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We investigated the in vivo localisation of fission yeast cyclin-dependent kinase cdc2p during mitosis and meiosis. Fusion to yellow fluorescent protein (YFP) revealed that cdc2-YFP is present in the cytoplasm at all stages of the cell cycle. Nuclear cdc2-YFP fluorescence oscillates with that of cdc13-YFP cyclin. At G1/S, at least one of cdc13p, cig1p or cig2p B-type cyclins is required for the accumulation of cdc2-YFP into the nucleus. Cdc2-YFP and cdc13-YFP are highly enriched on the spindle pole body of cells in late G2 or arrested at S phase. Both accumulate on the spindle pole bodies and the spindle in prophase and metaphase independently of the microtubule-associated protein dis1p. In anaphase, the cdc2p/cdc13p complex leaves the spindle prior to sister chromatid separation, and cdc13-YFP is enriched at the nuclear periphery before fluorescence disappears. If cdc13p cannot be recognized by the anaphase-promoting complex, cdc2-YFP and cdc13-YFP remain associated with the spindle. In mating cells, cdc2-YFP enters the nucleus as soon as the cells undergo fusion. During karyogamy and meiotic prophase, cdc2-YFP is highly enriched on the centromeres. In meiosis I, association of cdc2-YFP with the spindle and the spindle pole bodies shows differences to mitotic cells, suggesting different mechanisms of spindle formation. This study suggests that changes in cdc2p localisation are important for both mitosis and meiosis regulation.

Schizosaccharomyces pombe cells grow from both ends during most of interphase and divide symmetrically into two daughter cells. The pom1 gene, encoding a member of the Dyrk family of protein kinases, has been identified through a mutant showing abnormal cellular morphogenesis. Here we show that Pom1p kinase activity is cell cycle regulated in correlation with the state of cellular symmetry: the activity is high during symmetrical growth and division, but lower when cells grow at just one end. Point mutations in the catalytic domain lead to asymmetry during both cell growth and division, whilst cells overexpressing Pom1p form additional growing ends. Manipulations of kinase activity indicate a negative role for Pom1p in microtubule growth at cell ends. Pom1p is present in a large protein complex and requires its non-catalytic domain to localize to the cell periphery and its kinase activity to localize to cell ends. These data establish that Pom1p kinase activity plays an important role in generating cellular symmetry and suggest that there may be related roles of homologous protein kinases ubiquitously present in all eukaryotes.

Summary S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes. However, it has been suggested that changes in the total level of CDK kinase activity, rather than substrate specificity, drive the temporal ordering of S phase and mitosis. Here, we present a phosphoproteomics-based systems analysis of CDK substrates in fission yeast and demonstrate that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK. This is achieved by rising CDK activity and the differential sensitivity of substrates to CDK activity over a wide dynamic range. This is combined with rapid phosphorylation turnover to generate clearly resolved substrate-specific activity thresholds, which in turn ensures the appropriate ordering of downstream cell-cycle events. Comparative analysis with wild-type cells expressing multiple cyclin-CDK complexes reveals how cyclin-substrate specificity works alongside activity thresholds to fine-tune the patterns of substrate phosphorylation., Graphical Abstract, Highlights • Global analysis of CDK substrates and their phosphorylation dynamics in fission yeast • CDK substrate phosphorylation can be temporally ordered by a single cyclin-CDK • Substrate-specific activity thresholds set phosphorylation timing and cell-cycle order • Cyclin-substrate specificity provides a secondary, more minor, level of regulation, A systems analysis of CDK substrates in yeast shows that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK via rising CDK activity and the differential sensitivity of substrates to CDK activity.

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe,spp1+and spp2+(S. pombe primase 1 and 2).spp1+encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polα to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1+gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1+exhibit an allele-specific abnormal mitotic phenotype. Mutations ofspp1+reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polα–primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polα has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polα. Together, these have led us to propose that suboptimal cellular levels of polα–primase complex due to the allele-specific mutations of Spp1 might not allow Polα to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100-420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed.

Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2+, that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Δ cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.

Rod-shaped fission yeast cells grow in a polarized manner, and unlike budding yeast, the correct positioning of the growth sites at cell ends requires interphase microtubules. Here we describe a microtubule guidance mechanism that orients microtubules in the intracellular space along the long axis of the cell, guiding them to their target region at the cell ends. This mechanism involves tip1p, a CLIP170-like protein that localizes to distal tips of cytoplasmic microtubules. In the absence of tip1p, microtubular catastrophe is no longer restricted to cell ends but occurs when microtubules reach any region of the cellular cortex. Thus, tip1p enables microtubules to discriminate different cortical regions and regulates their dynamics accordingly.

Downregulation of cyclin-dependent kinase (Cdk)-mitotic cyclin complexes is important during cell cycle progression and in G(1) arrested cells undergoing differentiation. srw1p, a member of the Fizzy-related protein family in fission yeast, is required for the degradation of cdc13p mitotic cyclin B during G(1) arrest. Here we show that srw1p is not required for the degradation of cdc13p during mitotic exit demonstrating that there are two systems operative at different stages of the cell cycle for cdc13p degradation, and that srw1p is phosphorylated by Cdk-cdc13p only becoming dephosphorylated during G(1) arrest. We propose that this phosphorylation targets srw1p for proteolysis and inhibits its activity to promote cdc13p turnover.

The ability to generate spatial form is a fundamental characteristic of all living organisms, which has been much studied by successive generations of developmental biologists. In recent years increasing numbers of cell biologists have turned their attention to the mechanisms by which cells generate their spatial form. These include the mechanisms that position components in different places within the cell, that specify the position of these components, and that generate the overall shape of these components. These problems are entirely analogous to those studied by developmental biologists, although usually at the level of the whole organism, organ or tissue. Because the organization of all cells is basically similar, it is possible that the concepts and the underlying molecular mechanisms of cell morphogenesis may be highly conserved. In this article we consider the generation of spatial form within the fission yeast cell, focusing on emerging new concepts, which may be applicable to the morphogenesis of other cells.

The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast.

In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression. These mutations define a single gene which we call ded1. We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species. Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex. When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins. We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G(1) arrest and subsequent mating. Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis.

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle1. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin2,3,4,5,6,7,8,9,10. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis12. Other factors acting in this control have been postulated13 and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.

The regulation of cell polarity in the fission yeast Schizosaccharomyces pombe is apparent in the restriction of extensile growth to the two ends of a cylindrically shaped cell, and in a specific transition - termed 'new-end take-off' (NETO) - between monopolar and bipolar growth mid-way through the cell cycle [1]. Several genes have been identified that affect one or more aspects of cell polarity (reviewed in [2] [3]), and the molecular pathways regulating cell polarity in fission yeast appear to be conserved among eukaryotes [3] [4] [5] [6] [7] [8] [9], but it is less clear how the proteins involved organize polarity at the level of the entire cell. Here, we describe novel cytological markers of cell polarity in fission yeast and their unusual localization in the monopolar growth mutant orb2-34, which carries a non-lethal mutation in the essential gene shk1(+)/pak1(+)/orb2(+), which encodes a p21-activated kinase (PAK) family member [8] [9] [10] [11] [12]. Our results suggest that, in contrast to other monopolar-growing mutants, the monopolar phenotype of the orb2-34 mutant might not be due to a defect in activating end growth per se, but rather reflects a failure of one of the cell ends to maintain the molecular properties that identify an end. Thus, one role of the Shk1/Pak1 kinase in vivo might be to contribute to how a cell recognizes its ends as sites for growth.

In eukaryotes, the DNA replication checkpoint prevents entry into mitosis when DNA replication is incomplete and is crucial for maintaining genomic integrity. Much less is known about equivalent controls that operate during meiosis. Here, we show that a DNA replication checkpoint control operates during meiosis in fission yeast. The mitotic checkpoint Rad genes and the Cds1 protein kinase are required for the DNA replication checkpoint during meiosis, with Cds1 playing a more prominent role than it does during mitosis. When DNA replication is blocked, the checkpoint maintains Cdc2 tyrosine 15 phosphorylation keeping Cdc2 protein kinase activity low and preventing onset of meiosis I. Additionally, there is a second checkpoint acting during meiosis that is revealed if cells are prevented from maintaining Cdc2 tyrosine 15 phosphorylation when DNA replication is blocked. Such cells arrest with high Cdc2 protein kinase activity and separated spindle pole bodies, an arrest state similar to that observed in mitotic budding yeast cells when DNA replication is incomplete. This second checkpoint is meiosis specific and may reflect processes occurring only during meiosis such as increased recombination rates, an extended duration of nuclear division, or homolog chromosome pairing.

When cells exit from mitotic cell division, their sister chromatids lose cohesion and separate to opposite poles of the dividing cell, resulting in equational chromosome segregation. In contrast, the reductional segregation of the first stage of meiotic cell division (meiosis I) requires that sister chromatids remain associated through their centromeres and move together to the same pole. Centromeric cohesion is lost as cells exit from meiosis II and sister chromatids can then separate1,2,3,4. The fission yeast cohesin protein Rec8 is specific to and required for meiosis5,6,7,8. Here we show that Rec8 appears in the centromeres and adjacent chromosome arms during the pre-meiotic S phase. Centromeric Rec8 persists throughout meiosis I and disappears at anaphase of meiosis II. When the rec8 gene is deleted, sister chromatids separate at meiosis I, resulting in equational rather than reductional chromosome segregation. We propose that the persistence of Rec8 at centromeres during meiosis I maintains sister-chromatid cohesion, and that its presence in the centromere-adjacent regions orients the kinetochores so that sister chromatids move to the same pole. This results in the reductional pattern of chromosome segregation necessary to reduce a diploid zygote to haploid gametes.

Paul Nurse, winner of the 1998 Albert Lasker Basic Medical Research Award for his work on the cell cycle, began working at the Imperial Cancer Research Fund (ICRF) as Head of the Cell Cycle Control laboratory in 1984. Twelve years later he was appointed Director General of the ICRF and now describes the origins, structure and goal of the UK's largest cancer charity.

In fission yeast, cdc18p plays a critical role in bringing about the onset of S phase. We show that cdc18p expression is subject to a complex sequence of cell cycle controls which ensure that cdc18p levels rise dramatically as cells exit mitosis, before the appearance of CDK activity in G1. We find that transcription of cdc18, together with the transcription of other cdc10p/res1p targets, is first initiated as cells enter mitosis and continues even in cells arrested in mitosis with highly condensed chromatin. However, cdc18p cannot accumulate during mitosis because it is targeted for proteolysis by mitotic cdc2p-protein kinase-mediated phosphorylation. On exit from mitosis, the cdc2p mitotic kinase activity falls, stabilizing cdc18p, which then rapidly accumulates. This combination of mitotic transcription and CDK-mediated proteolysis ensures that progression through mitosis simultaneously prepares cells for DNA replication. During S phase, cdc18 transcription is then switched off, preventing the re-initiation of DNA synthesis until the completion of the next round of mitosis.

Yoshio Masui is a gentle, modest traditional biologist whose contributions have had a major influence on the recent advances that have taken place in understanding how the cell cycle is controlled. His work forms part of a long tradition of experimental zoologists beginning at the end of the last century, who have studied the process of cell division and its control. In this essay I want to place Yoshio Masui's contributions in perspective, as the successor to these early experimentalists, and as the forerunner of the molecular biologists now working on cell cycle control. For a fuller treatment of much of the same historical material, I recommend Yoshio Masui's own scholarly review (Masui (1992) Biochem Cell Biol 70, 920–945). After this I will briefly summarise the central role of cyclin dependent kinases (CDK's) in controlling the cell cycle, following Yoshio Masui's own example by emphasising their biological significance. Finally I will sketch out some of the unsolved problems of the future in this area of research as I see them.

To investigate the role of microtubules in regulating cell polarity in Schizosaccharomyces pombe, we have developed a system in which normally cylindrical fission yeast synchronously form branched cells at high frequency upon treatment with the microtubule-depolymerizing drug thiabendazole (TBZ). Branching depends on both elevated temperature and cell cycle state and occurs at high frequency only when TBZ is added to cells that have not yet passed through New-End Take-Off (NETO), the normal transition from monopolar to bipolar growth. This suggests that microtubules may be of greatest physiological importance for the maintenance of cell shape at specific points in the cell cycle. The localization of three different proteins normally found at cell ends—cortical F-actin, tea1, and an ral3 (scd2)–green fluorescent protein (GFP) fusion—is disrupted by TBZ treatment. However, these proteins can eventually return to cell ends in the absence of microtubules, indicating that although their localization to ends normally depends on microtubules, they may recover by alternative mechanisms. In addition, TBZ induces a shift in ral3–GFP distribution from cell ends to the cell middle, suggesting that a protein complex containing ral3 may be part of the cue that specifies the position of branch formation.

The alignment of homologous chromosomes during meiosis is essential for their recombination and segregation. Telomeres form and protect the ends of eukaryotic linear chromosomes, and are composed of tandem repeats of a simple DNA sequence and the proteins that bind to these repeats1,2,3. A role for telomeres in meiosis was suspected from observations of telomere clustering in meiotic cells4,5,6,7 and has now been supported experimentally by the dramatic rearrangement of telomere locations during premeiotic stages in fission yeast8,9. Here we show that the fission yeast telomere protein, Taz1 (ref. 10), is required for stable association between telomeres and spindle pole bodies during meiotic prophase. In the absence of Taz1, telomere clustering at the spindle pole bodies is disrupted, meiotic recombination is reduced, and both spore viability and the ability of zygotes to re-enter mitosis are impaired to a level that would be expected if chromosome segregation were occurring randomly. Such telomeric association mediated by telomere-specific proteins may also be important for proper chromosome alignment and recombination during meiosis in humans.

How cells generate and orientate polarized growth is of fundamental importance to understanding cell morphogenesis. The budding yeast Saccharomyces cerevisiae and the distantly related fission yeast Schizosaccharomyces pombe have both been used for genetic analysis of cell morphogenesis. Generation and maintenance of their cell shape require the formation of polarized growth sites and the correct localization of these growth sites on the cell surface with respect to other cellular structures. In this review, the authors discuss and compare the mechanisms used by the two yeasts to achieve polarized growth.

In fission yeast, passage through START and into S-phase requires cyclin-dependent kinase (CDK) activity and the periodic transcription of genes essential for S-phase ('S-phase transcription'). Here we investigate the control of this transcription in the mitotic cell cycle. We demonstrate that the periodicity of S-phase transcription is likely to be controlled independently of CDK activity. This contrasts with the equivalent system in budding yeast. Furthermore, the CDK function required for S-phase acts after the onset of S-phase transcription and after the accumulation of cdc18p, a critical target of this transcriptional machinery. We investigate the role of individual components of the S-phase transcriptional machinery, cdc10p, res1p, res2p and rep2p, and define a new role for res2p, previously demonstrated to be important in the meiotic cycle, in switching off S-phase transcription during G2 of the mitotic cycle. We show that the presence of the in vitro bandshift activity DSC1, conventionally thought to represent the active complex, requires res2p and correlates with inactive transcription. We suggest that S-phase transcription is controlled by both activation and repression, and that res2p represses transcription in G2 of the cell cycle as a part of the DSC1 complex.

The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up-regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.

As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a syntheticlethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

What is the best way to promote innovative research? How can we build research institutions of the future that will promote cross-disciplinary interactions among young creative minds? Despite the recent growth in scientific knowledge, conventional disciplinebased methods have not been sufficiently effective at developing new understanding and treatments. Researchers need to be encouraged to identify important questions and tackle them with multidisciplinary approaches. Contemporary biomedical research has to integrate biological, nonbiological, and clinical disciplines, and its application requires interactions with hospital and commercial partners. This can be facilitated by research institutions with an environment that supports strong interdisciplinary interactions between scientists: a place where laboratory biologists are encouraged to collaborate with clinical researchers to understand the medical implications of their work, with pharmaceutical companies for the translation of discoveries into treatments, and with physical scientists to expand their thinking and repertoire of experimental approaches. Such an institution must be continually open to new ideas and permeable to interactions with outside researchers and organizations. ![Figure][1] CREDIT: CITYSCAPE/THE FRANCIS CRICK INSTITUTE Another imperative is to support early career scientists in setting up their own independent research programs. This initial period, when young investigators are often at their most creative, frequently sees them rein in their ambition and originality, given the pressures of securing grant funding and establishing a laboratory group. Too often this results in derivative research goals and aspirations driven by incremental objectives. New principal investigators need a supportive research environment, secure research funding, and time to make important discoveries. New institutions have adopted different approaches to promote innovative and collaborative atmospheres, including the Janelia Farm Research Campus and more recently the Francis Crick Institute, opening in London in 2015. The Crick will be a large institute (120 groups) with a broad and diverse research agenda. Cross-disciplinary and clinical interactions will be forged with researchers from three university partners (University College London, Imperial College London, and King's College London) and the Wellcome Trust Sanger Institute, through the reciprocal interchange of small satellite groups of one to three researchers. And the institute will encourage exchanges and visits from other research institutions, including the for-profit sector and health care delivery organizations. Architecturally, the institute has been designed to resemble a chromosome, with the four arms meeting at a “centromere” that facilitates informal encounters and exchanges. The largely open-plan laboratories will juxtapose groups with different interests and encourage their mixing. Rather than traditional departments, Crick investigators will self-organize into interest groups focused around research questions or technical approaches. Researchers will be free to join as many or as few of these as they wish, and the groups will come and go as science develops. Moreover, around two-thirds of the Crick's research group leaders will be in the initial phase of their independent careers, with a smaller number of more senior investigators to mentor younger colleagues and help identify the best new recruits. These appointments will be of up to 12 years, supported by the Crick's funding partners (the Medical Research Council, Cancer Research UK, and the Wellcome Trust). Group leaders will then leave the institute to establish a research group elsewhere; the aim is to give researchers who are effective and remain in the United Kingdom a transition package to support their moves, creating a thriving network of highly trained researchers. Francis Crick commented that “by the time most scientists have reached age thirty they are trapped by their own expertise.” It is fitting that Crick's eponymous institute aims to develop a culture that escapes this constraint. [1]: pending:yes

Transcriptional control of DHHC palmitoyltransferase activity levels shapes cellular palmitoylation patterns and influences meiotic entry in fission yeast., Protein S-palmitoylation, a lipid modification mediated by members of the palmitoyltransferase family, serves as an important membrane-targeting mechanism in eukaryotes. Although changes in palmitoyltransferase expression are associated with various physiological and disease states, how these changes affect global protein palmitoylation and cellular function remains unknown. Using a bioorthogonal chemical reporter and labeling strategy to identify and analyze multiple cognate substrates of a single Erf2 palmitoyltransferase, we demonstrate that control of Erf2 activity levels underlies the differential modification of key substrates such as the Rho3 GTPase in vegetative and meiotic cells. We show further that modulation of Erf2 activity levels drives changes in the palmitoylome as cells enter meiosis and affects meiotic entry. Disruption of Erf2 function delays meiotic entry, while increasing Erf2 palmitoyltransferase activity triggers aberrant meiosis in sensitized cells. Erf2-induced meiosis requires the function of the Rho3 GTPase, which is regulated by its palmitoylation state. We propose that control of palmitoyltransferase activity levels provides a fundamental mechanism for modulating palmitoylomes and cellular functions., Author Summary Palmitoylation is a fatty acid modification, which increases the hydrophobicity of proteins, known to control the membrane trafficking and function of many proteins important for cellular homeostasis and disease. Being a reversible process, palmitoylation is proposed to be a major cellular regulator. While changes in global protein palmitoylation patterns are often associated with different physiological states, the mechanisms that regulate this posttranslational modification and their functional consequences have been elusive. Using improved methods for protein palmitoylation analysis in the genetically tractable fission yeast Schizosaccharomyces pombe, we discovered that the global protein palmitoylation pattern is significantly altered and correlates with entry into meiosis, a major developmental transition. We further showed that the precise level of a palmitoylating enzyme is important to shape the subset of lipid-modified proteins and influence the start of this cellular transition. As palmitoylated proteins are involved in eukaryotic cellular pathways, our results provide a crucial insight into how the quantitative control of palmitoylating enzymes may impact human physiology and disease.

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.

We have cloned a novel fission yeast gene, spd1, which causes G1 arrest when overexpressed. Deleting the gene results in cells being accelerated through G1 into S phase in certain circumstances when the G1-->S phase control is compromised. We have found that the encoded 14 kDa protein is cell cycle regulated, declining in level during S phase, and that p14spd1 physically associates with p34cdc2 in vivo when overexpressed, suggesting that p14spd1 may regulate S phase progression via an interaction with p34cdc2. We conclude that p14spd1 is a negative regulator of S phase, and that it may be part of the control ensuring an orderly onset of S phase or part of a G1-->S phase checkpoint control.

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.

In this article we consider the role of the cyclin-dependent protein kinase cdc2 in regulating progression through the fission yeast cell cycle. The onset of mitosis is governed by cdc2 in partnership with the B-type cyclin, cdc13. Recent evidence shows that the cdc2-cdc13 complex can also control the onset of S phase and, in addition, ensures that there is only one S phase per cell cycle. This leads us to propose a novel quantitative model in which different levels of cdc2 activity regulate cell-cycle progression: S phase is initiated when protein kinase activity increases from a very low to a moderate level; maintenance of this moderate level prevents re-initiation of S phase, and a further increase of activity to a high level initiates mitosis. Inactivation of the kinase activity at the end of mitosis resets the cell for a new cell cycle.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.

In fission yeast, we propose that the division plane may be positioned by the position of the premitotic nucleus, perhaps by a signal emanating from the nucleus. Gene products involved in the assembly of the ring and its temporal and spatial controls are beginning to be characterized. Some of these, such as mid1p, may be involved in signals that position the division site. In animal cells, the division site may be determined by analogous signals emanating from the mitotic asters (Rappaport, 1986). We speculate that the signals defined in fission yeast will help identify signals that determine the division plane in all kinds of eukaryotes.

First paragraph (this article has no abstract) For well over 100 years, cell biologists have been wondering what determines the size of cells. In modern times, we know all of the molecules that control the cell cycle and cell division, but we still do not understand how cell size is determined. To check whether modern cell biology has made any inroads on this age-old question, BMC Biology asked several heavyweights in the field to tell us how they think cell size is controlled, drawing on a range of different cell types. The essays in this collection address two related questions - why does cell size matter, and how do cells control it.

To identify new genes involved in the control of cell morphogenesis in the fission yeast Schizosaccharomyces pombe we have visually screened for temperature-sensitive mutants that show defects in cell morphology. We have isolated and characterized 64 mutants defining 19 independent genes, 10 of which have not been previously described. One class of mutants, defining 12 orb genes, become round and show a complete loss of cell polarity. A second class of mutants exhibits branched or bent morphologies. These mutants show defects in either selection of the growth site, defining two tea genes, or in the maintenance of growth direction, defining five ban genes. Immunofluorescence analysis of these morphological mutants shows defects in the organization of the microtubule and actin cytoskeleton. These defects include shortened, bundled, and asymmetrically localized microtubules and enlarged and mislocalized actin patches. Analysis of the mutant phenotypes has allowed us to order the genes into four groups according to their function during the cell cycle: genes required for the maintenance of cell polarity throughout the cell cycle; genes necessary only for the reestablishment of cell polarity after mitosis and not for maintaining cell polarity once it is established; genes essential for the transition from monopolar to bipolar growth and genes that severe as 'polarity markers'.

p25 rum1 from the fission yeast S. pombe is shown to act as a specific in vitro inhibitor of the p34 cdc2 /p56 cdc13 mitotic kinase. It is also shown that early G1 cells contain p25 rum1 which associates with and inhibits the mitotic kinase, and maintains p56 cdc13 mitotic B cyclin at a low level, ensuring that these cells do not undergo a premature lethal entry into mitosis. A high level of p25 rum1 in G2 cells inhibits the p34 cdc2 /p56 cdc13 kinase that removes the block preventing a further S phase and leads to repeated rounds of DNA replication. Thus, the cyclin-dependent kinase inhibitor p25 rum1 acting on the p34 cdc2 mitotic kinase, plays an important role in ensuring the correct sequence of S phase and mitosis during the cell cycle.

A key problem in the cell cycle is understanding what brings about the initiation of DNA replication and how this is linked with global cell cycle controls. The fission yeast gene cdc18 is required for DNA replication and is transcriptionally activated by the cdc10/res1/res2 control acting at START in late G1. We show here that overexpressing cdc18 is able to bring about repeated rounds of DNA synthesis in the absence of mitosis and of continuing protein synthesis. The level of the cdc18 -encoded protein p65 ced18 is periodic in the cell cycle, peaking at the G1 to S phase transition, and p65 cdc18 is located in the nucleus when cdc18 is overexpressed. We propose that p65 cdc18 acts at the initiation of DNA replication and plays a major role in controlling the onset of S phase.

Summary All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins’ physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin’s role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly., Graphical Abstract, Highlights • Ribozinoindoles are potent chemical inhibitors of eukaryotic ribosome assembly • Activity of four of Mdn1’s six ATPase sites is likely needed for cell growth • Ribozinoindoles inhibit recombinant full-length Mdn1’s ATPase activity in vitro • Assembly of Nsa1 particles, precursors of the 60S subunit, depends on Mdn1, Selective potent inhibitors of eukaryotic ribosome biogenesis are found through a chemical synthetic lethal screen.

A large number of dominant mutants have been generated in the fission yeast cdc2 gene, causing lethality when expressed in wild-type cells. The mutants interfere with distinct aspects of p34cdc2 function, producing one of four different phenotypes: mitotic arrest, multiple rounds of S phase in the absence of mitosis, premature mitosis or G2 arrest. The mitotic mutants DL41, DL45 and DL50 are characterized in this paper. Over-expression of DL41 or DL45 causes mitotic arrest, specifically interfering with sister chromatid separation, without preventing spindle elongation. This suggests a role for p34cdc2 in triggering sister chromatid separation at anaphase. DL41 and DL45 also cause abnormal septum formation, suggesting that p34cdc2 may also be involved in regulating this process in fission yeast. These mitotic aspects of p34cdc2 function may involve interaction with p13suc1, since increased expression of suc1 partially suppresses DL41 and DL45. Over-expression of DL50 causes premature mitotic entry in cells that have not completed S phase, resulting in lethality. DL41, DL45 and DL50 correspond to mutation of p34cdc2 residues predicted to be on the surface of the protein, identifying potential sites of interaction with mitotic regulators of p3cdc2, and these residues are conserved amongst cdc2 proteins found in other eukaryotes.

When cell cycle progression in fission yeast is disrupted, checkpoint controls ensure that the normal sequence of cell cycle events is maintained. Activation of a checkpoint relies on monitoring signals that might involve assembly of macromolecular structures essential for specific cell cycle processes. The past year has seen further elucidation of two new checkpoints operating during the cell cycle of Schizosaccharomyces pombe. One involves the product of the rum1 gene and prevents cells from entering mitosis from the pre-Start G1 interval. The second checkpoint operates during the later stages of the cell cycle and is essential for coupling the events of mitosis and cell division.

SummaryChemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a “closed” mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.

The fission yeast, Schizosaccharomyces pombe, has been used extensively for genetic studies but until now it has not been utilized as a host organism for DNA cloning. Here we describe a method for high-frequency transformation fo a leu 1(-) strain of this yeast with hybrid plasmids containing the Saccharomyces cerevisiae LEu 2(+) gene, a bacterial plasmid and either the S. cerevisiae 2 μm plasmid or autonomously replicating sequences (ars)(1) derived from S. pombe DNA. Some of the plasmids contain unique restriction sites which make them suitable for the isolation of S. pombe genes, and they can also be used for the exchange of DNA between S. pombe and S. cerevisiae.

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability., Author Summary Aneuploidy is a major cause of abortive development and is implicated in tumorigenesis in humans. Recent studies revealed that the increased need for protein degradation might account for the detrimental effects of aneuploidy on a cell. Here, we investigated the genetic systems responsible for aneuploid viability. Using a collection of gene deletions in fission yeast, we isolated mutants that affect aneuploid viability. We found that an evolutionarily conserved transcription regulator, the CCR4-NOT complex, and its related factors are required for aneuploid viability, suggesting that regulation of mRNA turnover is required to tolerate aneuploidy. In addition, homologous recombination repair is important for aneuploid viability.

How does a cell know if it's in G1 or G2 and should proceed to S phase or mitosis? This is a restatement of the question of how a cell ensures mitosis is dependent upon S phase, and S phase is dependent upon mitosis. Several gene products have been identified which play important roles in maintaining these interdependencies. Central to these controls are oscillations between different complexes of cyclins and cyclin-dependent kinases.

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.

The 110-kDa catalytic subunit of the phosphatidylinositol 3'-kinase (p110) is shown to be expressed in Schizosaccharomyces pombe as a functional protein, as judged by the accumulation of 3'-phosphorylated lipids in vivo and the extraction of 3'-kinase activity in vitro. On expression of p110, the cells fail to grow and lose viability. In contrast, while the Saccharomyces cerevisiae protein Vps34p can be expressed in S. pombe as a functional, extractable, phosphatidylinositol 3-kinase, expression of this protein fails to increase substantially 3'-phosphorylated lipids in vivo and does not induce a phenotype equivalent to that induced by p110. The results indicate that (over-)-accumulation of 3'-phosphorylated inositol lipids in S. pombe causes loss of viability.

Structural models for the eukaryotic cell cycle control protein p34 from human, S. pombe and S. cerevisiae have been derived from the crystallographic coordinates of the cAMP-dependent protein kinase (cAPK) catalytic subunit (active conformation) and compared with the structure of inactive CDK2 apoenzyme. Differences between the p34 and cAPK catalytic sites provide a possible explanation for their different substrate specificities. The p34 models localize Tyr15 and Thr14 close to the sites of catalysis and substrate recognition where their phosphorylation could inhibit p34 kinase activity either by blocking MgATP or substrate binding. The conserved sequences PSTAIRE and LYLIFEFL are both close to the catalytic site and accessible on the protein surface available to mediate interactions with other proteins. It is predicted that p34 has an active-site cleft composed almost entirely of sequences common to all protein kinases and sequences unique to the p34 protein family. Genetic and biochemical analyses of p34 have shown that it interacts extensively with a number of other proteins. The model allows the relative disposition of these sites of mutation to each other and to the sites of catalysis and substrate recognition to be appreciated. Surface regions on p34 that are important for function have been identified. These sites identify residues that may interact with p13suc1, cyclin, p107wee1 and p80cdc25.