Regulation of septum formation in Aspergillus nidulans by a DNA damage checkpoint pathway.

In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.     

Hypomorphic bimA(APC3) alleles cause errors in chromosome metabolism that activate the DNA damage checkpoint blocking cytokinesis in Aspergillus nidulans

The Aspergillus nidulans sepI(+) gene has been implicated in the coordination of septation with nuclear division and cell growth. We find that the temperature-sensitive (ts) sepI1 mutation represents a novel allele of bimA(APC3), which encodes a conserved component of the anaphase-promoting complex/cyclosome (APC/C). We have characterized the septation, nuclear division, cell-cycle checkpoint defects, and DNA sequence alterations of sepI1 (renamed bimA10) and two other ts lethal bimA(APC3) alleles, bimA1 and bimA9. Our observations that bimA9 and bimA10 strains had morphologically abnormal nuclei, chromosome segregation defects, synthetic phenotypes with mutations in the DNA damage checkpoint genes uvsB(MEC1/rad3) or uvsD(+), and enhanced sensitivity to hydroxyurea strongly suggest that these strains accumulate errors in DNA metabolism. We found that the aseptate phenotype of bimA9 and bimA10 strains was substantially relieved by mutations in uvsB(MEC1/rad3) or uvsD(+), suggesting that the presence of a functional DNA damage checkpoint inhibits septation in these bimA(APC3) strains. Our results demonstrate that mutations in bimA(APC3) lead to errors in DNA metabolism that indirectly block septation.     

LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.     

The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity.

Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature-sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila. Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion mutants of sepA exhibit temperature-sensitive growth and severe delays in septation at the permissive temperature, indicating that expression of another gene may compensate for the loss of sepA. Conidiophores formed by sepA mutants exhibit abnormal branching of the stalk and vesicle. These results suggest that sepA interacts with the actin cytoskeleton to promote formation of the actin ring during cytokinesis and that sepA is also required for maintenance of cellular polarity during hyphal growth and asexual morphogenesis.     

Polo-like kinase controls vertebrate spindle elongation and cytokinesis

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.     

Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.     

Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis

By examining cytological phenotypes of 587 temperature-sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 – cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1⁺ gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.     

Changing roles of aurora-B kinase in two life cycle stages of Trypanosoma brucei

Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei, depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.     

Identification and Characterization of Aspergillus Nidulans Mutants Defective in Cytokinesis

Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokinesis mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis.     

Spatial Uncoupling of Mitosis and Cytokinesis during Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe oryzae

To infect plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we report that appressorium development in the rice blast fungus Magnaporthe oryzae involves an unusual cell division, in which nuclear division is spatially uncoupled from the site of cytokinesis and septum formation. The position of the appressorium septum is defined prior to mitosis by formation of a heteromeric septin ring complex, which was visualized by spatial localization of Septin4:green fluorescent protein (GFP) and Septin5:GFP fusion proteins. Mitosis in the fungal germ tube is followed by long-distance nuclear migration and rapid formation of an actomyosin contractile ring in the neck of the developing appressorium, at a position previously marked by the septin complex. By contrast, mutants impaired in appressorium development, such as Δpmk1 and ΔcpkA regulatory mutants, undergo coupled mitosis and cytokinesis within the germ tube. Perturbation of the spatial control of septation, by conditional mutation of the SEPTATION-ASSOCIATED1 gene of M. oryzae, prevented the fungus from causing rice blast disease. Overexpression of SEP1 did not affect septation during appressorium formation, but instead led to decoupling of nuclear division and cytokinesis in nongerminated conidial cells. When considered together, these results indicate that SEP1 is essential for determining the position and frequency of cell division sites in M. oryzae and demonstrate that differentiation of appressoria requires a cytokinetic event that is distinct from cell divisions within hyphae.     

A mutation in the gene involved in sister chromatid separation causes a defect in nuclear mRNA export in fission yeast

Fission yeast ptr4-1 is one of the mRNA transport mutants that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature. We cloned the ptr4(+) gene and found that it is identical with the cut1(+) gene essential for chromosome segregation during mitosis. ptr4/cut1 has no defects in nucleocytoplasmic transport of a protein, indicative of a specific blockage of mRNA export by this mutation. A mutant of Cut2p cooperating with Cut1p in sister chromatid separation also showed defective mRNA export at the nonpermissive temperature. Our results suggest a novel linkage between the cell division cycle and nuclear mRNA export in eukaryotic cells.     

AIM-1: a mammalian midbody-associated protein required for cytokinesis.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.     

The Smc5-Smc6 complex is required to remove chromosome junctions in meiosis

Meiosis, a specialized cell division with a single cycle of DNA replication round and two consecutive rounds of nuclear segregation, allows for the exchange of genetic material between parental chromosomes and the formation of haploid gametes. The structural maintenance of chromosome (SMC) proteins aid manipulation of chromosome structures inside cells. Eukaryotic SMC complexes include cohesin, condensin and the Smc5-Smc6 complex. Meiotic roles have been discovered for cohesin and condensin. However, although Smc5-Smc6 is known to be required for successful meiotic divisions, the meiotic functions of the complex are not well understood. Here we show that the Smc5-Smc6 complex localizes to specific chromosome regions during meiotic prophase I. We report that meiotic cells lacking Smc5-Smc6 undergo catastrophic meiotic divisions as a consequence of unresolved linkages between chromosomes. Surprisingly, meiotic segregation defects are not rescued by abrogation of Spo11-induced meiotic recombination, indicating that at least some chromosome linkages in smc5-smc6 mutants originate from other cellular processes. These results demonstrate that, as in mitosis, Smc5-Smc6 is required to ensure proper chromosome segregation during meiosis by preventing aberrant recombination intermediates between homologous chromosomes.     

SEPH, a Cdc7p orthologue from Aspergillus nidulans, functions upstream of actin ring formation during cytokinesis

In the filamentous fungus, Aspergillus nidulans, multiple rounds of nuclear division occur before cytokinesis, allowing an unambiguous identification of genes required specifically for cytokinesis. As in animal cells, both an intact microtubule cytoskeleton and progression through mitosis are required for actin ring formation and contraction. The sepH gene from A. nidulans was discovered in a screen for temperature-sensitive cytokinesis mutants. Sequence analysis showed that SEPH is 42% identical to the serine-threonine kinase Cdc7p from fission yeast. Signalling through the Septation Initiation Network (SIN), which includes Cdc7p and the GTPase Spg1p, is emerging as a primary regulatory pathway used by fission yeast to control cytokinesis. A similar group of proteins comprise the Mitotic Exit Network (MEN) in budding yeast. This is the first direct evidence for the existence of a functional SIN-MEN pathway outside budding and fission yeast. In addition to SEPH, potential homologues were also identified in other fungi and plants but not in animal cells. Deletion of sepH resulted in a viable strain that failed to septate at any temperature. Interestingly, quantitative analysis of the actin cytoskeleton revealed that sepH is required for construction of the actin ring. Therefore, SEPH is distinct from its counterpart in fission yeast, in which SIN components operate downstream of actin ring formation and are necessary for ring contraction and later events of septation. We conclude that A. nidulans has components of a SIN-MEN pathway, one of which, SEPH, is required for early events during cytokinesis.     

Nuclear envelope fission is linked to cytokinesis in budding yeast

We have investigated the relationship between nuclear envelope fission and cytokinesis during mitotic cell division in budding yeast. By carrying out time-lapse and optical sectioning video microscopy analysis of cells that express green fluorescent protein (GFP)-tagged nuclear envelope and actomyosin ring components, we found that nuclear division is temporally coupled to cytokinesis. Light and electron microscopy analysis also showed that nuclear envelope fission and the division of the nucleoplasm are severely delayed in cytokinesis mutants, resulting in discoupling between the nuclear division cycle and the budding cycle. These results suggest that homotypic membrane fusion may be activated by components or the mechanical action of cytokinetic structures and presents a mechanism for the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during mitosis.     

Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe.

Topoisomerases catalyse changes in the topological state of DNA and are required for many aspects of DNA metabolism. While the functions of topoisomerases I and II in eukaryotes are well established, the role of topoisomerase III remains poorly defined. We have identified a gene in the fission yeast Schizosaccharomyces pombe, designated top3 (+), which shows significant sequence similarity to genes encoding topoisomerase III enzymes in other eukaryotic species. In common with murine TOP3 alpha, but in contrast to Saccharomyces cerevisiae TOP3, the S.pombe top3 (+)gene is essential for long-term cell viability. Fission yeast haploid spores containing a disrupted top3 (+)gene germinate successfully, but then undergo only a limited number of cell divisions. Analysis of these top3 mutants revealed evidence of aberrant mitotic chromosome segregation, including the 'cut' phenotype, where septation is completed prior to nuclear division. Consistent with the existence of an intimate association (originally identified in S.cerevisiae ) between topoisomerase III and DNA helicases of the RecQ family, deletion of the rqh1 (+)gene encoding the only known RecQ helicase in S.pombe suppresses lethality in top3 mutants. This conservation of genetic interaction between two widely diverged yeasts suggests that the RecQ family helicases encoded by the Bloom's and Werner's syndrome genes are likely to act in concert with topoisomerase III isozymes in human cells. Our data are consistent with a model in which the association of a RecQ helicase and topoisomerase III is important for facilitating decatenation of late stage replicons to permit faithful chromosome segregation during anaphase.     

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.     

Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.

DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures. We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52). At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells. delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations. At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome. delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth. Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division.     

fumble encodes a pantothenate kinase homolog required for proper mitosis and meiosis in Drosophila melanogaster

A number of fundamental processes comprise the cell division cycle, including spindle formation, chromosome segregation, and cytokinesis. Our current understanding of these processes has benefited from the isolation and analysis of mutants, with the meiotic divisions in the male germline of Drosophila being particularly well suited to the identification of the required genes. We show here that the fumble (fbl) gene is required for cell division in Drosophila. We find that dividing cells in fbl-deficient testes exhibit abnormalities in bipolar spindle organization, chromosome segregation, and contractile ring formation. Cytological analysis of larval neuroblasts from null mutants reveals a reduced mitotic index and the presence of polyploid cells. Molecular analysis demonstrates that fbl encodes three protein isoforms, all of which contain a domain with high similarity to the pantothenate kinases of A. nidulans and mouse. The largest Fumble isoform is dispersed in the cytoplasm during interphase, concentrates around the spindle at metaphase, and localizes to the spindle midbody at telophase. During early embryonic development, the protein localizes to areas of membrane deposition and/or rearrangement, such as the metaphase and cellularization furrows. Given the role of pantothenate kinase in production of Coenzyme A and in phospholipid biosynthesis, this pattern of localization is suggestive of a role for fbl in membrane synthesis. We propose that abnormalities in synthesis and redistribution of membranous structures during the cell division cycle underlie the cell division defects in fbl mutant cells.