hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans.

Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.     

Isolation and Characterization of Conditional-Lethal Mutations in the Tub1 α-Tubulin Gene of the Yeast Saccharomyces Cerevisiae

Microtubules in yeast are functional components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. We have isolated 70 conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae using a plasmid replacement technique. Of the 70 mutations isolated, 67 resulted in cold-sensitivity, one resulted in temperature-sensitivity, and two resulted in both. Fine-structure mapping revealed that the mutations were located throughout the TUB1 gene. We characterized the phenotypes caused by 38 of the mutations after shifts of mutants to the nonpermissive temperature. Populations of temperature-shifted mutant cells contained an excess of large-budded cells with undivided nuclei, consistent with the previously determined role of microtubules in yeast mitosis. Several of the mutants arrested growth with a sufficiently uniform morphology to indicate that TUB1 has at least one specific role in the progression of the yeast cell cycle. A number of the mutants had gross defects in microtubule assembly at the restrictive temperature, some with no microtubules and some with excess microtubules. Other mutants contained disorganized microtubules and nuclei. There were no obvious correlations between these phenotypes and the map positions of the mutations. Greater than 90% of the mutants examined were hypersensitive to the antimicrotubule drug benomyl. Mutations that suppressed the cold-sensitive phenotypes of two of the TUB1 alleles occurred in TUB2, the single structural gene specifying beta-tubulin.     

Mutations Affecting Embryonic F-Actin Reorganization also Affect Separation of Nuclei from their Sisters and from the Cortex in Drosophila Cleavage Embryos

We showed in Drosophila that nuclear migration was reduced all through cleavage stages in embryos with any one of the maternal-effect mutations, gs(1)N441 and gs(1)N26 , in which F-actin reorganization in cleavage embryos is disordered. Moreover, we determined nuclear positions in embryos at cycle 1 and 2 in the wild type and two mutants, gs(1)N441 and gs(1)N26 , in order to test if the nuclear migration is regulated within a nuclear cycle. At cycle 1, there was no difference in nuclear position among the strains that we observed. At cycle 2 the two sister nuclei had already migrated posteriorly in the wild type. However, migration was not detectable at cycle 2 in the mutants. Besides, the two sister nuclei were less-separated from each other, and orientation of the two nuclei with regard to the anteroposterior axis was random, different from the wild type. These results support the hypothesis that F-actin is involved in the regulation to separate cleavage nuclei from each other and from the egg cortex. This regulation is apparently required for posteriorward nuclear migration, and for synchronous nuclear arrival in the whole egg cortex.     

Events associated with the initiation of mitosis in fused multinucleate HeLa cells

Large multinucleate (LMN) HeLa cells with more than 10–50 nuclei were produced by random fusion with polyethylene glycol. The number of nuclei in a particular stage of the cell cycle at the time of fusion was proportionate to the duration of the phase relative to the total cell cycle. The fused cells did not gain generation time. Interaction of various nuclei in these cells has been observed. The nuclei initially belonging to the G1-or S-phase required a much longer time to complete DNA synthesis than in mononucleate cells. Some of the cells reached mitosis 15 h after fusion, whereas others required 24 h. The cells dividing early, contained a larger number of initially early G1-phase nuclei than those cells dividing late. The former very often showed prematurely condensed chromosome (PCC) groups. In cells with a large number of advanced nuclei the few less advanced nuclei could enter mitosis prematurely. On the other hand, the cells having a large number of nuclei belonging initially to late S-or G2-phase took longer to reach mitosis. These nuclei have been taken out of the normal sequence and therefore failed to synthesize the mitotic factors and depended on others to supply them. Therefore the cells as a whole required a longer period to enter mitosis. Although the nuclei became synchronized at metaphase, the cells revealed a gradation in prophase progression in the different nuclei. At the ultrastructural level the effect of advanced nuclei on the less advanced ones was evident with respect to chromosome condensation and nuclear envelope breakdown. Less advanced nuclei trapped among advanced nuclei showed PCC and nuclear envelope breakdown prematurely, whereas mitotic nuclei near interphase or early prophase nuclei retained their nuclear envelopes for a much longer time. PCC is closely related to premature breakdown of the nuclear envelope. Our observations clearly indicate that chromosome condensation and nuclear envelope breakdown are two distinct events. Kinetochores with attached microtubules could be observed on prematurely condensed chromosomes. Kinetochores of fully condensed chromosomes often failed to become connected to spindle elements. This indicates that the formation of a functional spindle is distinct from the other events and may depend on different factors.     

Onset of the DNA replication checkpoint in the early Drosophila embryo

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. We have reported before that increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. Here we show that the level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. We propose that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. Our results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles.     

The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay

The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell-cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, whereas inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, because cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of the DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continued unperturbed when cells delayed in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intranuclear organization.     

Distribution of nuclear matrix proteins in interphase CHO cells and rearrangements during the cell cycle. An ultrastructural study

The nuclear matrix contains a group of residual non-histone proteins which remain structurally organized after extensive extraction of isolated nuclei with a high salt buffer, nucleases and a non-ionic detergent. Electron microscopic examination shows that the nuclear matrix is composed of a pore-complex lamina, an intranuclear network and residual nucleoli. In CHO cells biochemical analyses performed by one-dimensional SDS-PAGE show three major nuclear matrix polypeptides with molecular weights between 60 and 70 kDa. Polyclonal antibodies produced against these polypeptides were used to determine their nuclear distribution. Using immunoblotting, these proteins were found in whole nuclei, nuclear matrix, and in the intranuclear network but not in the pore-complex lamina. In order to determine the relationship between these structural proteins and the organization of the nucleus, the proteins were localized in situ. Ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl K4M-embedded cells. In interphase nuclei all condensed chromatin clumps were labelled. The nucleolus and the interchromatin granules were never immunogold-stained. During mitosis, the label was found to be associated with the chromosomes. This study shows that unlike the lamins, these 60-70 kDa nuclear matrix proteins are associated with the condensed chromatin throughout the cell cycle.     

Interdependence of cell cycle events inSchizosaccharomyces pombe. Terminal phenotypes of cell division cycle mutants arrested during dna synthesis and nuclear division

Summary Temperature-sensitive cell division cycle (cdc) mutants of the fission yeastSchizosaccharomyces pombe, previously characterized as defective in nuclear division were examined by thin section electron microscopy. All of the mutants failed to enter mitosis, rather they accumulated at one of four distinct terminal phenotypes. Class one were arrested with a nucleus rectangular in cross-section and a laterally situated spindle pole body (SPB). The second group had spherical or rectangular nuclei with a single SPB. The sole member of the third group wascdc 27. K 3, which had a spherical crenated nucleus with a single SPB from which microtubules emerged and extended into the cytoplasm. Allelic variants ofcdc 25 comprised the fourth group all of which displayed aberrant nuclear morphologies. Utilizing this ultrastructural data together with a knowledge of the transition points of these mutants a model for the interdependence of certain cell cycle event is proposed in which the initiation of DNA synthesis is uncoupled from the replication and separation of the SPB. This paper also provides new information on SPB structure inS. pombe. This is discussed in connection with the transient assembly of both spindle and cytoplasmic microtubules.     

The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development

We describe in this report a novel class of mutants that should facilitate the identification of genes required for progression through the mitotic cell cycle during seed development in angiosperms. Three non-allelic titan ( ttn ) mutants with related but distinct phenotypes are characterized. The common feature among these mutants is that endosperm nuclei become greatly enlarged and highly polyploid. The mutant embryo is composed of a few giant cells in ttn1 , several small cells in ttn2 , and produces a normal plant in ttn3 . Condensed chromosomes arrested at prophase of mitosis are found in the free nuclear endosperm of ttn1 and ttn2 seeds. Large mitotic figures with excessive numbers of chromosomes are visible in ttn3 endosperm. The ttn1 mutation appears to disrupt cytoskeletal organization because endosperm nuclei fail to migrate to the chalazal end of the seed. How double fertilization leads to the establishment of distinct patterns of mitosis and cytokinesis in the embryo and endosperm is a central question in plant reproductive biology. Molecular isolation of TITAN genes should help to answer this question, as well as related issues concerning cell cycle regulation, chromosome movement and endosperm identity in angiosperms.     

Nuclear size control in fission yeast

Along-standing biological question is how a eukaryotic cell controls the size of its nucleus. We report here that in fission yeast, nuclear size is proportional to cell size over a 35-fold range, and use mutants to show that a 16-fold change in nuclear DNA content does not influence the relative size of the nucleus. Multi-nucleated cells with unevenly distributed nuclei reveal that nuclei surrounded by a greater volume of cytoplasm grow more rapidly. During interphase of the cell cycle nuclear growth is proportional to cell growth, and during mitosis there is a rapid expansion of the nuclear envelope. When the nuclear/cell (N/C) volume ratio is increased by centrifugation or genetic manipulation, nuclear growth is arrested while the cell continues to grow; in contrast, low N/C ratios are rapidly corrected by nuclear growth. We propose that there is a general cellular control linking nuclear growth to cell size.     

Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP- bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.     

Changes in DNA content of nuclei in rust uredospore germlings during the start of differentiation

Digital video microscopy in conjunction with the DNA-binding fluorescent probe 4′,6-diamidino-2-phenylindole was used to determine the relative DNA content of single nuclei in germ tubes of uredospores of the bean rust fungus (Uromyces phaseoli), a parasite of bean plants (Phaseolus vulgaris). The uredospores develop a series of infection structures in response to contact stimuli, and the first structure to appear, the appressorium, occupies the stomatal opening. This study was made to determine the time after the start of germination on an inductive surface that DNA replication and mitosis occur. It was found that the start of DNA replication detected by increased nuclear fluorescence power of some individual nuclei in the population was nearly coincident with mitosis between 2.0 and 2.5 h after the start of germination and that the appressorium was completed about 30 min later. The fluoresence power of nuclei was about the same before DNA replication began as at the end of mitosis; hence the germ tube nuclei were in the G₁ phase of the cell cycle before mitosis.     

Patterns of Nuclear and Nucleolar Growth in Synchronously Dividing Explants from Tubers of Helianthus tuberosus L.

The nucleus and nucleolus have been examined by phase contrastmicroscopy of isolated fixed nuclei from synchronously dividingcells of Helianthus tuberosus L. tuber explants grown in nutrientmedium on filter paper. The volumes of nuclei and nucleoli werecomputed from their areas and perimeters obtained by digitizingimages projected from film. The nuclei did not show a pattern of growth related to the Sphase but enlarged at times of both de-differentiation and differentiation.There was also rapid post-mitotic nuclear enlargement. The sizeattained by nuclei in the three successive divisions followingcell activation decreased progressively, but started to riseagain at the time of cell differentiation. The changes are discussed in relation to nuclear size regulation,the nuclear matrix and hypotheses relating nuclear growth toDNA, protein and water in the processes of de-differentiation,mitosis and differentiation. Nucleoli showed a clear fusion and growth cycle. The patternof fusion can be used to identify the position of a sample ofcells, though not any particular cell, within the cycle. Nucleolargrowth was different in the succeeding cell cycles that wereinduced in the de-differentiating tissue. Nucleolar enlargementwas slow in the first cycle and continued until mitosis. Therewas rapid nucleolar growth in the second cycle and none in latercycles until the time of cell differentiation. Nucleolar changes are discussed in relation to rRNA gene dosage,replication and polymerase availability. Helianthus tuberosus L. Jerusalem artichoke, isolated-nuclei, tissue culture, cell cycle, nucleolar cycle     

Temperature-sensitive mutants of Physarum polycephalum: viability, growth, and nuclear replication.

Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.     

γ-Tubulin regulates the anaphase-promoting complex/cyclosome during interphase

A cold-sensitive γ-tubulin allele of Aspergillus nidulans, mipAD159, causes defects in mitotic and cell cycle regulation at restrictive temperatures that are apparently independent of microtubule nucleation defects. Time-lapse microscopy of fluorescently tagged mitotic regulatory proteins reveals that cyclin B, cyclin-dependent kinase 1, and the Ancdc14 phosphatase fail to accumulate in a subset of nuclei at restrictive temperatures. These nuclei are permanently removed from the cell cycle, whereas other nuclei, in the same multinucleate cell, cycle normally, accumulating and degrading these proteins. After each mitosis, additional daughter nuclei fail to accumulate these proteins, resulting in an increase in noncycling nuclei over time and consequent inhibition of growth. Extensive analyses reveal that these noncycling nuclei result from a nuclear autonomous, microtubule-independent failure of inactivation of the anaphase-promoting complex/cyclosome. Thus, γ-tubulin functions to regulate this key mitotic and cell cycle regulatory complex.     

Nuclear reformation after mitosis requires downregulation of the Ran GTPase effector RanBP1 in mammalian cells

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.     

Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.     

Duration of the nuclear cycle in Tradescantia root tips at three temperatures as measured with H-thymidine

The duration of the subdivisions of the nuclear cycle in cells from the root tips of Tradescantia paludosa was determined by the labelled mitosis method. These times were found not to increase or decrease in direct proportion to each other at the three temperatures tested, 13, 21 and 30 C. The main difference between the nuclear cycles at 30 and 21 C was in the shortening of the pre-DNA synthetic period (G₁) at the higher temperature. A comparison between the nuclear cycle at 21 and 13 C showed that at the lower temperature mitosis and the post-DNA synthetic period (G₂) were approximately tripled in duration while DNA synthesis was doubled. Cell synchronization and radiosensitivity are discussed in relation to these findings.