to adjacent regions of DNA. Consistent with our prediction, the results indicated that the initiation of DNA cleavage in anti-Fas-treated Jurkat cells occurred within the BUR sites, which likely became accessible to endonucleases due to the degradation of BUR-binding proteins.     

CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis

DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic chromatin condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of chromatin condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic chromatin condensation.     

Multiple Pathways for Apoptotic Nuclear Fragmentation

We analyzed the ultrastructure of apoptotic nuclear fragmentation in U937 cells treated with many different apoptogenic agents. We found that this characteristic apoptotic feature can be achieved through multiple alternative pathways, depending on the apoptogenic inducer, leading to slightly different final nuclear morphologies. In most instances, the irregularly shaped nucleus of U937 rounds up; then, chromatin condenses at the nuclear periphery. Condensed chromatin can form protruding patches, which eventually bud from the nucleus in sealed vesicles through a process which is actin-dependent, since it could be blocked by cytochalasins. Alternatively, chromatin condenses in tiny, nonprotruding crescents, and a cleavage in the nuclear sap forms, beginning from the inner nuclear membrane and growing inward, thus splitting the nucleus. In U937 induced to apoptosis by hydrogen peroxide in the presence of ADP-ribosylation inhibitors, the nuclei fragment in many vesicles before chromatin even begins to condense: chromatin condensation probably occurs as a consequence. While all the apoptotic morphologies described above evolve from interphase cells, a peculiar apoptotic morphology, possibly deriving from mitotic cells, is detected upon oxidative stress, recalling the formation of micronuclei by clastogenic treatments; it shows partially membrane-bound chromatin patches, which look midway between condensed chromosomes and apoptotic condensed chromatin. The existence of these multiple pathways for nuclear fragmentation may indicate an evolutionary convergence, suggesting that this event may play an important physiological role in apoptosis.     

Pim-1 associates with protein complexes necessary for mitosis

The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HP1beta) and the cytoplasmic proteins dynein and dynactin via complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the 'kinase-dead' mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HP1beta, dynein and dynactin, a complex that is necessary for mitosis.