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.     

The nuclear form of glutathione peroxidase 4 is associated with sperm nuclear matrix and is required for proper paternal chromatin decondensation at fertilization

The nuclear isoform of the selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is expressed in haploid male germ cells, contains several cysteines and is able to oxidize protein thiols, besides glutathione. In this study we have investigated the subnuclear localization of this isoform in isolated mouse male germ cells at different steps of maturation. Immunoblotting and confocal microscopy analyses of subnuclear fractions showed that nGPx4 is localized to the nuclear matrix together with well known markers of this subnuclear compartment like lamin B and topoisomerase IIβ at all stages of germ cell differentiation. The peculiar nGPx4 distribution was confirmed by both biochemical and morphological analyses of COS-1 cells overexpressing Flag-tagged nGPx4. To test the functional role of nGPx4 in the process of chromatin assembly, sperm isolated from the caput and the cauda epididymides of wild-type (WT) and genetically deficient in nGPx4 (nGPx4-KO) mice were analyzed in an in vitro chromatin decondensation assay. Results showed that sperm from nGPx4-KO mice were more prone to decondense than those from WT mice at all stages of epididymal maturation, providing conclusive evidence that nGPx4 is required for a correct sperm chromatin compaction. We next addressed the issue of whether the lack of nGPx4 impacts on early events occurring at fertilization. Indeed, in vitro fertilization experiments showed an acceleration of sperm chromatin dispersion in oocytes fertilized by nGpx4-KO sperm compared with control. Overall these data indicate that the absence of nGPx4 leads to sperm nuclear matrix/chromatin instability that may negatively affect the embryo development.     

NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.     

Protein phosphatase 1 is essential for Greatwall inactivation at mitotic exit

Entry into mitosis is mediated by the phosphorylation of key cell cycle regulators by cyclin-dependent kinase 1 (Cdk1). In Xenopus embryos, the M-phase-promoting activity of Cdk1 is antagonized by protein phosphatase PP2A-B55. Hence, to ensure robust cell cycle transitions, Cdk1 and PP2A-B55 must be regulated so that their activities are mutually exclusive. The mechanism underlying PP2A-B55 inactivation at mitotic entry is well understood: Cdk1-activated Greatwall (Gwl) kinase phosphorylates Ensa/Arpp19, thereby enabling them to bind to and inhibit PP2A-B55. However, the re-activation of PP2A-B55 during mitotic exit, which is essential for cell cycle progression, is less well understood. Here, we identify protein phosphatase PP1 as an essential component of the PP2A-B55 re-activation pathway in Xenopus embryo extracts. PP1 initiates the re-activation of PP2A-B55 by dephosphorylating Gwl. We provide evidence that PP1 targets the auto-phosphorylation site of Gwl, resulting in efficient Gwl inactivation. This step is necessary to facilitate subsequent complete dephosphorylation of Gwl by PP2A-B55. Thus, by identifying PP1 as the phosphatase initiating Gwl inactivation, our study provides the molecular explanation for how Cdk1 inactivation is coupled to PP2A-B55 re-activation at mitotic exit.     

NuMA is required for the organization of microtubules into aster-like mitotic arrays

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.     

Maternal phosphatase inhibitor-2 is required for proper chromosome segregation and mitotic synchrony during Drosophila embryogenesis

Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase conserved among all eukaryotes, present as the essential GLC7 gene in yeast. Inhibitor-2 (I-2) is an ancient PP1 regulator, named GLC8 in yeast, but its in vivo function is unknown. Unlike mammals with multiple I-2 genes, in Drosophila there is a single I-2 gene, and here we describe its maternally derived expression and required function during embryogenesis. During oogenesis, germline expression of I-2 results in the accumulation of RNA and abundant protein in unfertilized eggs; in embryos, the endogenous I-2 protein concentrates around condensed chromosomes during mitosis and also surrounds interphase nuclei. An I-2 loss-of-function genotype is associated with a maternal-effect phenotype that results in drastically reduced progeny viability, as measured by reduced embryonic hatch rates and larval lethality. Embryos derived from I-2 mutant mothers show faulty chromosome segregation and loss of mitotic synchrony in cleavage-stage embryos, patchy loss of nuclei in syncytial blastoderms, and cuticular pattern defects in late-stage embryos. Transgenic expression of wild-type I-2 in mutant mothers gives dose-dependent rescue of the maternal effect on embryo hatch rate. We propose that I-2 is required for proper chromosome segregation during Drosophila embryogenesis through the coordinated regulation of PP1 and Aurora B.     

The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B’s subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14BΔ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.     

Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells

Nucleophosmin (NPM) is an abundantly expressed multifunctional nucleolar phosphoprotein. Here we show that depletion of NPM by RNA interference causes defects in cell division, followed by an arrest of DNA synthesis due to activation of a p53-dependent checkpoint response in HeLa cells. Depletion of NPM leads to mitotic arrest due to spindle checkpoint activation. The mitotic cells arrested by NPM depletion have defects in chromosome congression, proper mitotic spindle and centrosome formation, as well as defects in kinetochore-microtubule attachments. Loss of NPM thus causes severe mitotic defects and delayed mitotic progression. These findings indicate that NPM is essential for mitotic progression and cell proliferation.     

Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression

Members of the NIMA-related kinases (NRK) family are recently emerging as central regulators of various aspects of the cell cycle. However, the cellular roles of the mammalian NRK, Nek7, remain obscure. We show here that the endogenous Nek7 protein is enriched at the centrosome in a microtubule-independent manner. Overexpression of wt or kinase-defective Nek7 resulted in cells of rounder appearance, and higher proportions of multinuclear and apoptotic cells. Down-regulation of Nek7 using a small interfering RNA approach resulted in a significant increase in mitotic cells presenting multipolar spindle phenotype. These results suggest a role for Nek7 in regulating proper spindle assembly and mitotic progression.     

De novo fatty acid synthesis at the mitotic exit is required to complete cellular division

Although the regulation of the cell cycle has been extensively studied, much less is known about its coordination with the cellular metabolism. Using mass spectrometry we found that lysophospholipid levels decreased drastically from G₂/M to G₁ phase, while de novo phosphatidylcholine synthesis, the main phospholipid in mammalian cells, increased, suggesting that enhanced membrane production was concomitant to a decrease in its turnover. In addition, fatty acid synthesis and incorporation into membranes was increased upon cell division. The rate-limiting reaction for de novo fatty acid synthesis is catalyzed by acetyl-CoA carboxylase. As expected, its inhibiting phosphorylation decreased prior to cytokinesis initiation. Importantly, the inhibition of fatty acid synthesis arrested the cells at G₂/M despite the presence of abundant fatty acids in the media. Our results suggest that de novo lipogenesis is essential for cell cycle completion. This “lipogenic checkpoint” at G₂/M may be therapeutically exploited for hyperproliferative diseases such as cancer.     

Inhibition of Cdc42 during mitotic exit is required for cytokinesis

The role of Cdc42 and its regulation during cytokinesis is not well understood. Using biochemical and imaging approaches in budding yeast, we demonstrate that Cdc42 activation peaks during the G₁/S transition and during anaphase but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to down-regulate Cdc42 during mitotic exit impairs the normal localization of key cytokinesis regulators—Iqg1 and Inn1—at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase Ste20. Inhibition of Cdc42 and related Rho guanosine triphosphatases may be a general feature of cytokinesis in eukaryotes.     

NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gα_i1‐3 anchor the coiled‐coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Gα_i1‐3 are dispensable for NuMA‐dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP₂ (PIP₂) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP₂ prevents NuMA cortical localization during mitosis, and conversely, increasing PIP₂ levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.     

Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity

The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.     

Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified a number of phosphorylated residues by mass spectrometry and generated mutants in which these amino acids are replaced by Asp to mimic the effect of phosphorylation. Our results show that, among the eight phosphoryl groups experimentally detected, four are located at the flexible N terminus, and the rest are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

Pericentric chromatin is an elastic component of the mitotic spindle

BACKGROUND: Prior to chromosome segregation, the mitotic spindle bi-orients and aligns sister chromatids along the metaphase plate. During metaphase, spindle length remains constant, which suggests that spindle forces (inward and outward) are balanced. The contribution of microtubule motors, regulators of microtubule dynamics, and cohesin to spindle stability has been previously studied. In this study, we examine the contribution of chromatin structure on kinetochore positioning and spindle-length control. After nucleosome depletion, by either histone H3 or H4 repression, spindle organization was examined by live-cell fluorescence microscopy. RESULTS: Histone repression led to a 2-fold increase in sister-centromere separation and an equal increase in metaphase spindle length. Histone H3 repression does not impair kinetochores, whereas H4 repression disrupts proper kinetochore function. Deletion of outward force generators, kinesins Cin8p and Kip1p, shortens the long spindles observed in histone-repressed cells. Oscillatory movements of individual sister chromatid pairs are not altered after histone repression. CONCLUSIONS: The increase in spindle length upon histone repression and restoration of wild-type spindle length by the loss of plus-end-directed motors suggests that during metaphase, centromere separation and spindle length are governed in part by the stretching of pericentric chromatin. Chromatin is an elastic molecule that is stretched in direct opposition to the outward force generators Cin8p and Kip1p. Thus, we assign a new role to chromatin packaging as an integral biophysical component of the mitotic apparatus.