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PubMed2003

Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities.

In C. elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, we analyzed defects in embryos and larvae of mat-1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos developed to the multicellular stage but were slow to complete meiosis I and displayed aberrant meiotic chromosome separation. More severely affected embryos skipped meiosis II altogether and exhibited striking defects in meiotic exit. These latter embryos failed to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages were associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle progression and particular developmental defects.

Amino Acid Sequence↗
PubMed2003

Drosophila securin destruction involves a D-box and a KEN-box and promotes anaphase in parallel with Cyclin A degradation.

Sister chromatid separation during exit from mitosis requires separase. Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C. Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin PIM. Consistent with this proposal and with results from yeast and vertebrates, we show here that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A. However, we also show that PIM contains a KEN-box, which is required for mitotic degradation in addition to the D-box, and that sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals.

Amino Acid Motifs↗
PubMed1995

Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation.

Fission yeast nuc2+ gene encodes a protein of a tetratricopeptide repeat (TPR) family which is conserved throughout evolution. We previously showed that nuc2 is required for exit from the mitotic metaphase. In this study, we present evidence which shows that nuc2 has two additional roles in the cell cycle. We showed that the nuc2 mutant is sterile even at the permissive temperature and septation occurs in the absence of chromosome separation at the restrictive temperature. The nuc2 mutant fails to arrest at the G1 phase upon nitrogen starvation at the permissive temperature which is a prerequisite for conjugation. Upon starvation, however, the nuc2 mutant ceased division normally and induced starvation-dependent gene expression. Therefore, the nuc2 mutant is deficient only for failure to block DNA replication upon starvation. At the lower restrictive temperature, the nuc2 mutant showed a 'cut' phenotype where septation and cytokinesis takes place without the completion of mitosis. Ectopic overexpression of the nuc2+ gene caused multiple rounds of S and M phases in the complete absence of septum formation. We propose that nuc2 is a novel cell cycle regulator essential for three events; firstly for exit from mitosis, secondly for DNA replication restraint under nutrient starvation and thirdly for inhibition of septation and cytokinesis until the completion of mitosis.

Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

BIMAAPC3, a component of the Aspergillus anaphase promoting complex/cyclosome, is required for a G2 checkpoint blocking entry into mitosis in the absence of NIMA function.

Temperature sensitive (ts) nimA mutants of Aspergillus nidulans arrest at a unique point in G2 which is post activation of CDC2. Here we show that this G2 arrest is due to loss of nimA function and that it is dependent on BIMAAPC3, a component of the anaphase promoting complex/cyclosome (APC/C). Whereas nimA single mutants arrested in G2 with decondensed chromatin and interphase microtubule arrays, nimA, bimAAPC3 double mutants arrested growth with condensed chromatin and aster-like microtubule arrays. nimA, bimAAPC3 double mutants entered mitosis with kinetics similar to bimAAPC3 single mutants and wild-type cells, indicating a checkpoint-like role for BIMAAPC3 in G2. Even cells which had been depleted for NIMA protein and which contained insignificant levels of NIMA kinase activity entered mitosis on inactivation of bimAAPC3. BIMAAPC3 was present in a >25S complex containing BIMEAPC1, and bimAAPC3 mutants were sensitive to elevated CYCLIN B expression, consistent with BIMAAPC3 being a component of the APC/C. Inactivation of bimAAPC3 had little affect on the steady state levels of the B-type cyclin, NIMECyclin B. Our results indicate that BIMAAPC3, and most likely the APC/C itself, is activated in G2 in nimA mutants. We propose that APC/C activation is part of a novel, late G2 checkpoint, which responds to a defective process or structure in nimA mutants, and which prevents inappropriate entry into mitosis.

Alleles↗
PubMed2000

Association of human ubiquitin-conjugating enzyme CDC34 with the mitotic spindle in anaphase.

Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function. Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression. We find that CDC34 protein is constitutively expressed during all stages of the cell cycle. Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm. The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments. Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34's cell cycle function in Saccharomyces cerevisiae. Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with (beta)-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis. Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells.

Anaphase↗
PubMed2001

Fission yeast mfr1 activates APC and coordinates meiotic nuclear division with sporulation.

Meiosis is the developmental program by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. These two events are normally coordinated in such a way that spore formation is dependent upon completion of the meiotic nuclear divisions. Here we describe a meiosis-specific protein, mfr1, that is involved in this coordination. mfr1 is an activator of the anaphase-promoting complex (APC), which is necessary for the rapid degradation of the cdc13 cyclin at the end of meiosis II, prior to the formation of spores. An mfr1 null mutant completes meiosis II but remains with high levels of cdc13 and cdc2 kinase activity and has considerably delayed spore formation. By analogy with the mitotic cell cycle, where proteolysis and inactivation of cdc2 kinase are necessary to trigger mitotic exit and cytokinesis, we propose that at the end of meiosis rapid and timely proteolysis of cyclins is required to switch on the differentiation program that eventually leads to the formation of haploid gametes.

Amino Acid Sequence↗
PubMed2001

The Bub2-dependent mitotic pathway in yeast acts every cell cycle and regulates cytokinesis.

In eukaryotes an abnormal spindle activates a conserved checkpoint consisting of the MAD and BUB genes that results in mitotic arrest at metaphase. Recently, we and others identified a novel Bub2-dependent branch to this checkpoint that blocks mitotic exit. This cell-cycle arrest depends upon inhibition of the G-protein Tem1 that appears to be regulated by Bfa1/Bub2, a two-component GTPase-activating protein, and the exchange factor Lte1. Here, we find that Bub2 and Bfa1 physically associate across the entire cell cycle and bind to Tem1 during mitosis and early G1. Bfa1 is multiply phosphorylated in a cell-cycle-dependent manner with the major phosphorylation occurring in mitosis. This Bfa1 phosphorylation is Bub2-dependent. Cdc5, but not Cdc15 or Dbf2, partly controls the phosphorylation of Bfa1 and also Lte1. Following spindle checkpoint activation, the cell cycle phosphorylation of Bfa1 and Lte1 is protracted and some species are accentuated. Thus, the Bub2-dependent pathway is active every cell cycle and the effect of spindle damage is simply to protract its normal function. Indeed, function of the Bub2 pathway is also prolonged during metaphase arrests imposed by means other than checkpoint activation. In metaphase cells Bub2 is crucial to restrain downstream events such as actin ring formation, emphasising the importance of the Bub2 pathway in the regulation of cytokinesis. Our data is consistent with Bub2/Bfa1 being a rate-limiting negative regulator of downstream events during metaphase.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2001

MTOC formation during mitotic exit in fission yeast.

Microtubules polymerise from nucleation templates containing gamma tubulin. These templates are generally concentrated in discrete structures called microtubule organising centres (MTOCs). In Schizosaccharomyces pombe, an equatorial MTOC (EMTOC) forms mid-way through anaphase B and then disassembles during the final stages of cell separation. We show that the EMTOC was generated by recruiting gamma tubulin to the equatorial F-actin ring before it constricted to cleave the cell in two during cytokinesis. The EMTOC was not a continuous ring. It had a variable structure ranging from a horseshoe to a number of short bars. EMTOC integrity depended upon the integrity of the F-actin but not the microtubule cytoskeleton. EMTOC assembly required the activity of both the septation-inducing network (SIN) that regulates the onset of cytokinesis and the anaphase-promoting complex. Activation of the SIN in interphase cells induced F-actin ring formation and contraction and the synthesis of the primary septum but did not promote EMTOC assembly. In contrast, overproduction of the polo-like kinase, Plo1, which also induced multiple rounds of septation in interphase cells, induced EMTOC formation. Thus, the network governing EMTOC formation shared many of the regulatory elements that control cytokinesis but was more complex and revealed an additional function for Plo1 during mitotic exit.

Actins↗
PubMed2002

The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised.

In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularised embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos, only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction, but its subcellular localisation remains controversial. We constructed GFP fusions of two core APC/C subunits, Cdc16 and Cdc27. These fusion proteins were incorporated into the endogenous APC/C and were largely localised in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulated in the nucleus prior to nuclear envelope breakdown but only weakly associated with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly, we noticed that GFP-Cdc27 associated with mitotic chromosomes, whereas GFP-Cdc16 did not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90% in tissue culture cells led to a transient mitotic arrest that was both biochemically and morphologically distinct. Taken together, our results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localised and perform distinct functions.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2026

Viral inhibition of the anaphase promoting complex enhances replication by elevating nucleotide pools.

The anaphase promoting complex/cyclosome (APC/C) is a large, ubiquitin ligase and a central regulator of cell cycle progression. By targeting key substrates for degradation during mitosis and G1 phase, the APC/C coordinates metabolic fluctuations that occur during the cell cycle. A diverse range of viruses have convergently evolved mechanisms to bind and inhibit the APC/C; however, a molecular understanding for these interactions has never been demonstrated. Here, we use chicken anemia virus (CAV), a small single-stranded DNA virus encoding only three proteins, to demonstrate the importance of viral APC/C inhibition during replication. We show that the Vp3 protein of CAV inhibits the APC/C, causing a dramatic mitotic arrest during infection. The mutant virus lacking Vp3 is defective for replication and can be rescued by APC/C inhibition. Metabolomic profiling during CAV infection revealed that Vp3 expression mediates a broad increase in nucleotide pools. Moreover, viral inhibition of the APC/C resulted in stabilization of enzymes required for nucleotide biosynthesis. These findings suggest that the APC/C is a general target of many viruses to elevate nucleotide levels and facilitate viral genome replication.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2001

Down-regulation of the human CDC16 gene after exposure to ionizing radiation: a possible role in the radioadaptive response.

We have used the method of differential display of mRNAs to search for changes in gene expression associated with radioadaptation triggered by low doses of ionizing radiation in human lymphoblasts. We isolated a cDNA designated PB13 that was down-regulated as early as 1 h after irradiation with 4 Gy in cells adapted by a pre-exposure to a dose of 2 cGy, compared to 3 h in nonadapted cells (4 Gy alone). Northern analysis confirmed the differential expression of a 2.4-kb transcript that was repressed for at least 10 h after irradiation. The major part of the PB13 cDNA was identical to the human CDC16 mRNA. When using either PB13 or CDC16 cDNA as probes, similar radiation-induced alterations in gene expression were observed. Expression of the CDC16 gene was also repressed after oxidative stress with H(2)O(2). The CDC16 protein belongs to the anaphase-promoting complex (APC) that controls progression through mitosis. The repression of expression of the CDC16 gene by ionizing radiation could result in delayed progression of damaged cells through mitosis. This cycle delay would occur earlier in adapted cells and would allow a more rapid and efficient repair that could contribute to the tolerance to subsequent irradiation.

Adaptation, Biological↗
PubMed2001

The small GTPase Rac interacts with ubiquitination complex proteins Cullin-1 and CDC23.

Racs are involved in the regulation of important cellular processes including mitogenesis. We found that the E3 ubiquitination ligase subunit Cullin-1 interacts with constitutively active Rac3 but not with wild-type Rac3 in yeast. In mammalian cell lysates, Cullin-1 bound to V12Rac3, effector domain mutants V12L37Rac3 and V12H40Rac3, and insert domain deletion mutant V12Rac3DeltaIns(124-135). Cullin-1 also formed a clearly detectable complex with other activated Rac3-related proteins including Rac1, Rac2, Cdc42 and RhoA but not with the distantly related small GTPase Rap1. Since the proteasome is involved in cell cycle control through the programmed degradation of cell cycle proteins, the possible regulation of Rac levels during the cell cycle was examined. However, Rac was expressed at constant levels throughout the cell cycle, and a specific proteasome inhibitor had no effect on Rac protein levels. These combined results indicate that the binding of activated Rac to Cullin-1 does not affect Rac protein levels, nor does it mediate the regulation of mitogenesis by Rac. However, Rac-Cullin-1 interactions may serve to regulate other E3 ligase functions such as subcellular localization. Indeed, activated Rac3 and Cullin-1 co-localized to the perinuclear region of the cell. We also detected complex formation between Rac and the APC component CDC23. These results indicate that Rac may regulate specific proteolytic processes through directed subcellular localization of SCF or APC complexes.

Animals↗