Budding yeast relies on G1 cyclin specificity to couple cell cycle progression with morphogenetic development

Two models have been put forward for cyclin-dependent kinase (Cdk) control of the cell cycle. In the qualitative model, cell cycle events are ordered by distinct substrate specificities of successive cyclin waves. Alternatively, in the quantitative model, the gradual rise of Cdk activity from G1 phase to mitosis leads to ordered substrate phosphorylation at sequential thresholds. Here, we study the relative contributions of qualitative and quantitative Cdk control in Saccharomyces cerevisiae. All S phase and mitotic cyclins can be replaced by a single mitotic cyclin, albeit at the cost of reduced fitness. A single cyclin can also replace all G1 cyclins to support ordered cell cycle progression, fulfilling key predictions of the quantitative model. However, single-cyclin cells fail to polarize or grow buds and thus cannot survive. Our results suggest that budding yeast has become dependent on G1 cyclin specificity to couple cell cycle progression to essential morphogenetic events.

Cell-growth gene expression reveals a direct fitness cost of grazer-induced toxin production in red tide dinoflagellate prey

Induced prey defences against consumers are conspicuous in microbes, plants and animals. In toxigenic prey, a defence fitness cost should result in a trade-off between defence expression and individual growth. Yet, previous experimental work has failed to detect such induced defence cost in toxigenic phytoplankton. We measured a potential direct fitness cost of grazer-induced toxin production in a red tide dinoflagellate prey using relative gene expression (RGE) of a mitotic cyclin gene ( cyc ), a marker that correlates to cell growth. This approach disentangles the reduction in cell growth from the defence cost from the mortality by consumers. Treatments where the dinoflagellate Alexandrium catenella were exposed to copepod grazers significantly increased toxin production while decreasing RGE of cyc , indicating a defence-growth trade-off. The defence fitness cost represents a mean decrease of the cell growth rate of 32%. Simultaneously, we estimate that the traditional method to measure mortality loss by consumers is overestimated by 29%. The defence appears adaptive as the prey population persists in quasi steady state after the defence is induced. Our approach provides a novel framework to incorporate the fitness cost of defence in toxigenic prey–consumer interaction models.

Complex haploinsufficient interaction between APC11 and CYCLIN A1;2/TARDY ASYNCHRONOUS MEIOSIS in embryo development and seed germination in Arabidopsis

Background: Complex haploinsufficiency is characterized by individuals with two heterozygous loci producing a phenotype that is not seen in either of the corresponding single-locus heterozygous individuals. The mutants of the anaphase-promoting complex/cyclosome (APC/C) subunit gene APC11 and the mitotic cyclin gene CYCLIN A1;2/TARDY ASYNCHRONOUS MEIOSIS (TAM) in Arabidopsis thaliana are embryo-lethal and defective in meiosis, respectively, but their heterozygous single mutants do not exhibit defective embryo development and seed germination. Methods: Crosses between two heterozygous apc11 mutant alleles and two homozygous tam mutant alleles, and between two heterozygous apc11 mutant alleles and a TAM:TAM-GFP line were conducted. Phenotypes of the F1 seeds were analyzed by light microscopy. Results: We found that F1 embryos from the crosses between heterozygous apc11-1 (APC11/apc11-1) and homozygous tam-2 (tam-2/tam-2) or between APC11/apc11-2 and tam-2/tam-2 were morphologically normal but all the seeds failed to germinate. F1 embryos from the crosses between APC11/apc11-2 and tam-1/tam-1 (weaker allele than tam-2) produced morphologically normal seeds that germinated to form mature plants. However, F1 embryos from the crosses between APC11/apc11-1 and tam-1/tam-1 were abnormal and the seeds failed to germinate. Moreover, F1 embryos from the crosses between APC11/apc11-1 and a TAM:TAM-GFP line were arrested at early developmental stages while F1 embryos from the crosses between APC11/apc11-2 and the TAM:TAM-GFP line appeared fully developed but the seeds failed to germinate. Conclusions: Our observations indicate that the apc11 and tam mutants have an allele-dependent complex haploinsufficient relationship in embryo development and seed germination.

Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

Computational modelling of chromosome re-replication in mutant strains of fission yeast

Typically cells replicate their genome only once per division cycle, but under some circumstances, both natural and unnatural, cells synthesize an overabundance of DNA, either in a disorganized fashion (‘over-replication’) or by a systematic doubling of chromosome number (‘endoreplication’). These variations on the theme of DNA replication and division have been studied in strains of fission yeast, Schizosaccharomyces pombe, carrying mutations that interfere with the function of mitotic cyclin-dependent kinase (Cdk1:Cdc13) without impeding the roles of DNA-replication licensing factor (Cdc18) and S-phase cyclin-dependent kinase (Cdk1:Cig2). Some of these mutations support endoreplication, and some over-replication. In this paper, we propose a dynamical model of the interactions among the proteins governing DNA replication and cell division in fission yeast. By computational simulations of the mathematical model, we account for the observed phenotypes of these re-replicating mutants, and by theoretical analysis of the dynamical system, we provide insight into the molecular distinctions between over-replicating and endoreplicating cells. In case of induced over-production of regulatory proteins, our model predicts that cells first switch from normal mitotic cell cycles to growth-controlled endoreplication, and ultimately to disorganized over-replication, parallel to the slow increase of protein to very high levels.

Characterization and localization of cyclin B3 transcript in both oocyte and spermatocyte of the rainbow trout (Oncorhynchus mykiss)

B-type cyclins are regulatory subunits with distinct roles in the cell cycle. To date, at least three subtypes of B-type cyclins (B1, B2, and B3) have been identified in vertebrates. Previously, we reported the characterization and expression profiles of cyclin B1 and B2 during gametogenesis in the rainbow trout (Oncorhynchus mykiss). In this paper, we isolated another subtype of cyclin B, cyclin B3 (CB3), from a cDNA library of the rainbow trout oocyte. The full-length CB3 cDNA (2,093 bp) has an open reading frame (1,248 bp) that encodes a protein of 416 amino acid residues. The CB3 transcript was widely distributed in all the examined tissues, namely, eye, gill, spleen, brain, heart, kidney, stomach, skin, muscle, and, especially, gonad. Northern blot analysis indicated only one form of the CB3 transcript in the testis and ovary. In situ hybridization revealed that, in contrast to cyclin B1 and B2 transcripts, CB3 transcripts were localized in the oocytes, spermatocytes, and spermatogonia. These findings strongly suggest that CB3 plays a role not only as a mitotic cyclin in spermatogonial proliferation during early spermatogenesis but also during meiotic maturation of the spermatocyte and oocyte in the rainbow trout.

Response of the Green Alga Chlamydomonas reinhardtii to the DNA Damaging Agent Zeocin

DNA damage is a ubiquitous threat endangering DNA integrity in all living organisms. Responses to DNA damage include, among others, induction of DNA repair and blocking of cell cycle progression in order to prevent transmission of damaged DNA to daughter cells. Here, we tested the effect of the antibiotic zeocin, inducing double stranded DNA breaks, on the cell cycle of synchronized cultures of the green alga Chlamydomonas reinhardtii. After zeocin application, DNA replication partially occurred but nuclear and cellular divisions were completely blocked. Application of zeocin combined with caffeine, known to alleviate DNA checkpoints, decreased cell viability significantly. This was probably caused by a partial overcoming of the cell cycle progression block in such cells, leading to aberrant cell divisions. The cell cycle block was accompanied by high steady state levels of mitotic cyclin-dependent kinase activity. The data indicate that DNA damage response in C. reinhardtii is connected to the cell cycle block, accompanied by increased and stabilized mitotic cyclin-dependent kinase activity.

A kinetochore-based ATM/ATR-independent DNA damage checkpoint maintains genomic integrity in trypanosomes

DNA damage-induced cell cycle checkpoints serve as surveillance mechanisms to maintain genomic stability, and are regulated by ATM/ATR-mediated signaling pathways that are conserved from yeast to humans. Trypanosoma brucei, an early divergent microbial eukaryote, lacks key components of the conventional DNA damage-induced G2/M cell cycle checkpoint and the spindle assembly checkpoint, and nothing is known about how T. brucei controls its cell cycle checkpoints. Here we discover a kinetochore-based, DNA damage-induced metaphase checkpoint in T. brucei. MMS-induced DNA damage triggers a metaphase arrest by modulating the abundance of the outer kinetochore protein KKIP5 in an Aurora B kinase- and kinetochore-dependent, but ATM/ATR-independent manner. Overexpression of KKIP5 arrests cells at metaphase through stabilizing the mitotic cyclin CYC6 and the cohesin subunit SCC1, mimicking DNA damage-induced metaphase arrest, whereas depletion of KKIP5 alleviates the DNA damage-induced metaphase arrest and causes chromosome mis-segregation and aneuploidy. These findings suggest that trypanosomes employ a novel DNA damage-induced metaphase checkpoint to maintain genomic integrity.

The Mitotic Exit Network integrates temporal and spatial signals by distributing regulation across multiple components

GTPase signal transduction pathways control cellular decision making by integrating multiple cellular events into a single signal. The Mitotic Exit Network (MEN), a Ras-like GTPase signaling pathway, integrates spatial and temporal cues to ensure that cytokinesis only occurs after the genome has partitioned between mother and daughter cells during anaphase. Here we show that signal integration does not occur at a single step of the pathway. Rather, sequential components of the pathway are controlled in series by different signals. The spatial signal, nuclear position, regulates the MEN GTPase Tem1. The temporal signal, commencement of anaphase, is mediated by mitotic cyclin-dependent kinase (CDK) phosphorylation of the GTPase’s downstream kinases. We propose that integrating multiple signals through sequential steps in the GTPase pathway represents a generalizable principle in GTPase signaling and explains why intracellular signal transmission is a multi-step process. Serial signal integration rather than signal amplification makes multi-step signal transduction necessary.